( g ) Expression levels of Atg5 and Atg12 - Atg5 conjugate , the conversion of LC3 I into LC3 II , and p62 level in the hearts of Atg5 Tg mice . Whole - tissue extracts were prepared from 3 - month - old Atg5 Tg mice and WT littermates , and analysed by western blotting using the indicated antibodies . β - Actin served as a control . ( h ) Densitometric analysis of the signals on the blots shown in ( g ) . The bars represent the mean ± s . d . ( n = 3 ) .

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(g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).


( a ) Western blot analysis showing enhanced levels of Atg5 , Atg12 - Atg5 conjugate and LC3 conversion , and reduced p62 levels in the indicated tissues of 18 - month - old Atg5 Tg mice no . 25 ( T ) and WT mice ( W ) . ( b ) Densitometric analysis of the signals on the blots shown in ( a ) .

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(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a).


( c ) Electron microscopy images of autophagosomes ( arrows ) and late - autophagic compartments ( arrowheads ) in the liver tissues of 18 - month - old Atg5 Tg mice no . 25 and WT mice . Scale bars are indicated . ( d ) The numbers of autophagosomes and autolysosomes per cell ( n = 50 ) in the electron microscopic images shown in ( c ) were counted . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( e ) Fluorogenic comparison of basal autophagy activity between GFP - LC3 Tg mice and Atg5 / GFP - LC3 double Tg mice . Whole bodies of 8 - week - old male ( left ) and female ( right ) GFP - LC3 Tg ( LC3 ) , and LC3 / LC3 - LC3Tg mice ( Atg5 / LC3 ) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC ( Kodak Molecular Imaging Software , Version 4 . 0 ) .

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(e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and LC3/LC3-LC3Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0).


( f ) Western blot analysis showing autophagy markers in the liver of GFP - LC3 mice . GFP - LC3 mice were injected for 5 days with siRNA - Atg5 or Atg5 - HA cDNA through the tail vein . Whole - liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies . β - Actin served as a control .

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(f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.


( a ) Representative physical pictures of 18 - month - old WT and Atg5 Tg mice fed with a regular diet . ( b ) Body weight chart of ageing - induced obesity in WT and Atg5 Tg mice . The mice were fed with a regular diet for 24 months ( n = 180 - - 250 ) and their body weights were monitored monthly . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


. ( c ) Average daily oral food intake normalized to body weight , measured on regular diet . The food intake of mice ( n = 18 - 24 ) was measured for 2 weeks at 3 - , 6 - , 12 - and 18 - month - old age .

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. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age.


d ) Quantification of gonadal fat pads from 18 - month - old mice ( n = 11 ) . * * * P0 . 0001 versus control ; Student ' s t - test . ( e , f ) Quantification of visceral ( e ) and subcutaneous ( f ) fat pads of 18 - month - old mice ( n = 6 ) . * * * P0 . 0001 versus control ; Student ' s t - test .

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d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test.


( g ) Enhanced glucose tolerance of Atg5 Tg mice . WT and Atg5 Tg mice were starved overnight and then given with an i . p injection of glucose ( 1 mg g − 1 body weight ) . The results are the mean ± s . d . of 12 - week - old WT and Atg5 Tg mice ( n = 12 ) . * P0 . 01 , * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.


( h ) Enhanced insulin sensitivity of Atg5 Tg mice . WT and Atg5 Tg mice were starved for 6 h and then i . p injected with 0 . 75 IU soluble insulin . * P0 . 01 , * * P0 . 001 versus control ; Student ' s t - test .

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(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.


( i ) Plasma levels of triglycerol ( TG ) in WT and Atg5 Tg mice . The mice were fasted for 3 h before measurement . Young mice ( 4 - week old , n = 8 ) ; old mice ( 18 - month old , n = 8 ) . * * * P0 . 0001 versus control ; Student ' s t -

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(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test


( j ) Plasma leptin levels in WT and Atg5 Tg mice ( n = 8 - 12 ) . * * * P0 . 0001 versus control ; Student ' s t - test . (

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(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. (


( k ) GSH / GSSG ratio in the liver ( n = 4 males , three times trial ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.


( l ) Respiration rates of mitochondria in WT and Atg5 Tg MEFs ( n = 2 ) .

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(l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2).


( m ) Wire - hang endurance test of Atg5 Tg and WT mice ( n = 10 ) . All the bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( a ) Comparison of Atg12 - Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis . MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13 , after which cell extracts were examined by western blot analysis using anti - Atg5 and anti - β - actin antibodies .

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(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies.


( b ) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no . 6 by rapamycin and / or Baf . A1 . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf . A1 , and cell extracts were then analysed by western blotting . The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β - actin .

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(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.


( c , d ) Increased resistance of Atg5 Tg MEFs to oxidative stress . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated for 24 h with 50 , 100 , 200 and 300 μM H2O2 ( c ) or 300 μM H2O2 in the absence or presence of 5 mM 3 - MA or 20 nM Baf . A1 ( Baf . A1 ) ( d ) . Cell viability was assessed by propidium iodide staining . The values are the mean ± s . e . m . ( n = 3 ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.


( e ) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress . Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope . Scale bars , 50 μm .

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(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.


( f ) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf . A1 , and cell extracts were analysed by western blotting . About 10 and 30 μg of proteins were used for LC3 ( upper panel ) and p62 ( bottom panel ) detection , respectively . β - Actin served as a control .

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(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.


Representative images for the TIF analysis shown in ( C ) . Shown are POT1Y223C / - hESCs and an unedited wild - type hESC clone ( WT / WT ) isolated in parallel , as well as a POT1 WT / Q623H hESC and a contemporaneously isolated hECS clones ( WT / WT ) . Scale bars = 10µm . Box and whisker plot ( showing min , max and average ) quantifying telomere - dysfunction induced foci ( TIF ) in two POT1 Y223C / - hESC lines , one POT1 Q623H / Q623H , and one POT1WT / Q623H hESC lines compared to wild - type unedited control hESCs . Cells were analyzed 3 months post targeting . Cells were stained for TRF1 and γ - H2Ax . POT1Y223C / - cells have increased TIFs compared to an unedited wild - type cell clone , isolated and cultured in parallel ( P < 0 . 01 , two - tailed Mann - Whitney test , one biological replicate ) .

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Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).


Images of telomere and centromere FISH on metaphase spreads of cell lines as in ( B ) . Cy3 Tel - C PNA probes were used . Arrows indicate chromosomal abnormalities . E ) Quantification of chromosomal abnormalities as determined by FISH analysis in ( D ) .

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Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).


B ) Quantification of telomere length data from two STELA experiments using the same genomic DNA , shown in ( A ) and ( Appendix Figure 2C ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


B ) TIF analysis of wild - type , POT1WT / Y223C hESCs , and the indicated controls using antibodies against TRF1 and γ - H2AX . Scale bars = 10µm . Lentiviral expression of TPP1 ∆ PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response . TPP1 ∆ PBD infected cells were analyzed 72h post infection and without selection .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O

B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.


E ) Quantification of two STELA experiments , including that shown in ( D ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


D ) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

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D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


C ) Representative genotyping outcomes of hCD34 + cells nucleofected sgRNA - 1 , xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS . Data information : The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


B ) Telomere restriction fragment assay for the five subjects shown in ( A ) . Mean telomere length is indicated .

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B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


( A ) Representative micrographs of human skin sections stained with hematoxylin - eosin ( H & E ) ( left ) and anti - IL - 38 antibody ( right ) from normal patients ( n = 11 ) and tumors of cSCC patients ( n = 13 ) . Scale bars represent 100 μm . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . ( B ) Relative expression of IL - 38 in human normal tissues ( n = 9 ) and cSCC ( n = 18 ) were analyzed using Geo Datasets ( GSE98767 ) . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O O O O O B-ORGANISM B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. (B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( C and D ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 32 weeks to induce skin tumors . ( C ) Representative micrographs of mouse normal skin ( n = 6 ) and tumor ( n = 6 ) sections stained with anti - IL - 38 antibody . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . Scale bars represent 100 μm . ( D ) Relative expression levels of Il - 38 in normal skin ( n = 5 ) and tumors ( n = 5 ) of mice were quantified using qPCR . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-DISEASE I-DISEASE O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( F ) Representative western blot bands indicating IL - 38 in mouse normal skin ( n = 3 ) and DMBA / TPA - induced tumors ( n = 3 ) . The graph shows the quantification of mean IL - 38 expression in tissues . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( B ) Representative photographs of Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA for 32 weeks . White triangles indicate the tumors on the back of mice . ( C Average tumor number ( C ) per mouse from Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA . . Error bars represent the mean ± SD ( C ) All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test (

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice. (C Average tumor number (C) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C


( A ) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f ( n = 5 ) mice after treated with DMBA for 24h . Red triangles indicate the γH2AX + positive cells . Scale bars represent 100 μm . The graph shows the number of γH2AX + cells per high - powered field . Data information : Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunohistochemical staining micrographs of γH2AX in the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of γH2AX+ cells per high-powered field. Data information: Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( A The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( A ) Representative micrograph sections stained with hematoxylin - eosin ( H & E ) from the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f mice ( n = 5 ) Scale bars represent 100 μm . The graph shows average epidermal thickness .

O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE B-DISEASE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-TISSUE O O

(A The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph sections stained with hematoxylin-eosin (H&E) from the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal thickness.


B ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( B ) Representative micrograph sections stained with anti - Ki67 antibody from the skin of Il - 38f / f ( n = 6 ) and K14Cre / + - Il - 38f / f mice ( n = 6 ) . Scale bars represent 100 μm . The graph shows average numbers of Ki67 + cells per high - powered field .

O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE B-DISEASE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

B) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (B) Representative micrograph sections stained with anti-Ki67 antibody from the skin of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.


( G and H ) Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) after overexpression ( G ) or knockdown ( H ) of IL - 38 . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) after overexpression (G) or knockdown (H) of IL-38. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test. NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( I and J ) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL - 38 overexpression ( I ) or knockdown ( J ) . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O

(I and J) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL-38 overexpression (I) or knockdown (J). NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( A and B ) Pull - down assay of IL - 38 ( 1 - 152 aa ) ( A ) or IL - 38 ( 20 - 152 aa ) ( B ) with IL - 1Rrp2 - Fc , IL - 1RAPL1 - Fc , IL1RAcp - Fc , and IgG - Fc fusion proteins .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O

(A and B) Pull-down assay of IL-38 (1-152 aa) (A) or IL-38 (20-152 aa) (B) with IL-1Rrp2-Fc, IL-1RAPL1-Fc, IL1RAcp-Fc, and IgG-Fc fusion proteins.


( C ) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector ( left ) or an expression vector encoding IL - 1Rrp2 ( right ) were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) or IL - 36β ( 200 ng / ml ) at different times .

O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(C) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector (left) or an expression vector encoding IL-1Rrp2 (right) were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) or IL-36β (200 ng/ml) at different times.


( D ) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 at different concentrations .

O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(D) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 at different concentrations.


( F ) Cell extracts prepared from 293T cells transfected with HA - tagged full - length or deletion mutants of IL - 1Rrp2 , were probed using anti - HA or anti - β - actin antibodies . Oe , overexpression .

O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(F) Cell extracts prepared from 293T cells transfected with HA-tagged full-length or deletion mutants of IL-1Rrp2, were probed using anti-HA or anti-β-actin antibodies. Oe, overexpression.


( G ) Cell extracts prepared from 293T cells transfected with full - length or deletion mutants of IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) at different times .

O O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(G) Cell extracts prepared from 293T cells transfected with full-length or deletion mutants of IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) at different times.


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after IL - 1Rrp2 interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after IL-1Rrp2 interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after JNK interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after JNK interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Expression of Tardbp , total Sort1 and exon 17b Sort1 mRNAs quantified by Q - PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to the value of scrambled shRNA ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Tardbp, total Sort1 and exon 17b Sort1 mRNAs quantified by Q-PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to the value of scrambled shRNA (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of cell lysates and culture supernatants showing TDP - 43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Uninfected cultures were used as an additional control . β - actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants , respectively . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blots of cell lysates and culture supernatants showing TDP-43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Uninfected cultures were used as an additional control. β-actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants, respectively. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses , immunolabled for BDNF ( green ) , SCG2 ( red ) and counterstained with DAPI ( blue ) in orthogonal views ( dashed lines ) showing colocalization of BDNF and SCG2 in the soma ( white arrows ) .

O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O

Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).


Colocalization between BDNF and SCG2 - positive vesicles expressed as percentage of total BDNF immunoreactivity ± SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colocalization between BDNF and SCG2-positive vesicles expressed as percentage of total BDNF immunoreactivity ±SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Colocalization between BDNF and SCG2 - positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF / SCG2 colocalization expressed as percentage of total BDNF immunoreactivity ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colocalization between BDNF and SCG2-positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF/SCG2 colocalization expressed as percentage of total BDNF immunoreactivity (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion in cultured hippocampal neurons infected with scrambled ( left ) or Tardbp ( right ) shRNA lentiviruses together with either EGFP , Sort1 shRNA or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion in cultured hippocampal neurons infected with scrambled (left) or Tardbp (right) shRNA lentiviruses together with either EGFP, Sort1 shRNA or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of TDP - 43 ( green ) and NeuN ( red ) immunohistochemistry in hippocampal CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Arrows denote areas of depleted TDP - 43 immunoreactivity in the mutant .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.


Percentage of TDP - 43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus ( HC [ - CA1 ] ) of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-TISSUE O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of TDP-43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus (HC [-CA1]) of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


TDP - 43 intensity in TDP - 43 / NeuN double - positive cells in CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of Tardbp mRNA in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Tardbp mRNA in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of micro - dissected CA1 and the remaining hippocampus showing expression of TDP - 43 , full - length and truncated ( soluble ) Sortilin , and GAPDH in Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Bands corresponding to soluble Sortilin are indicated ( black arrows ) . GAPDH immunoblotting was used to control for equal loading . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blots of micro-dissected CA1 and the remaining hippocampus showing expression of TDP-43, full-length and truncated (soluble) Sortilin, and GAPDH in Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Bands corresponding to soluble Sortilin are indicated (black arrows). GAPDH immunoblotting was used to control for equal loading. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


TDP - 43 expression in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TDP-43 expression in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of exon 17b Sort1 and total Sort1 mRNAs in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SD relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of exon 17b Sort1 and total Sort1 mRNAs in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SD relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of full - length ( left ) and truncated ( soluble , right ) Sortilin protein in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of full-length (left) and truncated (soluble, right) Sortilin protein in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx / fx mouse .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O B-ORGANISM O

Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx/fx mouse.


Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition , 20 neurons per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition, 20 neurons per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


High - magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx / fx mouse . Black arrows denote selected dendritic spines .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O

High-magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx/fx mouse. Black arrows denote selected dendritic spines.


Spine density in apical and basal dendrites of CA1 pyramidal neurons of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD ( N = 3 mice per condition , 15 sections per mouse ; * , p < 0 . 05 ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spine density in apical and basal dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD (N=3 mice per condition, 15 sections per mouse; *, p<0.05; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of a CamKIIaCRE ; Tardbpfx / fx hippocampus after stereotaxic injection of EGFP - expressing lentivirus immunostained for EGFP ( green ) and NeuN ( red ) , and counterstained with DAPI ( blue ) . The injected area within CA1 is indicated in the merged panel .

O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O

Representative photomicrographs of a CamKIIaCRE;Tardbpfx/fx hippocampus after stereotaxic injection of EGFP-expressing lentivirus immunostained for EGFP (green) and NeuN (red), and counterstained with DAPI (blue). The injected area within CA1 is indicated in the merged panel.


Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in micro - dissected CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue virus . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice injected with EGFP virus , after normalization to Gapdh mRNA . ( N = 3 mice per condition ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in micro-dissected CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice injected with EGFP virus, after normalization to Gapdh mRNA. (N=3 mice per condition). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Spine density in apical dendrites of CA1 pyramidal neurons of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue virus . Results are presented as average ± SEM ( N = 3 mice per condition , 15 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spine density in apical dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM (N=3 mice per condition, 15 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Percentage of change in field excitatory postsynaptic potential ( fEPSP ) recorded from hippocampal area CA1 after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded from hippocampal area CA1 after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice stereotactically injected with EGFP - expressing lentivirus or Sortilin rescue virus ( red ) superimposed to the same data shown in panel ( a ) ( black ) . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O O O B-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice stereotactically injected with EGFP-expressing lentivirus or Sortilin rescue virus (red) superimposed to the same data shown in panel (a) (black). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice under control conditions ( black ) or with BDNF ( 200ng / ml ) perfused from t = 0 ( green ) . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O O O B-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice under control conditions (black) or with BDNF (200ng/ml) perfused from t=0 (green). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Percentage of total time spent in the target quadrant or in the remaining three quadrants of the Barnes maze for Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice at 3 different probe times : 20 - 30 min after first training session ( short - term memory ) and 24h or 14 days after last training session ( long - term or remote memory , respectively ) . Results are presented as average ± SEM ( N = 8 mice per condition ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of total time spent in the target quadrant or in the remaining three quadrants of the Barnes maze for Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice at 3 different probe times: 20-30 min after first training session (short-term memory) and 24h or 14 days after last training session (long-term or remote memory, respectively). Results are presented as average ± SEM (N=8 mice per condition; * p < 0.05, ** p < 0.01, *** p < 0.001). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of cultured hippocampal neurons immunostained for TDP - 43 ( green ) , FLAG ( red ) and counterstained with DAPI ( blue ) in control conditions ( untransduced ) or infected with lentivirus expressing FLAG - tagged human TDP - 43 12xQN construct .

O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O

Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct.


TDP - 43 intensity in cell nuclei of untransduced or TDP - 43 12xQN - infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . Student ' s t - test ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using Student ' s t - test ( B )

B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TDP-43 intensity in cell nuclei of untransduced or TDP-43 12xQN-infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). Student's t-test; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using Student's t-test (B)


Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to levels in untransduced neurons ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . 1 - way ANOVA with Tukey ' s post - hoc ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc

B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to levels in untransduced neurons (N=3 independent experiments; n=3 wells per condition in each experiment). 1-way ANOVA with Tukey's post-hoc; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis


BDNF secretion induced by KCl treatment as assessed by in - situ BDNF ELISA in cultured hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc

B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion induced by KCl treatment as assessed by in-situ BDNF ELISA in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis


BDNF secretion in cultured hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc

B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis


Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM B-GENEPROD B-ORGANISM O O O O O O O B-ORGANISM B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of cell lysates showing total TDP - 43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . The antibody used here detects both mouse and human TDP - 43 proteins . GAPDH immunoblotting was used to control for equal loading . Molecular weights are noted in kDa . Bands corresponding to mouse and human TDP - 43 are indicated . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM B-GENEPROD B-ORGANISM O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blots of cell lysates showing total TDP-43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. The antibody used here detects both mouse and human TDP-43 proteins. GAPDH immunoblotting was used to control for equal loading. Molecular weights are noted in kDa. Bands corresponding to mouse and human TDP-43 are indicated. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM B-GENEPROD B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; ***, p<0.001 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM B-GENEPROD B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of TARDBP ( left ) , total SORT1 ( center ) and exon 17b SORT1 ( right ) mRNAs quantified by Q - PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 ( grey bars ) or disease - associated Val337 ( black bars ) TARDBP alleles . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of TARDBP (left), total SORT1 (center) and exon 17b SORT1 (right) mRNAs quantified by Q-PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 (grey bars) or disease-associated Val337 (black bars) TARDBP alleles. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion in stem - cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles , infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF secretion in stem-cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles, infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment. Results are presented as average ± SEM (N=3 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Asynchronously growing human U - 2 OS cells were treated with 0 . 5 Gy of IR , allowed to recover for the indicated time periods , and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC .

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Asynchronously growing human U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC.


Representative images from the QIBC analysis in ( A ) . Scale bars , 10 μm .

O O O O B-EXP_ASSAY O O O O O O O O O O O O

Representative images from the QIBC analysis in (A). Scale bars, 10 μm.


Cells were treated with 0 . 5 Gy of IR in absence or presence of 0 . 4 M sorbitol , fixed 1h later , and 53BP1 assembly at DSBs was analyzed by QIBC . Treatments as in ( C ) ; RIF1 accumulation at DSBs was analyzed by QIBC .

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Cells were treated with 0.5 Gy of IR in absence or presence of 0.4 M sorbitol, fixed 1h later, and 53BP1 assembly at DSBs was analyzed by QIBC. Treatments as in (C); RIF1 accumulation at DSBs was analyzed by QIBC.


U - 2 OS cells were treated with 0 . 5 Gy of IR , allowed to recover for the indicated time periods , stained for 53BP1 and MDC1 , and analyzed by QIBC . 53BP1 foci intensities are plotted against MDC1 foci intensities , and representative images are shown .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O

U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, stained for 53BP1 and MDC1, and analyzed by QIBC. 53BP1 foci intensities are plotted against MDC1 foci intensities, and representative images are shown.


U - 2 OS cells stably expressing GFP - MDC1 or GFP - 53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals . Representative images are provided and kinetics of GFP - MDC1 and GFP - 53BP1 foci formation and dissolution are shown as single cell tracks . Bold lines represent averages from n = 29 for GFP - MDC1 and n = 18 for GFP - 53BP1 cells .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

U-2 OS cells stably expressing GFP-MDC1 or GFP-53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals. Representative images are provided and kinetics of GFP-MDC1 and GFP-53BP1 foci formation and dissolution are shown as single cell tracks. Bold lines represent averages from n=29 for GFP-MDC1 and n=18 for GFP-53BP1 cells.


U - 2 OS cells were treated with 1 Gy of IR and fixed at the indicated time - points . Mean 53BP1 foci intensity was analyzed from more than 1500 cells per time - point . Mean ( solid line ) and standard deviation from the mean ( dashed lines ) are indicated . The same cells as in ( A ) were analyzed for mean MDC1 foci intensity . Mean ( solid line ) and standard deviation from the mean ( dashed lines ) are indicated .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O

U-2 OS cells were treated with 1 Gy of IR and fixed at the indicated time-points. Mean 53BP1 foci intensity was analyzed from more than 1500 cells per time-point. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated. The same cells as in (A) were analyzed for mean MDC1 foci intensity. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated.


GFP - 53BP1 cells were treated with 25 ng / ml NCS to induce DNA breaks and imaged at 30 minutes intervals . Examples of GFP - 53BP1 fusions ( green arrowheads and magnified regions ) and fissions ( blue arrowheads and magnified regions ) are shown .

B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

GFP-53BP1 cells were treated with 25 ng/ml NCS to induce DNA breaks and imaged at 30 minutes intervals. Examples of GFP-53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown.


53BP1 - RFP cells , in which the endogenous 53BP1 gene locus had been engineered by CRISPR / Cas9 to express 53BP1 - mScarlet from the natural promoter , were treated with NCS ( 25 ng / ml ) and imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads and magnified regions ) and fissions ( blue arrowheads and magnified regions ) are shown on the left and in higher magnification on the right .

B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

53BP1-RFP cells, in which the endogenous 53BP1 gene locus had been engineered by CRISPR/Cas9 to express 53BP1-mScarlet from the natural promoter, were treated with NCS (25 ng/ml) and imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown on the left and in higher magnification on the right.


GFP - 53BP1 cells were left untreated , or treated with APH ( 0 . 5 μM ) or ATRi ( 1 μM ) to induce replication stress - associated heritable DNA lesions , and cells were imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads and magnified regions ) are shown .

B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O

GFP-53BP1 cells were left untreated, or treated with APH (0.5 μM) or ATRi (1 μM) to induce replication stress-associated heritable DNA lesions, and cells were imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) are shown.


Light - induced optoDroplet formation of Cry2 - mCherry - 53BP1 , Cry2 - mCherry - 53BP1 W1495A , and Cry2 - mCherry - MDC1 . Cells were imaged at 15 seconds intervals . Representative images of optoDroplet formation before and 6 minutes after light induction are shown . Quantifications from single - cell QIBC analysis of 2 - 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

Light-induced optoDroplet formation of Cry2-mCherry-53BP1, Cry2-mCherry-53BP1 W1495A, and Cry2-mCherry-MDC1. Cells were imaged at 15 seconds intervals. Representative images of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mCherry - 53BP1 W1495A optoDroplet fusion at 15 seconds time resolution . Fusing optodroplets are highlighted by green arrowheads and in the magnified insets .

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Cry2-mCherry-53BP1 W1495A optoDroplet fusion at 15 seconds time resolution. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.


Light - induced optoDroplet formation of the indicated Cry2 - mCherry - 53BP1 constructs . Cells were imaged at 15 seconds intervals . Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown . Quantifications from single - cell QIBC analysis of 2 - 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated . Red bars indicate the part of 53BP1 that was expressed as Cry2 - mCherry fusion .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O

Light-induced optoDroplet formation of the indicated Cry2-mCherry-53BP1 constructs. Cells were imaged at 15 seconds intervals. Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion.


Cry2 - mCherry - 53BP1 1203 - 1972 W1495A optoDroplets undergo fusion events upon light induction . Fusing optodroplets are highlighted by green arrowheads and in the magnified insets .

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Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.