( B - D ) Quantification of normalized bouton number ( ( B ) , total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) , normalized axon length ( C ) and normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * Data information : In ( B - D bars are labelled with the number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates.


( B , C ) Western Blot showing the protein enrichments upon pulldown of the same probes as in ( A ) incubated with increasing concentrations of purified recombinant GST - Ythdf ( B ) and His - NT - Fmr1 proteins ( C ) . Quantification of the m6A / A signal intensity is shown below the blot as median + / - SEM of the triplicates . Both proteins bind more efficiently upon methylation .

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(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.


( D ) Results of fluorescence polarization ( FP ) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites , with or without the methylation , incubated with GST - Ythdf and His - NT - Fmr1 . The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8 . Binding constants ( kd values ) were determined by fitting a Michaelis - Menten non - linear regression onto the relative FP values in GraphPad Prism 8 , if applicable . The respective binding constants are given in the table next to the graphs .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.


( B - D ) Quantification of normalized bouton number ( ( B ) , total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) , normalized axon length ( C ) and normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * ) . Data information : In ( B - D , , bars are labelled with the number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In (B-D, , bars are labelled with the number of replicates.


( B ) Co - immunoprecipitation experiment in S2R + cells co - expressing FlagMyc - tagged Ythdf and Myc - tagged Fmr1 . Ythdf was used as a bait via its Flag tag . The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA .

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(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.


Pulldown of biotinylated RNA probes of ( A ) repetitive GGACU sequences incubated with 4 pmol His - NT - Fmr1 and increasing concentrations of GST - Ythdf The indicated P - values were determined by unpaired , two - tailed Student ' s t - test .

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Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf The indicated P-values were determined by unpaired, two-tailed Student's t-test.


( A ) Representative Western blot analysis of protein extracts of late pupae heads ( 85 - 95h ) from control , Ythdf # 0 / Ythdf # 0 and Fmr1 ∆ 50 / Fmr1 ∆ 50 flies . The membranes were probed with anti - Profilin or anti - Actin antibodies . ( B ) Quantification of Profilin protein levels ( mean ± SD ) obtained from three independent protein - extraction and Western blot analysis as in ( A ) . Quantification was performed using Fiji . p < 0 . 005 = * * ; p < 0 . 05 = * , measured with unpaired t - test .

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(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies. (B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.


( D ) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real - time PCR analysis . Bars show average ± SD of biological triplicates . Bars are labelled with the number of replicates .

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(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates. Bars are labelled with the number of replicates.


( F ) Quantification of normalized bouton number ( total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) of NMJ 6 / 7 in hemisegment A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 01 = * * ; p < 0 . 0001 = * * * * ) . Bars are labelled with the number of replicates .

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(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****). Bars are labelled with the number of replicates.


E - G ) Representative Western blot analysis of the protein level ( E ) and quantification of the translation efficiency of S2R + cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD ( F ) or Ythdf KD ( G ) . LacZ dsRNA was used for the control KD . The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level . mCherry served as a normalization control for both the protein and mRNA level . Bars show mean ± s . e . m of biological triplicates . P values were determined with a Student ' s t - test . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ) .

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E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).


( B ) Pol II ChIP - seq was performed in six different tissues shown in the left panel ( scale bar - 100 µm ) . The middle and the right panels show the read count normalized Pol II ChIP - seq tracks ( RPM ) from the six tissues at individual genes . For each gene , grey and red tracks indicate non - expressing tissues and expressing tissue , respectively . The middle panel shows the Pol II profile at two non - paused genes , which have Pol II only in the expressing tissues . The expression is limited to specific tissues as shown in the in situ images from BDGP . The right panel shows the Pol II profile at a paused gene , which has Pol II in all observed tissues , although the expression is limited to specific tissues as shown in the in situ images from BDGP . Paused Pol II is generally highest in the tissue with the highest expression . We also found systematic differences between samples , thus some tissues have generally higher enrichments than others , presumably because they are easier to crosslink .

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(B) Pol II ChIP-seq was performed in six different tissues shown in the left panel (scale bar - 100 µm). The middle and the right panels show the read count normalized Pol II ChIP-seq tracks (RPM) from the six tissues at individual genes. For each gene, grey and red tracks indicate non-expressing tissues and expressing tissue, respectively. The middle panel shows the Pol II profile at two non-paused genes, which have Pol II only in the expressing tissues. The expression is limited to specific tissues as shown in the in situ images from BDGP. The right panel shows the Pol II profile at a paused gene, which has Pol II in all observed tissues, although the expression is limited to specific tissues as shown in the in situ images from BDGP. Paused Pol II is generally highest in the tissue with the highest expression. We also found systematic differences between samples, thus some tissues have generally higher enrichments than others, presumably because they are easier to crosslink.


( C , D ) Normalized gene expression levels ( read count normalized for each cell , log2 ) in different tissues , from the scRNA - seq experiment , for a TATA group gene , and a highly paused group gene , are shown . ( C ) The TATA gene , Ccp84Aa , shows noisy expression in the epidermis , without detectable background expression in non - expressing tissues . ( D ) The highly paused gene , Gip , shows very robust expression in crystal cells , but has high background expression in the non - expressing tissues .

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(C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues.


C , D ) ATAC - seq chromatin accessibility was measured in different tissues isolated from 14 - 17 h embryos using INTACT . ( C ) Read - count normalized ATAC ( RPM ) signal at individual genes from the TATA group ( Ccp84Aa ) and highly paused gene group ( ect ) are shown . ( D ) Read - count normalized ATAC signals ( RPM ) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS . Paused genes show higher average accessibility across all tissues compared to the TATA genes ( Wilcoxon two - sided test , * P < 2 . 2 * 10 - 16 ) .

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C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).


( B ) Scatterplots represent SILAC / SUMO - IP quantification after EGF treatment from three biological replicates . Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells ( dark blue dots ) or is significantly more present in one of the samples ( red and yellow dots ) .

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(B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots).


( C ) Left panel : Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation ( significant hits with P < 0 . 0001 ) , as well as the three non - changing proteins SUMO2 , RanGAP1 and TRIM28 . Right panel : Proteins highlighted in bold in the left panel were validated by SUMO - IP / immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation .

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(C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation.


( D ) Time course experiment : serum starved HeLa cells were treated with 100 nM EGF , samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies . IRF2BP1 , IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment .

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(D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.


( B ) Identification of the endogenous SUMO - sites in IRF2BP1 ( left panel ) and IRF2BP2 ( right panel ) . HeLa cells were transfected with HA - tagged wildtype ( WT ) or mutant ( KR ) proteins , endogenous SUMO - IPs were performed and the HA - signal was analyzed by immunoblotting . Mutation of the C - terminal SUMO - site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells .

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(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells.


( E ) Stable HeLa cells expressing pIRES - empty vector , IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non - targeting ( nt ) siRNA . Exogenous siRNA - resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1 . * refers to an unspecific band .

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(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band.


( F ) Wt and mutant IRF2BP1 localizes in the nucleus . After knockdown of endogenous IRF2BP1 , stable IRF2BP1 ( WT or K579R ) cell lines were immunostained for IRF2BP1 . Exogenous IRF2BP1 variants show a similar nuclear localization . Scale bar = 10 µm .

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(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm.


( G ) IRF2BP1 wt and mutant associate with chromatin to a similar extent . HeLa cells were lysed in 0 . 075 % NP40 ( Input ) . After centrifugation , the nuclei were incubated and vortexed with a nuclear extract ( NE ) buffer containing 170 mM NaCl . The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations , first 290 mM , then 420 mM . Wildtype IRF2BP1 , the SUMO - deficient K579R mutant and the SUMOylated wildtype form ( * ) all behave similarly .

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(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.


( A ) Stable IRF2BP1 cell lines ( knocked - down for endogenous IRF2BP1 ) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour . A subset of 38 EGF - dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines ( at least 1 . 5 - fold ) . Among them are DUSP1 ( arrow ) , ATF3 , Fos and Egr2 . Each microarray was performed in triplicates , the bars show log2 values of the fold changes for wt cells ( black bars ) and for KR cells ( grey bars ) .

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(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars).


( B ) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells . Gene architecture of human DUSP1 . The primers at - 243 / - 67 ( " - 67 " ) , - 473 / - 224 ( " - 224 " ) and - 1170 / - 961 ( " - 916 " ) relative to the TSS were used for ChIP experiments . IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides - 243 and - 67 .

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(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.


( C ) ChIP / qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between - 243 and - 67 . Data show mean ( bar ) and individual data points from 2 biological replicates .

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(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.


( D ) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF . Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked - down for endogenous IRF2BP1 , followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region - 243 and - 67 . Data show means + / - SEM from 3 biological replicates .

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(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.


( E ) Q - PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / - SEM from 3 biological replicates .

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(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.


( F ) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q - PCR ( left panel ) , data show means + / − SEM from 5 biological replicates ( left panel ) , and IRF2BP1 protein levels . As expected , IRF2BP1 V578A is not SUMOylated ( right panel ) . * refers to an unspecific band .

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(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.


( G ) Q - PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / − SEM from 4 biological replicates .

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(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.


( A ) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1 . Uba2 and beta - actin serve as independent loading controls .

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(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.


( C ) Wound healing assay : HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days , grown to 90 % confluency and analysed for wound closure with or without addition of 100 ng / ml EGF . Data show means of the relative wound density + / - SEM from 4 biological replicates .

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(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.


( D ) Proliferation assay : HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium . One representative biological experiment is shown with means + / - SEM from 5 technical replicates .

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(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.


( E ) IRF2BP1 , IRF2BP2 , IRF2BP1 + IRF2BP2 were knocked - down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis ; non - targeting siRNAs were used as a control . IRF2BP1 and IRF2BP2 have distinct and overlapping functions . Experiments were done in triplicates .

(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates.


( G ) Validation of two candidate genes : IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells , and cell lysates were analyzed by immunoblotting with the indicated antibodies .

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(G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies.


( H ) FACS - based analysis of EGFR surface expression : IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells , cells were serum starved for 16 hours , stained with fluorescent anti - EGFR antibodies and analyzed by flow cytometry . Shown are means of 3 independent experiments + / - SEM .

(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.


( C ) All eight subunits of the CCT chaperonin complex were enriched in U2OS - A3A samples . Proteins detected by MS are displayed by fold enrichment ( log2 ) from U2OS - A3A cells compared to parental cells ( x - axis ) , and by protein abundance within the U2OS - A3A samples ( y - axis ) . Significantly enriched proteins ( p < 0 . 05 by one - tailed t - test , n = 3 ) are denoted in dark blue . Components of the CCT complex are highlighted and labeled .

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(C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled.


( A ) A3A co - precipitates with CCT complex subunits . U2OS and HepaRG cells with dox - inducible A3A - HA transgenes were treated with dox prior to immunoprecipitation of HA . HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex .

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(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.


( C ) A3A interacts with CCT in leukemia cells . HA immunoprecipitation in hematopoietic cell lines with inducible A3A - HA was analyzed by immunoblot . HA , CCT1 , and CCT5 antibodies were used to evaluate co - precipitation of CCT subunits with A3A .

(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.


( A ) CCT co - precipitates with A3A but not A3B or A3G . 293T cells were transfected with empty vector ( V ) , A3A - HA , A3B - HA , or A3G - HA . CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA .

(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.


( B ) A3A , but not other APOBEC3 enzymes , interacts with CCT complex subunits . After transfection of 293T cells with HA - tagged APOBEC3 expression plasmids , IP was performed with anti - HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits . Arrows mark location of each APOBEC3 protein on HA immunoblot .

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(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.


( F ) CCT co - precipitates with endogenous A3A . Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1 . Chemiluminescent exposure times described as short and long are indicated beside the blot . IgG antibody was used as a control throughout .

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(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.


( A ) Knockdown of one CCT complex member results in depletion of other CCT subunits . Following siCCT4 or siCCT7 transfection , cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1 , CCT4 , CCT5 , and CCT7 . GAPDH was used as a loading control .

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(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.


( B ) Decreased cell viability upon CCT depletion and A3A expression . CCT4 ( left ) and CCT7 ( right ) subunits were depleted by siRNA transfection in U2OS cells with dox - inducible A3A transgenes . Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit , but no alteration in A3A expression . Scr indicates non - targeted siRNA , used as control . Ku - 86 is a loading control . Following siRNA transfection to deplete CCT subunits , U2OS - A3A cells were treated with dox . Viability was determined by colorimetric change after addition of a water - soluble tetrazolium salt . Percent viability was normalized to untreated controls . Statistical analysis was performed using a paired two - tailed t - test , n = 3 biological replicates ; error bars , SEM .

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(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.


( D ) Viability of cells with catalytically inactive A3A . U2OS - A3A - C106S cells were induced with dox to express the catalytically inactive A3A mutant ( C106S ) , depleted of CCT4 by siRNA , or combination . Cells were subsequently evaluated by live - dead staining assessed by FACS . Bar chart shows quantitation of FACS results averaged over three biological replicates . Statistical analysis was performed using a paired two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting . Ku - 86 is a loading control .

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(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.


( E ) Increased DNA damage signaling upon CCT depletion and A3A expression . Depletion of CCT7 in K562 - A3A cells was achieved by siRNA targeting . Expression of A3A was induced by dox treatment . Intracellular staining with anti - γH2AX antibody was analyzed by flow cytometry . Bar chart shows quantification of FACS results obtained over three biological replicates . Statistical analysis was performed using a two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment . Ku - 86 is a loading control . * * p - value < 0 . 01 , * * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 , n . s . non - significant .

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(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.


( C ) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity . Breast , bladder , and cervical cancer samples from the TCGA database were divided into CCT - mutated and non - mutated genomes , as above . The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average . Comparison by Wilcoxon rank sum test yielded p - values denoted .

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(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.


( A ) Representative photomicrographs of MAP2 ( green ) and cleaved caspase - 3 ( red ) immunocytochemistry of embryonic cortical neurons from wild type ( upper panels ) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h . Scale bar 15µm .

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(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.


( B ) Quantification of active ( cleaved ) caspase - 3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers ( 2 . 5 , 5 , or 10 µM ) . Data are expressed as percentage cleaved caspase - 3 and MAP2 double positive cells over the total number of MAP2 positive cells .

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(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells.


( C ) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers ( 2 . 5 , or 10 µM ) . Data are expressed as mean neurite length in individual neurons , relative to wild type vehicle ( set to 100 units ) .

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(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units).


( A ) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type , 5xFAD and p75NTR knock - out ( KO ) mice . Scale bar , 20μm .

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(A) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type, 5xFAD and p75NTR knock-out (KO) mice. Scale bar, 20μm.


Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type ( WT ) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading . ( C ) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH .

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Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading. (C) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH.


( D ) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice of the indicated ages . Scale bar , 400μm . ( E ) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by Aβ plaques ( mean ± SEM , N = 5 mice per group ) . * , P < 0 . 05 and * * , P < 0 . 01 vs 5XFAD . # , P < 0 . 05 vs 5XFAD / KO ( one - way ANOVA followed by post hoc test ) . ( F ) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Color codes are as in panel ( E ) . Histogram shows mean ± SEM , N = 4 mice per group ; * , P < 0 . 05 KO vs . knock - in genotypes .

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(D) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice of the indicated ages. Scale bar, 400μm. (E) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N=5 mice per group). *, P<0.05 and **, P<0.01 vs 5XFAD. #, P<0.05 vs 5XFAD/KO (one-way ANOVA followed by post hoc test). (F) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N=4 mice per group; *, P<0.05 KO vs. knock-in genotypes.


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers ( G ) refers to the soluble fraction after Tris - buffered saline

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extraction


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers ( H ) to the soluble fraction after RIPA buffer extraction of the Tris - buffered saline pellet ,

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet,


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils ( I ) to the soluble fraction after formic acid treatment of the RIPA pellet .

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.


( A ) Immunostaining for Glial fibrillary acidic protein ( GFAP ) , a marker of astrocytes , and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 300μm . Right hand panels show high magnification of the indicated areas . Scale bar , 50μm . ( B ) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining ( mean ± SEM , N = 5 mice per group ) .

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(A) Immunostaining for Glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 50μm. (B) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N=5 mice per group).


( C ) Immunostaining for Ionized calcium binding adaptor molecule 1 ( Iba1 ) , a marker of microglial cells , and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A . Scale bar , 300μm . Right hand panels show high magnification of the indicated areas . Scale bar , 10μm . ( D ) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining ( mean ± SEM , N = 5 mice per group ) .

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(C) Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 10μm. (D) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N=5 mice per group).


( E ) Immunostaining of reticulon 3 ( RTN3 ) , a marker of dystrophic neurites , and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 40μm . ( F ) Quantification of RTN3 - positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining ( mean ± SEM , N = 5 mice per group ) .

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(E) Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 40μm. (F) Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N=5 mice per group).


( G ) MitoSox staining , a mitochondrial superoxide indicator , and DAPI in coronal sections through the hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A mice . Scale bar , 60μm . ( H ) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows MitoSox mean fluorescence intensity in arbitrary units ( mean ± SEM , N = 5 mice per group ) .

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(G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).


( A ) Percentage of change in field excitatory postsynaptic potential ( fEPSP ) recorded after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from 6 month old wild type , 5xFAD and 5xFAD / p75NTR mutant mice , as indicated . Results are presented as mean % change normalized to t = 0 ± SEM . N = 6 ( wild type ) , 5 ( 5xFAD ) , 9 ( 5xFAD / KO ) , 7 ( 5xFAD / ΔDD ) and 8 ( 5xFAD / C259A ) slices from 3 mice per genotype , respectively . ( B ) Quantification of fEPSP ( mean % change ± SEM ) in the indicated genotypes at 3 time points . * , P < 0 . 05 ; * * , P < 0 . 01 vs . 5xFAD ( two - way ANOVA followed by post hoc test ) . N numbers as in ( A ) .

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(A) Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6 month old wild type, 5xFAD and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t=0 ± SEM. N=6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD) and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. (B) Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *, P<0.05; **, P<0.01 vs. 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A).


( C ) Behavior in the novel object recognition ( NOR ) test of 6 month old wild type , 5XFAD and 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean recognition index ± SEM during training , and 30min , 24h and 14 days after training , corresponding to measures of short term , long term and remote memory , respectively . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ( two - way ANOVA followed by post hoc test ) . N = 12 ( wild type , 5xFAD / ΔDD and 5xFAD / C259A ) , 10 ( 5xFAD ) and 8 ( 5xFAD / KO ) mice per genotype , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Behavior in the novel object recognition (NOR) test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30min, 24h and 14 days after training, corresponding to measures of short term, long term and remote memory, respectively. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD (two-way ANOVA followed by post hoc test). N=12 (wild type, 5xFAD/ΔDD and 5xFAD/C259A), 10 (5xFAD) and 8 (5xFAD/KO) mice per genotype, respectively.


( D ) Training latency in the Barnes maze test of 6 month old wild type , 5XFAD and 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two - way ANOVA followed by post hoc test ) . N = 14 ( wild type and 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO and 5xFAD / C259A ) mice per genotype , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Training latency in the Barnes maze test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively.


N = 14 ( wild type and 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO and 5xFAD / C259A ) mice per genotype , respectively . ( E ) Percentage of time spent ( mean ± SEM ) in the target quadrant of the Barnes maze test 30min , 24h and 14 days after training . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two way ANOVA followed by post hoc test ) .

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

N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. (E) Percentage of time spent (mean ± SEM) in the target quadrant of the Barnes maze test 30min, 24h and 14 days after training. *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two way ANOVA followed by post hoc test).


( A ) Western blot analysis of CTF beta ( CTFβ ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti - APP - CTF antibody Arrows point to phospho - C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH . ( B ) Quantification ( mean ± SEM ) of phospho - C99 / C99 CTFβ species , normalized to GAPDH and expressed relative to levels in 5xFAD mice . * , P < 0 . 05 5xFAD vs , 5xFAD / KO ; * * * , P < 0 . 001 5xFAD vs . 5xFAD / ΔDD and 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P = 0 . 056 5xFAD / ΔDD vs . 5xFAD / KO . N = 5 mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of CTF beta (CTFβ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti-APP-CTF antibody Arrows point to phospho-C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH. (B) Quantification (mean ± SEM) of phospho-C99/C99 CTFβ species, normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs, 5xFAD/KO; ***, P<0.001 5xFAD vs. 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P=0.056 5xFAD/ΔDD vs. 5xFAD/KO. N=5 mice per group.


( C ) Western blot analysis of soluble APP alpha ( sAPPα ) in the soluble Tris - buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH . ( D ) Quantification ( mean ± SEM ) normalized to GAPDH and expressed relative to levels in 5xFAD mice . * , P < 0 . 05 5xFAD vs . 5xFAD / KO , 5xFAD / ΔDD and 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P < 0 . 05 5xFAD / ΔDD vs . 5xFAD / KO . N = 9 mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of soluble APP alpha (sAPPα) in the soluble Tris-buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH. (D) Quantification (mean ± SEM) normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs. 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P<0.05 5xFAD/ΔDD vs. 5xFAD/KO. N=9 mice per group.


( A ) Internalization of triple mutant hAPP in wild type , p75NTR knock - out ( KO ) , ΔDD and C259A hippocampal neurons ( 15min time point ) . Counterstaining for p75NTR and DAPI are also shown . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O 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) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm.


( B ) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type , p75NTR knock - out ( KO ) , ΔDD and C259A mice . Shown is mean ± SEM of percentage internalization of total surface APP ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 knock - in vs . KO mutants ; * * , P < 0 . 01 , WT vs . all other genotypes ( 2 - way ANOVA ) . ( C ) Linear transformation of hAPP internalization kinetics shown in ( B ) . IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type, p75NTR knock-out (KO), ΔDD and C259A mice. Shown is mean ± SEM of percentage internalization of total surface APP (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in vs. KO mutants; **, P<0.01, WT vs. all other genotypes (2-way ANOVA). (C) Linear transformation of hAPP internalization kinetics shown in (B). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.


( D ) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF . Shown is mean ± SEM of percentage internalization of total surface p75NTR ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 ± NGF ( 2 - way ANOVA ) . ( E ) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF. Shown is mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 ± NGF (2-way ANOVA). (E) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.


( F ) Internalization of endogenous p75NTR in wild type hippocampal neurons ( 30min time point ) . Counterstaining for MAP2 and DAPI are also shown . Scale bar , 10μm .

O O O O O O O O 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) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.


( G ) Kinetics of internalization of p75NTR in hippocampal neurons from wild type , ΔDD and C259A mice . Show are mean ± SEM of percentage internalization of total surface p75NTR ( set to 100 % ) . N = 3 independent experiments each performed in duplicate ; * , P < 0 . 05 knock - in mutants vs . WT ( 2 - way ANOVA ) . ( H ) Linear transformation of p75NTR internalization kinetics shown in ( G ) . IMAX denotes maximal internalization in % . T1 / 2 denotes time for half maximal internalization in minutes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of internalization of p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in mutants vs. WT (2-way ANOVA). (H) Linear transformation of p75NTR internalization kinetics shown in (G). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.


( I ) ( A ) Co - immunoprecipitation between p75NTR and β - adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated . Results shown are representative from 2 independent experiments . Molecular weights are in kDa . IP : immunoprecipitation ; IB : immunoblotting ; WCL , whole cell lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (A) Co-immunoprecipitation between p75NTR and β-adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated. Results shown are representative from 2 independent experiments. Molecular weights are in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.


( A ) Co - immunoprecipitation between hAPP and p75NTR in hippocampal extracts from 6 month 5xFAD and p75NTR mutant mice as indicated . Results shown are representative from 3 independent experiments . Molecular weights are indicated in kDa . IP : immunoprecipitation ; IB : immunoblotting ; WCL , whole cell lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation between hAPP and p75NTR in hippocampal extracts from 6 month 5xFAD and p75NTR mutant mice as indicated. Results shown are representative from 3 independent experiments. Molecular weights are indicated in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.


( B ) Proximity ligation assay ( PLA ) between triple mutant hAPP and p75NTR in hippocampal neurons from wild type and C259A mice ( first and second rows , respectively ) . The third row ( no APP ) shows control PLA reaction in neurons that did not receive hAPP lentivirus . The fourth row ( KO ) shows control PLA reaction in neurons from p75NTR knock - out mice infected with hAPP lentivirus . Counterstaining for MAP2 is also shown . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proximity ligation assay (PLA) between triple mutant hAPP and p75NTR in hippocampal neurons from wild type and C259A mice (first and second rows, respectively). The third row (no APP) shows control PLA reaction in neurons that did not receive hAPP lentivirus. The fourth row (KO) shows control PLA reaction in neurons from p75NTR knock-out mice infected with hAPP lentivirus. Counterstaining for MAP2 is also shown. Scale bar, 10μm.


( C ) Quantification of total PLA signals in wild type and p75NTR mutant neurons in the presence or absence of hAPP lentivirus , as indicated . Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments . ( D ) Quantification of cell surface PLA signals in wild type and p75NTR mutant neurons in the presence or absence of triple mutanthAPP lentivirus , as indicated . Live neuron cultures were fed with anti - mouse p75NTR and anti - hAPP antibodies on ice , washed after staining , fixed , and developed with PLA reaction . Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total PLA signals in wild type and p75NTR mutant neurons in the presence or absence of hAPP lentivirus, as indicated. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments. (D) Quantification of cell surface PLA signals in wild type and p75NTR mutant neurons in the presence or absence of triple mutanthAPP lentivirus, as indicated. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed after staining, fixed, and developed with PLA reaction. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments.


( E ) Internalization of hAPP / p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice . Live neuron cultures were fed with anti - mouse p75NTR and anti - hAPP antibodies on ice , washed and plates placed at 37 ° C for different periods of time to allow internalization . Internalization was stopped by acid wash , followed by fixation and PLA reaction . Counterstaining for MAP2 and DAPI are also shown . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Internalization of hAPP/p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed and plates placed at 37°C for different periods of time to allow internalization. Internalization was stopped by acid wash, followed by fixation and PLA reaction. Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.


( F ) Kinetics of co - internalization of hAPP and p75NTR in hippocampal neurons from wild type , ΔDD and C259A mice . Show are mean ± SEM of percentage internalization of total surface PLA signal ( set to 100 % ) . ( G ) Linear transformation of p75NTR internalization

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of co-internalization of hAPP and p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface PLA signal (set to 100%). (G) Linear transformation of p75NTR internalization kinetics


( A ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , BACE1 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main images . Scale bar , 5μm . ( B ) Quantification of the proportion of BACE1 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % BACE co - localized with hAPP . N = 3 independent experiments , each performed in duplicate ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( C ) Quantification of the proportion of internalized hAPP that co - localized with BACE1 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with BACE1 . N = 3 independent experiments , each performed in duplicate ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).


( D ) Micrographs of p75NTR after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( E ) Quantification of the proportion of Rab11 that co - localized with p75NTR after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with p75NTR . n = 4 ; * , P < 0 . 05 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) . ( F ) Quantification of the proportion of internalized p75NTR that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized p75NTR co - localized with Rab11 . n = 4 ; * * , P < 0 . 01 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).


( G ) Micrographs of triple mutant hAPP after 15min internalization ( red ) , Rab11 immunocytochemistry ( green ) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus . DAPI and MAP2 staining are shown in the left column . Insets show higher ( 3 . 5x ) magnification of the areas indicated in the main merged images . Scale bar , 5μm . ( H ) Quantification of the proportion of Rab11 that co - localized with hAPP after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % Rab11 co - localized with hAPP . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) . ( I ) Quantification of the proportion of internalized hAPP that co - localized with Rab11 after 6 and 15 min of internalization at 37 ° C . Results are expressed as mean ± SEM of % internalized hAPP co - localized with Rab11 . n = 4 ; * , P < 0 . 05 vs . WT ( one - way ANOVA followed by post hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test).


Tfeb expression in the vasculature ( mice n = 10 ) . ( A ) Representative images of retinal vessels ( p5 ) ( A ) ( scale bar : 50 µm ) of Tfeb - EGFP mice stained with anti - iB4 ( A ) and anti -

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

Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) of Tfeb-EGFP mice stained with anti-iB4 (A) and anti-GFP


Tfeb expression in the vasculature ( mice n = 10 ) . Representative images of glomerular , arterial ( scale bars : 10 µm ) and interstitial vessels ( scale bar : 50 µm ) of kidney ( p17 ) of Tfeb - EGFP mice stained with anti - CD31 ( B ) and anti -

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Tfeb expression in the vasculature (mice n=10). Representative images of glomerular, arterial (scale bars: 10 µm) and interstitial vessels (scale bar: 50 µm) of kidney (p17) of Tfeb-EGFP mice stained with anti-CD31 (B) and anti-GFP


Alterations in the embryonic vasculature in TfebEC - / - mice at E10 . 5 ( mice n = 25 ) . Representative images of whole - mount embryos and yolk sacs ( i , ii ) ( scale bars : 0 . 5 mm ) . Vessels of the head ( iii ) , ocular ( iv ) and intrasomitic regions ( v ) were stained with anti - endomucin Ab ( scale bars : 100 µm ) .

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Alterations in the embryonic vasculature in TfebEC-/- mice at E10.5 (mice n=25). Representative images of whole-mount embryos and yolk sacs (i, ii) (scale bars: 0.5 mm). Vessels of the head (iii), ocular (iv) and intrasomitic regions (v) were stained with anti-endomucin Ab (scale bars: 100 µm).


Alteration of retinal vascular maturation in TfebiEC - / - at p5 . Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti - iB4 Ab ( scale bars : 50 µm ) . Bar graphs indicate the percent of vascular area versus total area of the retina , the percent of vascular density , the number of branches per field , vessel diameter , the number of filopodia per field and the length of filopodia ( mice n = 10 , mean ± SEM ; * * * p < 0 . 0001 versus control mice by Student ' s t - test ) .

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Alteration of retinal vascular maturation in TfebiEC-/- at p5. Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti-iB4 Ab (scale bars: 50 µm). Bar graphs indicate the percent of vascular area versus total area of the retina, the percent of vascular density, the number of branches per field, vessel diameter, the number of filopodia per field and the length of filopodia (mice n=10, mean±SEM; ***p< 0.0001 versus control mice by Student's t-test).


Alteration of the glomerular ultrastructure in TfebiEC - / - mice at p17 ( mice n = 5 ) . Representative transmission electron micrographs of renal tissue . Magnification : i ) x6000 , ii ) x20000 ) , iii - iv ) x10000 , v - vi ) x25000 ; scale bars : 1µm ) . Asterisks indicate the accumulation of extracellular matrix in mesangium ; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae , respectively .

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Alteration of the glomerular ultrastructure in TfebiEC-/- mice at p17 (mice n=5). Representative transmission electron micrographs of renal tissue. Magnification: i) x6000, ii) x20000), iii-iv) x10000, v-vi) x25000; scale bars: 1µm). Asterisks indicate the accumulation of extracellular matrix in mesangium; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae, respectively.


Reduced EC proliferation in the retina ( p5 ) of in TfebiEC - / - mice . Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC - / - mice stained with anti - iB4 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percentage of EC Ki - 67 + nuclei versus total nuclei co - localized with iB4 +

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Reduced EC proliferation in the retina (p5) of in TfebiEC-/- mice. Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC-/- mice stained with anti-iB4 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percentage of EC Ki-67+ nuclei versus total nuclei co-localized with iB4+ vessels


Reduced EC proliferation in the kidney ( p17 ) of in TfebiEC - / - mice . ( B ) Representative images of vessels of the kidney in control and TfebiEC - / - mice stained with anti - CD31 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percent of EC Ki - 67 + nuclei versus total nuclei co - localized with CD31 + vessels ( mice n = 6 , mean ± SEM ; * * p < 0 . 001 versus control mice by Student ' s t - test ) . Podocin staining is shown to glomerular localization .

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Reduced EC proliferation in the kidney (p17) of in TfebiEC-/- mice. (B) Representative images of vessels of the kidney in control and TfebiEC-/- mice stained with anti-CD31 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percent of EC Ki-67+ nuclei versus total nuclei co-localized with CD31+ vessels (mice n=6, mean±SEM; **p<0.001 versus control mice by Student's t-test). Podocin staining is shown to glomerular localization.


TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . ( C ) DNA incorporation of EdU was detected by flow cytometry . The percentage of proliferating cells is indicated ( n = 4 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA ECs by Student ' s t - test ) .

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TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student's t-test).


TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . DNA content was determined by propidium iodide staining and assessed by fluorescences - activated cell sorter analysis ( representative experiment out of 4 with similar results ) .

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TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results).


TFEB silencing reduced human EC morphogenesis . Representative images of tube - like - structure of scr - shRNA and sh - TFEB human ECs stained with phalloidin - 555 . Bar graph indicates the percentage of phalloidin + area in sh - TFEB and scr - shRNA ECs ( scale bars : 0 . 25 mm ; n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA by Student ' s t - test ) .

TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student's t-test).


Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr - shRNA and sh - TFEB ECs . Red : up - regulated genes ; green : down - regulated genes .

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Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr-shRNA and sh-TFEB ECs. Red: up-regulated genes; green: down-regulated genes.


Heatmap , Red : up - regulated genes ; green : down - regulated genes .

Heatmap, Red: up-regulated genes; green: down-regulated genes.


qPCR Data are expressed as relative fold - change compared with the expression in scr - shRNA cells after normalization to the housekeeping gene

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qPCR Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBP


immunoblots showing the differentially expressed cell cycle related genes between scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with specific Abs . The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α -

immunoblots showing the differentially expressed cell cycle related genes between scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with specific Abs. The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α-tubulin


Modulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC - / - mice . Data are expressed as relative fold - change compared with the expression in ECs of control mice after normalization to the housekeeping gene

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Modulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC-/- mice. Data are expressed as relative fold-change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBP


Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for CDK4 . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) .

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Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD).


Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs . Analysis of TFEB modulation of CDK4 promoter in human ECs . Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding

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Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. Analysis of TFEB modulation of CDK4 promoter in human ECs. Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding site


Modulation of Rb protein in sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with phospho - Rb and total Rb Abs . The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh - TFEB vs scr - shRNA and the ratio between phospho - Rb and total Rb in the different

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Modulation of Rb protein in sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with phospho-Rb and total Rb Abs. The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh-TFEB vs scr-shRNA and the ratio between phospho-Rb and total Rb in the different conditions


Increase of Vegfr2 expression in the vasculature of TfebEC - / - mice . Representative immunostaining images of TfebEC - / - embryonic vessels ( E10 . 5 ) stained with anti - endomucin and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in endomucin + vessel areas ( embryos n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus control embryos by Student ' s t - test ) .

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Increase of Vegfr2 expression in the vasculature of TfebEC-/- mice. Representative immunostaining images of TfebEC-/- embryonic vessels (E10.5) stained with anti-endomucin and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in endomucin+ vessel areas (embryos n=6, mean±SEM; ***p< 0.0001 versus control embryos by Student's t-test).


Increase of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images ( i ) and detail ( ii ) of the vascular front and vascular plexus of the retina ( p5 ) of control and TfebiEC - / - mice with anti - iB4 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in iB4 + vessel

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Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and TfebiEC-/- mice with anti-iB4 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in iB4+ vessel areas


Increase of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images of the glomerulus ( p17 ) of control and TfebiEC - / - mice with anti - podocin , anti - CD31 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graphs indicate the Vegfr2 mean intensity in CD31 + vessel

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Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images of the glomerulus (p17) of control and TfebiEC-/- mice with anti-podocin, anti-CD31 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graphs indicate the Vegfr2 mean intensity in CD31+ vessel areas


VEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC - / - mice ( D ) Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

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VEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC-/- mice (D) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and in human sh - TFEB Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

VEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and in human sh-TFEB Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with anti - VEGFR2 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

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VEGFR2 expression is regulated by TFEB. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with anti-VEGFR2 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


Analysis of TFEB binding to the VEGFR2 promoter in human ECs . ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for VEGFR2 .

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Analysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for VEGFR2.


Silenced TFEB alters the localization of VEGFR2 . FACS analysis of surface VEGFR2 expression on human scr - shRNA and sh - TFEB ECs . Bar graph shows the ratio between total and PM

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Silenced TFEB alters the localization of VEGFR2. FACS analysis of surface VEGFR2 expression on human scr-shRNA and sh-TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2


Silenced TFEB alters the localization of VEGFR2 . Representative TIRF and epifluorescence images of human scr - shRNA and sh - TFEB ECs after staining with anti - VEGFR2 Ab ( scale bars : 10 µm ) . Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by

Silenced TFEB alters the localization of VEGFR2. Representative TIRF and epifluorescence images of human scr-shRNA and sh-TFEB ECs after staining with anti-VEGFR2 Ab (scale bars: 10 µm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRF