e - h , Ectopic expression of GFP - tagged NetB in the eye disc induces high levels GFP - NetB protein in the fat body . Single confocal z - section are shown images ( e and g ) . Data information : Scale bar , 20 μm f - h 50

e-h, Ectopic expression of GFP-tagged NetB in the eye disc induces high levels GFP-NetB protein in the fat body. Single confocal z-section are shown images (e and g). Data information: Scale bar, 20 μm f-h 50 μm


j - k , Ectopic expression of GFP - tagged NetB in the eye disc leads to detection of GFP signals in the hemolymph . Data information : Scale bar , 20 μm j - k )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j-k, Ectopic expression of GFP-tagged NetB in the eye disc leads to detection of GFP signals in the hemolymph. Data information: Scale bar, 20 μm j-k)


a , Knockdown of InR ( InR ) or expressing a dominant - negative form of InR ( InR - DN ) in the fat body using CG - Gal4 increases survival over oncogenic Ras expression in the imaginal disc . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test ( a NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a, Knockdown of InR (InR) or expressing a dominant-negative form of InR (InR-DN) in the fat body using CG-Gal4 increases survival over oncogenic Ras expression in the imaginal disc. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (a NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


c , qRT - PCR demonstrates that TMLHE expression in the fat body is reduced by Ras expression in the eye disc and reversed by insulin inhibition in the fat body . d , qRT - PCR demonstrates that NetB knockdown in the eye disc increases TMLHE expression in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test ( c and d ) NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, qRT-PCR demonstrates that TMLHE expression in the fat body is reduced by Ras expression in the eye disc and reversed by insulin inhibition in the fat body. d, qRT-PCR demonstrates that NetB knockdown in the eye disc increases TMLHE expression in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (c and d) NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


e , Knockdown of unc - 5 , but not other NetB receptors ( Fra or Dscam1 ) , in the fat body increases TMLHE expression . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test e ) . NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Knockdown of unc-5, but not other NetB receptors (Fra or Dscam1), in the fat body increases TMLHE expression. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test e). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


b , Local expression of oncogenic Ras in the eye disc decreases the amount of carnitine in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b, Local expression of oncogenic Ras in the eye disc decreases the amount of carnitine in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.


c , TMLHE knockdown in the fat body aggravates organismal survival over the oncogenic stress . d , In the absence of GMR - RasV12 , inhibition of TMLHE in the fat body is not sufficient to induce organismal death . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, TMLHE knockdown in the fat body aggravates organismal survival over the oncogenic stress. d, In the absence of GMR-RasV12, inhibition of TMLHE in the fat body is not sufficient to induce organismal death. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.


g , Feeding of acetyl - CoA precursor ( acetate ) makes GMR - RasV12 flies survive even with TMLHE knockdown in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g, Feeding of acetyl-CoA precursor (acetate) makes GMR-RasV12 flies survive even with TMLHE knockdown in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.


e , qRT - PCR with mRNA from the adult gut confirms higher expression of NetB in esg ts > RasV12 flies after oncogenic Ras induction for 10 days . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The statistical significance was determined by two - tailed unpaired t - test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, qRT-PCR with mRNA from the adult gut confirms higher expression of NetB in esg ts>RasV12 flies after oncogenic Ras induction for 10 days. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


f - g , Oncogenic Ras expression in the adult gut induces NetB protein , which was detected by GFP signal using CPTI - 000748 , a protein trap of NetB . Ras was induced for 10 days . Single confocal z - section images are shown . h , Quantification of GFP signals in f and g . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The statistical significance was determined by two - tailed unpaired t - test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f-g, Oncogenic Ras expression in the adult gut induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Ras was induced for 10 days. Single confocal z-section images are shown. h, Quantification of GFP signals in f and g. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001. Scale bar, 50 μm.


i , Knockdown of NetB in the adult gut of esg ts > RasV12 flies enhances survival . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The experiments were repeated independently at least twice with similar results The statistical significance was determined by log - rank ( Mantel - Cox ) test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

i, Knockdown of NetB in the adult gut of esg ts>RasV12 flies enhances survival. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


l , Knockdown of TMLHE in the fat body of adult flies shortened lifespan of esgts > RasV12 flies but not of control flies . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The experiments were repeated independently at least twice with similar results The statistical significance was determined by log - rank ( Mantel - Cox ) test l ) . NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

l, Knockdown of TMLHE in the fat body of adult flies shortened lifespan of esgts>RasV12 flies but not of control flies. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test l). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


Figure 1 - The origin of N - terminal proteoforms . A . 2 , 578 proteoforms were identified including 2 , 135 dbTIS and 443 aTIS . For the majority of proteins we found a unique translation initiation site ( grey ) . Additionally , 335 N - terminal peptides pointed to proteins with multiple N - termini created by alternative translation initiation ( blue ) , whereas 81 N - termini were generated by alternative iMet processing ( green ) . B - C . Proteomics - derived translation initiation events were verified at the transcript level using Ribo - Seq data obtained in Jurkat cells and HCT 116 cells ( data unpublished ) . Although in this study , the degree of in vivo Nt - acetylation could not be directly determined , all retained N - termini were compliant with the rules of N - terminal processing from previous experiments ( Van Damme et al , 2014 ) . In the case N - termini were found to be in vivo Nt - acetylated ( previous MS / MS - based evidence ( Van Damme et al , 2014 ) ) this information is indicated . dbTIS and aTIS peptides were further mapped to protein N - termini annotated in Swiss - Prot Isoform , UniProt TrEMBL and Ensembl databases and neo - N - termini ( proteolytic products ) of the knowledgebase TopFind 3 . 0 . The number of matching N - termini is indicated in dark grey . N - terminal peptides supported by at least one source of metadata pointing to translation ( Nt - acetylation , Ribo - Seq , Swiss - Prot Isoforms , TrEMBL or Ensembl ) were classified as highly confident TIS ( indicated in green ) . D . Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript ( leaky scanning ) contributes to the majority of alternative TIS identified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 - The origin of N-terminal proteoforms. A. 2,578 proteoforms were identified including 2,135 dbTIS and 443 aTIS. For the majority of proteins we found a unique translation initiation site (grey). Additionally, 335 N-terminal peptides pointed to proteins with multiple N-termini created by alternative translation initiation (blue), whereas 81 N-termini were generated by alternative iMet processing (green). B-C. Proteomics-derived translation initiation events were verified at the transcript level using Ribo-Seq data obtained in Jurkat cells and HCT 116 cells (data unpublished). Although in this study, the degree of in vivo Nt-acetylation could not be directly determined, all retained N-termini were compliant with the rules of N-terminal processing from previous experiments (Van Damme et al, 2014). In the case N-termini were found to be in vivo Nt-acetylated (previous MS/MS-based evidence (Van Damme et al, 2014)) this information is indicated. dbTIS and aTIS peptides were further mapped to protein N-termini annotated in Swiss-Prot Isoform, UniProt TrEMBL and Ensembl databases and neo-N-termini (proteolytic products) of the knowledgebase TopFind 3.0. The number of matching N-termini is indicated in dark grey. N-terminal peptides supported by at least one source of metadata pointing to translation (Nt-acetylation, Ribo-Seq, Swiss-Prot Isoforms, TrEMBL or Ensembl) were classified as highly confident TIS (indicated in green). D. Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript (leaky scanning) contributes to the majority of alternative TIS identified.


Figure 2 - Examples of translation initiation events confirmed by Ribo - Seq in Jurkat cells . A - B . Alternative translation initiation due to leaky scanning . C . Alternative splicing . D . The preferential expression of an N - terminally truncated proteoform confirmed by Ribo - Seq data . E - F . Evidence for alternative translation initiation occurring at two consecutive AUG codons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 - Examples of translation initiation events confirmed by Ribo-Seq in Jurkat cells. A-B. Alternative translation initiation due to leaky scanning. C. Alternative splicing. D. The preferential expression of an N-terminally truncated proteoform confirmed by Ribo-Seq data. E-F. Evidence for alternative translation initiation occurring at two consecutive AUG codons.


A . Jurkat cells were pre - labeled with light or medium L - Arg isotopes . A label swap was performed of cells growing in medium Arg , whereas light cells were cultured in the same medium . Cells were harvested at different time points and equal proteome amounts were mixed , followed by N - terminal COFRADIC fractionation and LC - MS / MS analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis.


C . The distribution of turnover times ( N = 1 , 894 ) in our experiment was not unimodal , with a sizable number of quickly turned over proteins , with the median protein turnover rate being 21 . 6 h . The doubling time of Jurkat cells ( 24 h ) is indicated in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. The distribution of turnover times (N=1,894) in our experiment was not unimodal, with a sizable number of quickly turned over proteins, with the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.


A . Degradation profiles of AIMP2 , MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N - terminal proteoforms that differ in stability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability.


B . MS spectra of the N - termini of two proteoforms originating from the aminoacyl tRNA synthase complex - interacting multifunctional protein 2 ( AIMP2 ) : a dbTIS indicative N - terminus with processed iMet and starting at the second amino acid Pro , an aTIS indicative peptide starting at position 3 and retaining its iMet . As evident from the MS spectra shown , these N - terminal proteoforms displayed largely different turnover rates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates.


A . Jurkat cells were treated with 100 µg / ml CHX for 0 , 0 . 5 , 1 . 5 , 4 , 8 , 12 or 24 hours . Protein degradation was monitored by Western blotting and stabilities of several short - lived endogenous proteins were confirmed using antibodies ( including lamin B , securin , β - catenin , GCIP interacting protein p29 ) and compared to stable proteins ( such as GAPDH and actin ) . Proteoform - specific bands were used to calculate protein half - lives which were in a good agreement with turnover times obtained from

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILAC


B - C . Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E . Selective C - terminal V5 - tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse - chase experiments were performed as described for Jurkat cells . Degradation of overexpressed proteoforms was monitored by an anti - V5 antibody and compared to the turnover of stable proteins such as actin and tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.


( A , B ) Overexpression of wildtype full - length human tau ( hTau , also termed tau441 or tau40 or tau2N4R ) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II , in which 96 transcription factors ( Appendix Table S1 and S2 ) were monitored . The empty vector was transfected as a control ( Ctrl ) .

(A, B) Overexpression of wildtype full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Table S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl).


( C - F ) Expression of hTau ( probed by HT7 ) increased total and the phosphorylated STAT1 at Tyr701 ( pY - STAT1 ) in cell whole extracts ( C , D ) and the nuclear fraction ( E , F ) measured by Western blotting ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 vs Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-F) Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in cell whole extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 vs Ctrl.


( G ) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control ( eGFP ) ( n = 5 ) . Scale bar , 10 μm . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . , * * , p < 0 . 01 vs Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n=5). Scale bar, 10 μm. Data information: Data were presented as mean ± SD (Mann-Whitney test). , **, p<0.01 vs Ctrl.


( H ) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction ( Nu ) measured by Western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting.


( I ) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 , * * * , p < 0 . 001 vs Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 , ***, p<0.001 vs Ctrl.


( J ) Overexpression of hTau increased STAT1 - DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay ( EMSA ) . * , indicates STAT1 / DNA complex .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.


( A , B ) AAV - hTau - eGFP ( AAV - hTau ) or the empty vector AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , the increased levels of STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) AAV-hTau-eGFP (AAV-hTau) or the empty vector AAV-eGFP (1.13×1013 v.g./ml) was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, the increased levels of STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( C , D ) The increased STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction of 12 - month - old hTau transgenic mice measured by Western blotting ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) The increased STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction of 12-month-old hTau transgenic mice measured by Western blotting (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( E , F ) The representative images of STAT1 and pY - STAT1 in the brain of AD patients probed by co - immunohistochemical staining and quantitative analysis ( hematoxylin stains nuclei , purple ; DAB stains the target proteins , brown ; n = 5 - 6 slices ) . Arrowheads indicated typical nuclear staining of STAT1 / pY - STAT1 . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n=5-6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1. Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( G ) The increased AT8 ( pS202 / pT205 ) , STAT1 and pY - STAT1 in cortex total extracts of AD patients measured by Western blotting ( n = 3 , Student ' s t test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The increased AT8 (pS202/pT205), STAT1 and pY-STAT1 in cortex total extracts of AD patients measured by Western blotting (n=3, Student's t test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( H ) STAT1 mRNA was analyzed by qPCR in AAV - hTau or AAV - eGFP infected hippocampal tissues ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) STAT1 mRNA was analyzed by qPCR in AAV-hTau or AAV-eGFP infected hippocampal tissues (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by immunohistochemical staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by immunohistochemical staining.


AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by Western

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by Western blotting


( C ) Downregulation of STAT1 ameliorated hTau - induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze ( MWM ) test ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for C * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Downregulation of STAT1 ameliorated hTau-induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze (MWM) test (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's post hoc test for C *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( D - G ) Downregulation of STAT1 ameliorated hTau - induced spatial memory deficit shown by the decreased latency to reach the platform quadrant ( D ) , the increased crossing time in the platform site ( E ) and time spent in the target quadrant ( F ) measured at day 6 by removed the platform in MWM test ; no motor dysfunction was seen ( G ) ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-G) Downregulation of STAT1 ameliorated hTau-induced spatial memory deficit shown by the decreased latency to reach the platform quadrant (D), the increased crossing time in the platform site (E) and time spent in the target quadrant (F) measured at day 6 by removed the platform in MWM test; no motor dysfunction was seen (G) (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. , one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( H ) Downregulation of STAT1 ameliorated hTau - induced contextual memory deficits measured at 24 h during contextual fear conditioning test ( n = 8 each group ) . Data information : Data were presented as mean ± s . e . m . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Downregulation of STAT1 ameliorated hTau-induced contextual memory deficits measured at 24 h during contextual fear conditioning test (n=8 each group). Data information: Data were presented as mean ± s.e.m. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( I - L ) One month after the virus infection , paired - pulse ratio ( PPR ) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice ( I ) . The IO curve of fEPSP recorded on acute hippocampal slices ( J ) . The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice ( K ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( L ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for I - K , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I-L) One month after the virus infection, paired-pulse ratio (PPR) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice (I). The IO curve of fEPSP recorded on acute hippocampal slices (J). The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice (K). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (L). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for I-K, one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( M - P ) One month after the virus infection , whole cell patch clamp was used to measure the function of NMDA ( at + 40 mV ) and AMPA ( at - 70 mV ) receptors on acute brain slices ( 400 μm ) . The insets show representative sample traces of EPSCs in virus infected neurons ( M ) . The reduced NMDA and unchanged AMPA currents with a reduced NMDA / AMPA ratio were seen in hTau infected neurons , while knockdown of STAT1 restored the hTau - induced NMDA currents ( N - P ) . ( n = 12 neurons from 4 animals for eGFP group ; n = 11 neurons from 4 animals for hTau group ; n = 13 neurons from 4 animals for hTau + CRE group ) . Data information : Data were presented as mean ± SD one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M-P) One month after the virus infection, whole cell patch clamp was used to measure the function of NMDA (at +40 mV) and AMPA (at -70 mV) receptors on acute brain slices (400 μm). The insets show representative sample traces of EPSCs in virus infected neurons (M). The reduced NMDA and unchanged AMPA currents with a reduced NMDA/AMPA ratio were seen in hTau infected neurons, while knockdown of STAT1 restored the hTau-induced NMDA currents (N-P). (n=12 neurons from 4 animals for eGFP group; n=11 neurons from 4 animals for hTau group; n=13 neurons from 4 animals for hTau+CRE group). Data information: Data were presented as mean ± SD one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( A , B ) Overexpression of AAV - hTau decreased the protein levels of GluN1 , GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice , compared with the AAV - eGFP vector control . ( C , D ) Simultaneous downregulation of STAT1 by infusing AAV - Cre in hippocampal CA3 of STAT1flox / flox mice abolished the hTau - induced inhibition in expression of NMDARs protein . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Overexpression of AAV-hTau decreased the protein levels of GluN1, GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice, compared with the AAV-eGFP vector control. (C, D) Simultaneous downregulation of STAT1 by infusing AAV-Cre in hippocampal CA3 of STAT1flox/flox mice abolished the hTau-induced inhibition in expression of NMDARs protein. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.


( E , F ) Overexpression of AAV - hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1 , GluN2A and GluN2B detected by qRT - PCR in the hippocampal CA3 . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) Overexpression of AAV-hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1, GluN2A and GluN2B detected by qRT-PCR in the hippocampal CA3. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.


( A ) Overexpression of AAV - hTau increased binding of STAT1 to the promoter regions of GluN1 , GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay ( ChIP ) ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Overexpression of AAV-hTau increased binding of STAT1 to the promoter regions of GluN1, GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay (ChIP) (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


( B ) Overexpression of hTau or wildtype STAT1 ( WT - STAT1 ) inhibits the transcription activity of NMDARs compared with the empty vector control ( Ctrl ) measured by luciferase activity assay in HEK293 cells ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Overexpression of hTau or wildtype STAT1 (WT-STAT1) inhibits the transcription activity of NMDARs compared with the empty vector control (Ctrl) measured by luciferase activity assay in HEK293 cells (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN1 ( C ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN1 (C) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2B ( E ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2B (E) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2A ( G , I ) . The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2A (G, I). The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


( A , B ) Overexpression of hTau in HEK293 cells for 48 h increased the activity - dependent phosphorylation of JAK2 , JNK1 and ERK1 compared with the empty vector control ( Ctrl ) measured by Western blotting ( n = 3 , student ' s t test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3, student's t test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of ERK1 ( C , D ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts ( C and the nuclear fraction ( D measured by Western blotting ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

Pharmacological inhibition of ERK1 (C, D) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C and the nuclear fraction (D measured by Western blotting (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of JNK1 ( E , F ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts E ) and the nuclear fraction F ) measured by Western blotting ( n = 3 ) . The alteration of pS - STAT1 ( Ser727 ) confirms the efficacy of JNK1 inhibitors . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pharmacological inhibition of JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts E) and the nuclear fraction F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of JAK2 ( G , H ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts ( G and the nuclear fraction ( H ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pharmacological inhibition of JAK2 (G, H) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G and the nuclear fraction (H (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


knockdown JAK2 by siRNA ( I , J ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts I ) and the nuclear fraction J ) ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts I) and the nuclear fraction J) (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


( K , L ) The phosphorylated JAK2 level increased in the hippocampus of 12 - month - old hTau transgenic mice ( K ) , and the hippocampus of C57 mice infected with AAV - hTau ( 1 . 13 × 1013 v . g . / ml ) ( L ) ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K, L) The phosphorylated JAK2 level increased in the hippocampus of 12-month-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×1013 v.g./ml) (L) (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( A ) The representative fluorescence image confirms expression of AAV - hTau and AAV - Y701F - STAT1 . Scale bar , 200 μm , or 100 μm for the enlarged .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (A) The representative fluorescence image confirms expression of AAV-hTau and AAV-Y701F-STAT1. Scale bar, 200 μm, or 100 μm for the enlarged.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( B ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial learning deficits shown by the decreased escape latency during water maze training ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (B) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial learning deficits shown by the decreased escape latency during water maze training (n=7-10 each group). Data information: Data were presented as mean ± s.e.m (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( C - E ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial memory impairment shown by the decreased latency to reach the platform ( C ) , the increased crossing time in the platform site ( D ) and time spent in the target quadrant ( E ) measured at day 6 by removed the platform ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (C-E) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial memory impairment shown by the decreased latency to reach the platform (C), the increased crossing time in the platform site (D) and time spent in the target quadrant (E) measured at day 6 by removed the platform (n=7-10 each group). Data information: Data were presented as mean ± s.e.m , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( F ) Expression of Y701F - STAT1 did not change the swimming speed of the mice in water maze task ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (F) Expression of Y701F-STAT1 did not change the swimming speed of the mice in water maze task (n=7-10 each group). Data information: Data were presented as mean ± s.e.m two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


( G - I ) Simultaneous expression of Y701F - STAT1 did not induced any further change on basal synaptic transmission ( I / O curve ) compared with expression of hTau alone , recorded in hippocampal CA3 ( G ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( I ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-I) Simultaneous expression of Y701F-STAT1 did not induced any further change on basal synaptic transmission (I/O curve) compared with expression of hTau alone, recorded in hippocampal CA3 (G). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (I). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs protein expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4).


Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs protein ( K ) expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein (K) expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs mRNA expression measured by qRT - PCR in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs mRNA expression measured by qRT-PCR in hippocampal CA3 of C57 mice (n=4). Data information: Data were presented as mean ± SD , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


A , B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG - A for 16 h at 37 ° C . Anti - BDCA2 - mediated IFNα inhibition in whole - blood assays ( A ) . IFNα levels were detected using human IFNα ELISA . Graph depicts average of duplicate wells of one representative donor ( n = 10 ) . IC50 of 24F4A - mediated IFNα inhibition ( B ) in whole blood ( circles ) ( n = 10 ) and PBMC ( n = 18 ) from healthy human donors ( squares ) or SLE patients ( triangles ) ( n = 11 ) . Horizontal bar represents the mean IC50 for each sample type . Error bars represent SD of IC50 between donors .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.


C Anti - BDCA2 - mediated internalization in whole - blood assays . Whole blood was treated with increasing concentrations of 24F4A for 16 h . Mean fluorescence intensity ( MFI ) of BDCA2 was determined with a non - cross - blocking anti - BDCA2 mAb ( 2D6 ) . Shown is a representative plot of 10 experiments conducted .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.


A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0 , 1 , 3 , 6 , 9 , and 24 h . FACS analysis was performed to determine BDCA2 levels on pDCs . Fluorescence minus one ( FMO ) represents background staining of BDCA2 . Shown is a representative plot of three independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.


B Isolated pDCs were treated with 10 μg / ml 24F4A - AF647 at 4 ° C or at 37 ° C and analyzed at the indicated time points . Subcellular localization of BDCA2 ( red ) compared to LAMP1 ( green ) was assessed using confocal microscopy . Phalloidin was used to delineate the cell membrane . Yellow represents co - localization of BDCA2 and LAMP1 . Shown is a representative image of four experiments conducted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.


C , D Whole blood was either treated with 24F4A for 1 h at 37 ° C ( pre - incubation ) or left untreated . PBMC were isolated from each condition . PBMC from the untreated whole blood were subsequently treated with 10 μg / ml 24F4A , and cells from all conditions were stimulated with CpG - A for 16 h . Plot represents mean of duplicate wells . Flow cytometry was used to measure BDCA2 levels ( MFI ) . Shown is a representative donor of eight donors tested .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.


E Whole blood was pre - treated for 0 , 1 , 3 , 6 , and 9 h with 10 μg / ml of 24F4 or the isotype control and then stimulated with CpG - A for additional 16 h . IFNα levels were measured by ELISA . Shown is a representative plot of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . A - C Prior to in vivo dosing , baseline surface levels of BDCA2 for both the vehicle ( Ai ) and 1 mg / kg ( Bi ) animals ( red , dotted line ) were established by staining with fluorescently labeled 24F4A ( direct method ) . Maximal binding of 24F4A to BDCA2 was also established pre - dose in the vehicle ( Aii ) and 1 mg / kg ( Bii ) animals ( red , solid line ) by treating whole blood with 10 μg / ml of 24F4A at 4 ° C and then detecting bound 24F4A with a fluorescently labeled anti - human IgG1 ( indirect method ) . The direct method was used to stain whole blood from both the vehicle ( Aiii ) and 1 mg / kg 24F4A ( Biii ) animals 6 h post - dose ( red , dotted line ) . In a separate stain , the indirect method was used to detect bound 24F4A in the vehicle ( Aiv ) and 1 mg / kg ( Biv ) treated animals ( black , solid line ) . ( C ) Percent BDCA2 internalization relative to pre - dose BDCA2 levels 6 h post - dose with vehicle , 10 mg / kg , or 1 mg / kg 24F4A . Graph shows mean ± standard deviation for each group ( n = 3 ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . D PK / PD relationship between 24F4Aserum concentrations ( red triangle , left axis ) and BDCA2 expression on pDCs ( black squares , right axis , normalized to pre - dose levels ) from the 1 mg / kg group ( i - iii ) . Serum 24F4A was measured by ELISA . ( iv ) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . E Whole blood from vehicle - or 1 mg / kg 24F4A - treated monkeys was stimulated with CpG - A , and induction of IFN - I was measured by MxA bioassay at various time points pre - and post - treatment . Horizontal black lines represent the model - based estimates of the geometric mean of IFN - I in pre - and post - dose samples . Duplicate symbols represent independent replicates of the MxA bioassay for that time point . Statistical analysis was performed using a two - way mixed - effects analysis of variance ( ANOVA ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).


A - F Isolated human pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( black squares ) , or 10 μg / ml of the isotype control ( black triangle ) and stimulated with 5 μM of R848 ( A and B ) , 1 μM of CpG - A ( C and D ) , or immune complexes ( E and F ) for 16 h at 37 ° C . IFNα concentrations in cultured supernatants were determined using ELISA . Representative plots for each stimulation ( A , C and E ) and the average IC50 of 24F4A and 24F4A - ef for each stimulation condition ( B , D and F ) are shown ( n = 3 ) . Horizontal bars represent mean IC50 , and error bars represent SD of the mean from three independent experiments .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.


G Isolated human pDCs were treated with 10 μg / ml of 24F4A ( black line ) , 24F4A - ef ( gray shaded histogram ) , or the isotype control ( gray dotted line ) for 10 min at 37 ° C . Flow cytometry was used to evaluate pSYK and pPLCγ2 . Shown is a representative plot of two independent experiments .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.


H Isolated pDCs treated with 10 μg / ml of anti - CD32a ( AT10 ) and stimulated with CpG - A , R848 , or immune complexes . Error bars represent SD of percent inhibition of IFNα from three independent experiments .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.


A - F Isolated pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( squares ) , Chi - 6G6 ( triangle ) , or isotype control ( diamond ) for 16 h at 37 ° C and stimulated with CpG - A . Flow cytometry was performed to evaluate the MFI of ( A ) BDCA2 and ( C ) CD32a on the surface of pDCs . Percent internalization of BDCA2 ( B ) and CD32a ( D ) on pDCs treated with 10 μg / ml of 24F4A , 24F4A - ef , Chi - 6G6 , or isotype control . Error bars represent SD of percent internalization of five experiments conducted . ( E ) Percent CD32a downmodulation on isolated pDCs treated with 10 μg / ml of an anti - CD40 mAb . Error bars represent SD of percent downmodulation of two independent experiments . FMO represents background staining of CD32a ( inverted triangle ) . ( F ) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi - 6G6 . Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells . Shown is a representative plot of five independent experiments conducted .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.


( A ) mH2A1 . 2 and different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , and P0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).


( C ) A scatter diagram shows the change trend of mH2A1 . 2 and different neurogenesis markers . n = 5 mice , independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.


( E ) Electroporation of mH2A1 . 2 - shRNA results in abnormal cell distribution in the developing neocortex . Electroporation was performed at E13 . 5 , and the mouse was harvested at E16 . 5 . Scale bar represents 50 μm . ( F ) Graph shows the percentage of GFP - positive cells distributed in the VZ / SVZ , IZ and CP . n = 7 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. (F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.


( D ) Abnormal cell distribution was observed in the mH2A1 . 2 silenced neocortex . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( E ) Graphs of the percentage of GFP - positive cells in the VZ / SVZ , IZ and CP . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( F ) Brain sections of WT and KO mice at E16 . 5 were immunostained with the mitotic marker pH3 and DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + cells of the cortex . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( H ) Representative images of E16 . 5 coronal brain sections were immunostained for PAX6 . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) Quantification of GFP and Pax6 double positive cells in the VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( A ) Representative images of E16 . 5 coronal brain sections immune stained for Tbr2 and BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of pulse labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + cells per 100μm2 surface of VZ / SVZ . The number of TBR2 + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + cells per 100μm2 surface of VZ / SVZ . The number of BrdU + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( E ) Cell cycle exit was decreased in KO mice . E16 . 5 brain sections were stained with anti - BrdU and anti - Ki67 in WT mice and KO mice that were administered BrdU ( 100 mg / kg ) for 24 h and harvested at E16 . 5 . Scale bar represents 20 μm . ( F ) Percentage of cell cycle exit ( BrdU + Ki67 − / BrdU + ) in WT mice and KO mice . n = 7 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( G ) Western blot analysis showed that the protein expression levels of neural stem cell marker , including TBR2 , PCNA , and PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of the normalized density of TBR2 , PCNA , and PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT. (H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( K ) Coronal brain slices of E16 . 5 WT mice and KO mice were immunostained using anti - TUJ1 . Scale bar represents 20 μm . ( L ) Relative TUJ1 + cells in the VZ of WT mice and KO mice . TUJ1 was reduced in cortical . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm. (L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( M ) The apoptosis levels between WT and KO brains showed no significant difference . E16 . 5 brain sections of WT and KO mice were subjected to TUNEL assay . Scale bar represents 50 μm . ( N ) Graph shows the numbers of apoptotic cells per field . n = 4mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm. (N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( A ) Representative images of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into the E13 . 5 cerebral cortices of WT and KO mice to label neural progenitor cells . After 24 h , the GFP + cells were isolated and cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that the total dendritic length of neurons is decreased when mH2A1 . 2 is deleted . n = 25 cells from four samples . ( C ) Statistics show that the number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 cells from four samples . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).


( F ) The brain sections were immunostained with CUX1 and CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + cells in the upper layer . CUX1 + cells were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + cells in the deep layer . CTIP2 + cells were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


( A ) Representative tracing path of WT mice and KO mice in the open field test . ( B ) The total traveling distance was not different between WT and KO mice . ( C ) KO mice spent less time in the center than WT mice . Date information : All mice were male , 8 - 12 weeks old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . Analysis was performed by researcher blinded to the experimental conditions .

(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.