Figure 6 ( E , F ) Endogenous ubiquitination of Usp9x ( E ) and CaN ( F ) in primary CD4 + T cells treated with DMSO or Gonib32 , then stimulated with ( + ) or without ( - ) PMA / ionomycin ( P / I , 10 / 100 ng / ml ) . ( G , H ) Immunoblot analysis of the interaction of Usp9x and CaN , NF - AT and CaN ( G ) , and NFATc1 / 2 in nuclear extract ( NE ) and cytoplasmic extract ( CE ) ( H ) in primary CD4 + T cells treated with Gonib32 , then stimulated with ( + ) or without ( - ) P / I ( 10 / 100 ng / ml ) . ECAR measurements of glycolysis and proliferation analysis in lean and obese naïve CD4 + T cells treated with DMSO or Gonib32 ( 2 μM ) upon α - CD3 / CD28 ( 1 / 1 µg / ml ) stimulation for 24 h ( ECAR ) ( I , J ) ( n = 3 , biological replicates ) . The statistical results are presented as a bar graph ( J ( M - O ) BM12 / SJL mice ( recipient , R ) were immunized with CD4 + T cells from lean and obese mice ( donors , D ) , then were injected with Gonib32 ( 50 mg / kg ) or vehicle control ( 5 % DMSO + 95 % saline ) once a day for 3 weeks . Anti - dsDNA , anti - ssDNA , and anti - histone IgG or IgM in serum were measured using ELISA ( M ) . Immunofluorescent analysis of anti - nuclear antibody in serum ( top ) and IgG deposition in the kidney ( bottom ) ( n = 4 or 5 , biological replicates ) . Data are presented as immunofluorescent images ( N ) and quantification bar graphs ( O ) . All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(E, F) Endogenous ubiquitination of Usp9x (E) and CaN (F) in primary CD4+ T cells treated with DMSO or Gonib32, then stimulated with (+) or without (-) PMA/ionomycin (P/I, 10/100 ng/ml).(G, H) Immunoblot analysis of the interaction of Usp9x and CaN, NF-AT and CaN (G), and NFATc1/2 in nuclear extract (NE) and cytoplasmic extract (CE) (H) in primary CD4+ T cells treated with Gonib32, then stimulated with (+) or without (-) P/I (10/100 ng/ml).ECAR measurements of glycolysis and proliferation analysis in lean and obese naïve CD4+ T cells treated with DMSO or Gonib32 (2 μM) upon α-CD3/CD28 (1/1 µg/ml) stimulation for 24 h (ECAR) (I, J) (n = 3, biological replicates). The statistical results are presented as a bar graph (J(M-O) BM12/SJL mice (recipient, R) were immunized with CD4+ T cells from lean and obese mice (donors, D), then were injected with Gonib32 (50 mg/kg) or vehicle control (5% DMSO + 95% saline) once a day for 3 weeks. Anti-dsDNA, anti-ssDNA, and anti-histone IgG or IgM in serum were measured using ELISA (M). Immunofluorescent analysis of anti-nuclear antibody in serum (top) and IgG deposition in the kidney (bottom) (n = 4 or 5, biological replicates). Data are presented as immunofluorescent images (N) and quantification bar graphs (O). All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


Figure 1A . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of neovascular age related macular degeneration ( NV AMD ) patient with active lesions ( 1 ) , cystoid macular edema ( 2 ) , subretinal fibrin deposits ( 3 ) and serous retinal detachment ( 4 ) . B . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of control patient with a medium sized , stage 3 , full thickness macular hole ( 5 ) . C - G . Vitreous humor analyzed by ELISA for VEGF - A ( C ) ; n = 7 ( Ctrl ) , 9 ( NV AMD ) , SEMA3A ( D ) ; n = 10 ( Ctrl ) , 10 ( NV AMD ) , TGFβ ( E ) ; n = 8 ( Ctrl ) , 7 ( NV AMD ) , PDGF - BB ( F ) ; n = 5 ( Ctrl ) , 6 ( NV AMD ) , PGF ( G ) : n = 5 ( Ctrl ) , 5 ( NV AMD ) . Dots represent concentrations of individual patient samples .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Optical coherence tomography (OCT) horizontal B-scan and thickness map of neovascular age related macular degeneration (NV AMD) patient with active lesions (1), cystoid macular edema (2), subretinal fibrin deposits (3) and serous retinal detachment (4). B. Optical coherence tomography (OCT) horizontal B-scan and thickness map of control patient with a medium sized, stage 3, full thickness macular hole (5).C-G. Vitreous humor analyzed by ELISA for VEGF-A (C); n = 7 (Ctrl), 9 (NV AMD), SEMA3A (D); n = 10 (Ctrl), 10 (NV AMD), TGFβ (E); n = 8 (Ctrl), 7 (NV AMD), PDGF-BB (F); n = 5 (Ctrl),6 (NV AMD), PGF (G): n = 5 (Ctrl), 5 (NV AMD). Dots represent concentrations of individual patient samples.


Figure 2H . Representative confocal images of IBA - 1 - stained mononuclear phagocytes on choroidal flat mounts from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D7 and D14 . Examples of macrophage quantification ( yellow stars ) are presented in side panels . Scale bar : 20μm . I , J . Total number of IBA - 1 - positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 ( I ) and D14 ( J ) ; n = 19 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 25 burns ( D7 LysM - Cre / Nrp1fl / fl ) n = 37 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 23 burns ( D7 LysM - Cre / Nrp1fl / fl ) , 3 - 5 mice with ~ 4 burns per eye . K . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and IB4 - stained laser impact area from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 . Scale bar : 20μm . L - N . Quantification of area of FITC - dextran - labeled CNV ( L ) , isolectin B4 ( IB4 ) - stained laser impact area ( M ) and the ratio of FITC / IB4 per laser - burn ( N ) relative to LysM - Cre / Nrp1 + / + at D14 ; n = 23 burns ( LysM - Cre / Nrp1 + / + ) , n = 27 burns ( LysM - Cre / Nrp1fl / fl ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2H. Representative confocal images of IBA-1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7 and D14. Examples of macrophage quantification (yellow stars) are presented in side panels. Scale bar: 20μm. I, J. Total number of IBA-1-positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 (I) and D14 (J); n = 19 burns (D7 LysM-Cre/Nrp1+/+), n = 25 burns (D7 LysM-Cre/Nrp1fl/fl) n = 37 burns (D7 LysM-Cre/Nrp1+/+), n = 23 burns (D7 LysM-Cre/Nrp1fl/fl), 3-5 mice with ~4 burns per eye.K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4 )-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).


Figure 3A - D . mRNA expression of inflammation markers relative to LysM - Cre / Nrp1 + / + in mouse RPE - choroid - sclera complexes at D3 for Il1b ( A ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Il6 ( B ) ; n = 7 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Vegfa ( C ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Tnf ( D ) ; n = 5 ( LysM - Cre / Nrp1 + / + ) , n = 10 ( LysM - Cre / Nrp1fl / fl ) . K , L . mRNA expression relative to LysM - Cre / Nrp1 + / + of inflammation markers in mouse BMDMs for Il1b ( K ) ; n = 4 ( LysM - Cre / Nrp1 + / + ) , n = 5 ( LysM - Cre / Nrp1fl / fl ) and Il6 ( L ) ; n = 3 ( LysM - Cre / Nrp1 + / + ) , n = 2 ( LysM - Cre / Nrp1fl / fl ) P . Heatmap ( left ) and enrichment plot ( right ) of GO Angiogenesis gene set enrichment analysis ( GSEA ) of wildtype and LysM - Cre / Nrp1fl / fl peritoneal macrophages ; n = 2 . NES , normalized enrichment score ; FDR , false discovery rate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-D. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D3 for Il1b (A); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Il6 (B); n = 7 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Vegfa (C); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Tnf (D); n = 5 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl).K, L. mRNA expression relative to LysM-Cre/Nrp1+/+ of inflammation markers in mouse BMDMs for Il1b (K); n = 4 (LysM-Cre/Nrp1+/+), n = 5 (LysM-Cre/Nrp1fl/fl) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)P. Heatmap (left) and enrichment plot (right) of GO Angiogenesis gene set enrichment analysis (GSEA) of wildtype and LysM-Cre/Nrp1fl/fl peritoneal macrophages; n = 2. NES, normalized enrichment score; FDR, false discovery rate.


Figure 4E . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and isolectin B4 ( IB4 ) - stained laser impact area from Vehicle and NRP1 - derived trap treated wildtype mice . Scale bar : 20μm . F - H . Quantification of area of FITC - dextran - labeled CNV ( F ) , IB4 - stained laser impact area ( G ) and the ratio of FITC / IB4 per laser - burn ( H ) relative to Vehicle at D14 ; n = 24 burns ( Vehicle ) , n = 26 burns ( NRP1 - derived trap ) . J - L . Quantification of area of FITC - dextran - labeled CNV ( J ) , isolectin B4 ( IB4 ) - stained laser impact area ( K ) and the ratio of FITC / IB4 per laser - burn ( L ) relative to LysM - Cre / Nrp1 + / + + Vehicle in Vehicle and NRP1 - derived trap treated LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 ; n = 16 burns ( LysM - Cre / Nrp1 + / + + Vehicle ) , n = 16 burns ( LysM - Cre / Nrp1 + / + + Trap ) , n = 13 burns ( LysM - null / Nrp1fl / fl + Vehicle ) , n = 19 burns ( LysM - Cre / Nrp1fl / fl + Trap ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).


Figure 1A Up , Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD - / - fibroblasts ( Left ) or in wt and Nfe2l2 - / - fibroblasts ( Right ) . Two representative clones , with their corresponding controls , are shown per genotype . H2B was used as negative control . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . n ≥ 3 independent experiments . B Up , Representative western blot from kidney whole cell extracts showing H2AX protein levels in age - matched wt and junD - / - mice . Mice are 2 months old ( young ) and 18 months old ( old ) mice , respectively . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 5 mice per age and per genotype . C Up , Representative western blot from kidney whole cell extracts , showing H2AX protein levels in 24 months old wt and junD - / - mice , either untreated ( - ) or treated with the anti - oxidant agent , N - acetyl - cysteine ( NAC ) . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 4 mice per treatment and per genotype . D , E Left , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and Kap1 phosphorylation ( P - Kap1 ) in wt and junD - / - fibroblasts ( D ) or in wt and Nfe2l2 - / - fibroblasts ( E ) , after H2O2 exposure for the indicated times ( hours , h ) . Right , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown on the left ) . n = 3 independent experiments . F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , following 2 Gy of γ - irradiation ( F ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 independent experiments . G , I Up , Representative γ - H2AX immunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after γ - irradiation ( 2Gy , 45 min ) ( G ) . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification . F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , after camptothecin ( CPT ) treatment ( H ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 independent experiments . G , I Up , Representative γ - H2AXimmunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after after camptothecin ( CPT ) treatment for 1h ( I ) . . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments.B Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype.C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.G,I Up, Representative γ-H2AX immunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after γ-irradiation (2Gy, 45 min) (G). Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.G,I Up, Representative γ-H2AXimmunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.


Figure 2A Bar plot showing the doubling time of wt and junD - / - fibroblasts . B Representative cell cycle distribution of asynchronous wt and junD - / - fibroblasts . C Bar plots showing the percentage of wt and junD - / - fibroblasts in G1 , S and G2 phases of the cell cycle , following serum stimulation for the indicated times ( hours , h ) , after 48h serum starvation . D Representative views of alkaline comet assays from wt and junD - / - fibroblasts either untreated or after 10Gy γ - irradiation for the indicated times ( minutes , min ) . E Alkaline comet assays from wt and junD - / - fibroblasts at basal state . Left , Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts . Right , Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments . At basal state , 16 % of junD - / - cells exhibit tail moment higher than 5 , while it is the case for only 3 % of wt cells . F Alkaline comet assays from wt and junD - / - fibroblasts after 10Gy γ - irradiation . Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts , following 10Gy γ - irradiation for the indicated times ( minutes , min ) . G Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments , following 10Gy γ - irradiation for the indicated times ( minutes , min ) . H Surviving fraction of wt and junD - / - fibroblasts 7 days after 2Gy or 5Gy γ - irradiation , as assessed by clonogenic assay . Surviving fraction is expressed as percentage ( % ) of number of colonies after irradiation normalized with respect to untreated conditions . I Surviving fraction of wt and junD - / - fibroblasts either transfected with an empty vector ( + Ctl ) or H2AX - expressing ( + H2AX ) vector . Surviving fraction is expressed as percentage ( % ) of alive cells , 72 hours post - transfection .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Bar plot showing the doubling time of wt and junD-/- fibroblasts.B Representative cell cycle distribution of asynchronous wt and junD-/- fibroblasts.C Bar plots showing the percentage of wt and junD-/- fibroblasts in G1, S and G2 phases of the cell cycle, following serum stimulation for the indicated times (hours, h), after 48h serum starvation.D Representative views of alkaline comet assays from wt and junD-/- fibroblasts either untreated or after 10Gy γ-irradiation for the indicated times (minutes, min).E Alkaline comet assays from wt and junD-/- fibroblasts at basal state. Left, Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts. Right, Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments. At basal state, 16% of junD-/- cells exhibit tail moment higher than 5, while it is the case for only 3% of wt cells.F Alkaline comet assays from wt and junD-/- fibroblasts after 10Gy γ-irradiation. Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts, following 10Gy γ-irradiation for the indicated times (minutes, min).G Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments, following 10Gy γ-irradiation for the indicated times (minutes, min).H Surviving fraction of wt and junD-/- fibroblasts 7 days after 2Gy or 5Gy γ-irradiation, as assessed by clonogenic assay. Surviving fraction is expressed as percentage (%) of number of colonies after irradiation normalized with respect to untreated conditions.I Surviving fraction of wt and junD-/- fibroblasts either transfected with an empty vector (+ Ctl) or H2AX-expressing (+ H2AX) vector. Surviving fraction is expressed as percentage (%) of alive cells, 72 hours post-transfection.


Figure 3A Up , Representative western blot from chromatin - free fraction or whole cell extracts , as indicated , showing H2AX protein levels in wt and junD - / - fibroblasts . Different amounts of loaded proteins and exposures ( low - or high - exp ) are presented , H2AX being easier to detect when associated with chromatin . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) . B Up , Representative western blot from chromatin - free fraction extracts showing H2AX protein levels in wt and junD - / - fibroblasts either untreated ( - ) or after 8h treatment ( + ) with proteasome inhibitor MG132 . Jun is used as positive control for treatment efficiency . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) . C Bar plot showing stabilization of H2AX protein in wt and junD - / - fibroblasts , as assessed by densitrometry analysis of western blots ( as shown in B ) . Data are expressed as percentage ( % ) of H2AX protein levels after treatment with MG132 with respect to untreated conditions . D Representative western blots from whole cell extracts showing HA - tagged ubiquitin ( Left ) or Flag - tagged H2AX ( Right ) protein levels . wt and junD - / - fibroblasts were co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were then immunoprecipitated with Flag - specific antibody and incubated either with HA - specific antibody ( Left ) or Flag - specific antibody ( Right ) . Two different exposures ( low or high exp ) are presented . * stands for the immunoglobulins . E Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in wt and junD - / - fibroblasts . Cells were first transfected with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and next treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in wt and junD - / - fibroblasts , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part . F Left , Representative western blots showing H2AX - WT protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in junD - / - fibroblasts co - transfected with Flag - tagged H2AX - WT construct and either non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , RNF8 ( siRNF8 ) or BMI1 ( siBMI1 ) . Efficiency of each siRNA has been verified and reached 60 % of inhibition , in average , as shown for RNF168 ( Up ) . Protein extracts were processed following cycloheximide ( CHX ) treatment for the indicated times ( hours , h ) . Right , Bar graph showing the percentage ( % ) of decrease of H2AX - WT protein levels after 4h treatment with CHX ( t = 4 ) with respect to untreated conditions ( t = 0 ) in junD - / - fibroblasts transfected with specific siRNA , as indicated . G Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels from whole cell extracts in junD - / - fibroblasts transfected either with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and co - transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) . Cells were treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) before protein extraction . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in junD - / - fibroblasts transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part . H Representative western blot showing RNF168 , Flag - tagged H2AX ( H2AX - FLAG ) and endogenous H2AX ( H2AX ) protein levels from whole cell extracts in wt and junD - / - fibroblasts . Cells were first co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were next immunoprecipitated with Flag - specific antibody and incubated either with RNF168 - specific antibody ( Up ) or H2AX - specific antibody ( Down ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Up, Representative western blot from chromatin-free fraction or whole cell extracts, as indicated, showing H2AX protein levels in wt and junD-/- fibroblasts. Different amounts of loaded proteins and exposures (low- or high-exp) are presented, H2AX being easier to detect when associated with chromatin. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).B Up, Representative western blot from chromatin-free fraction extracts showing H2AX protein levels in wt and junD-/- fibroblasts either untreated (-) or after 8h treatment (+) with proteasome inhibitor MG132. Jun is used as positive control for treatment efficiency. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).C Bar plot showing stabilization of H2AX protein in wt and junD-/- fibroblasts, as assessed by densitrometry analysis of western blots (as shown in B). Data are expressed as percentage (%) of H2AX protein levels after treatment with MG132 with respect to untreated conditions.D Representative western blots from whole cell extracts showing HA-tagged ubiquitin (Left) or Flag-tagged H2AX (Right) protein levels. wt and junD-/- fibroblasts were co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were then immunoprecipitated with Flag-specific antibody and incubated either with HA-specific antibody (Left) or Flag-specific antibody (Right). Two different exposures (low or high exp) are presented. * stands for the immunoglobulins.E Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels (revealed using Flag-specific antibody) from whole cell extracts in wt and junD-/- fibroblasts. Cells were first transfected with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and next treated with cycloheximide (CHX) for the indicated times (hours, h). Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in wt and junD-/- fibroblasts, as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.F Left, Representative western blots showing H2AX-WT protein levels (revealed using Flag-specific antibody) from whole cell extracts in junD-/- fibroblasts co-transfected with Flag-tagged H2AX-WT construct and either non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), RNF8 (siRNF8) or BMI1 (siBMI1). Efficiency of each siRNA has been verified and reached 60% of inhibition, in average, as shown for RNF168 (Up). Protein extracts were processed following cycloheximide (CHX) treatment for the indicated times (hours, h). Right, Bar graph showing the percentage (%) of decrease of H2AX-WT protein levels after 4h treatment with CHX (t=4) with respect to untreated conditions (t=0) in junD-/- fibroblasts transfected with specific siRNA, as indicated.G Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels from whole cell extracts in junD-/- fibroblasts transfected either with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and co-transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168). Cells were treated with cycloheximide (CHX) for the indicated times (hours, h) before protein extraction. Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in junD-/- fibroblasts transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.H Representative western blot showing RNF168, Flag-tagged H2AX (H2AX-FLAG) and endogenous H2AX (H2AX) protein levels from whole cell extracts in wt and junD-/- fibroblasts. Cells were first co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were next immunoprecipitated with Flag-specific antibody and incubated either with RNF168-specific antibody (Up) or H2AX-specific antibody (Down).


Figure 4A Up , Representative views of H2AXimmunostaining from 117 BC patients of Luminal - A ( LumA , N = 33 ) , HER2 ( N = 37 ) and Triple - Negative ( TN , N = 47 ) subtypes . Scale bars = 100 μm . Down , representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts ( Left ) or Epithelium ( Right ) cells . Scale bars = 25 μm . B Scatter plots of H2AX histological score ( Hscore = staining intensity ( 0 to 4 ) x % of positive cells quantified from H2AXimmunostaining ( as shown in A ) in Fibroblasts ( Left ) or Epithelial ( Right ) cells from LumA , HER2 and TNbreasttumours . C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells ( as shown in B ) , in HER2 ( left ) and TN ( right ) breasttumours . D Representative views of smooth muscle actin ( SMA ) immunostaining from BC patients ( N = 29 ) of Luminal - A ( LumA , N = 10 ) , HER2 ( N = 10 ) and TN ( N = 9 ) subtypes . Scale bars = 100 μm . Zoom views are focused on the fibroblast compartment . Scale bars = 25 μm . E Scatter plots of SMA histological score ( Hscore ) evaluated from SMA immunostaining ( as shown in D ) . F Correlation plot between SMA and H2AX Hscores in stromal compartment . G Scatter plot of the mitotic index , evaluated by pathologists , in LumA , HER2 , and TN BC patients , as indicated . H Absence of correlation between mitotic index and epithelial H2AX Hscore , in LumA HER2 and TN BC .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 4A Up, Representative views of H2AXimmunostaining from 117 BC patients of Luminal-A (LumA, N=33), HER2 (N=37) and Triple-Negative (TN, N=47) subtypes. Scale bars = 100 μm. Down, representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts (Left) or Epithelium (Right) cells. Scale bars = 25 μm.B Scatter plots of H2AX histological score (Hscore = staining intensity (0 to 4) x % of positive cells quantified from H2AXimmunostaining (as shown in A) in Fibroblasts (Left) or Epithelial (Right) cells from LumA, HER2 and TNbreasttumours.C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells (as shown in B), in HER2 (left) and TN (right) breasttumours.D Representative views of smooth muscle actin (SMA) immunostaining from BC patients (N=29) of Luminal-A (LumA, N=10), HER2 (N=10) and TN (N=9) subtypes. Scale bars = 100 μm. Zoom views are focused on the fibroblast compartment. Scale bars = 25 μm.E Scatter plots of SMA histological score (Hscore) evaluated from SMA immunostaining (as shown in D).F Correlation plot between SMA and H2AX Hscores in stromal compartment.G Scatter plot of the mitotic index, evaluated by pathologists, in LumA, HER2, and TN BC patients, as indicated.H Absence of correlation between mitotic index and epithelial H2AX Hscore, in LumA HER2 and TN BC.


Figure 5B Left , Representative views of H2AX and γ - H2AX immunostaining in TN tumours ( N = 22 ) before and after successive cycles of chemotherapy . Scale bars = 100 μm . Zoom views are focused on the epithelial cells . Scale bars = 25 μm . Right , Scatter plots of H2AX , γ - H2AX histological scores ( Hscores ) and γ - H2AX / H2AX ratio evaluated from immunostaining in the epithelium , before and after chemotherapy . C Correlation plot between Cisplatin IC50 ( half maximal inhibitory concentration ) and H2AX protein level defined by western blots in BC cell lines ( MCF7 , BT474 , MDA361 , SKBr3 , MDA453 , MDA231 , HCC70 ) . Correlation coefficients σ and P - value are based on Spearman ' s rank correlation test . D Up , Representative western - blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) . Actin is used as internal control for protein loading . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . E Bar plot showing the percentage ( % ) of apoptotic cells as assessed by flow - cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) , and next treated with cisplatin for 72 hrs . F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients ( N = 22 ) . Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy ( as evaluated in B ) , for each patient ( values in Log2 ) . Only patients who have experienced an incomplete response have been taken into account . After treatment H2AX staining was evaluated only on residual tumour cells . The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes . G Kaplan - Meier curves showing specific overall survival ( Left ) and progression - free survival ( Right ) of TN patients with respect to major ( N = 15 ) or minor ( N = 7 ) H2AX protein level decrease , observed upon chemotherapy . Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease ( respectively 2 / 3 and 1 / 3 of patients ) . H Scatter plots showing the number of cleaved caspase 3 - positive cells / mm2 in TN BC , categorized according to major - or minor - H2AX protein level decrease after chemotherapy . I Scatter plots showing variation of NRF2 histological score in patients with major - H2AX decrease before and after chemotherapy . J Box plots showing expression levels of representative NRF2 - target genes in TN tumours with minor - or major - H2AX decrease , as indicated . Lines indicate Means and whiskers show 10 - 90 percentiles . ACOT7 : Acyl - CoA Thioesterase 7 ; PGD : Phosphogluconate Dehydrogenase . AHR : Aryl Hydrocarbon Receptor ; JAG1 : Jagged1 . P - values are from t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Left, Representative views of H2AX and γ-H2AX immunostaining in TN tumours (N = 22) before and after successive cycles of chemotherapy. Scale bars = 100 μm. Zoom views are focused on the epithelial cells. Scale bars = 25 μm. Right, Scatter plots of H2AX, γ-H2AX histological scores (Hscores) and γ-H2AX/H2AX ratio evaluated from immunostaining in the epithelium, before and after chemotherapy.C Correlation plot between Cisplatin IC50 (half maximal inhibitory concentration) and H2AX protein level defined by western blots in BC cell lines (MCF7, BT474, MDA361, SKBr3, MDA453, MDA231, HCC70). Correlation coefficients σ and P-value are based on Spearman's rank correlation test.D Up, Representative western-blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX). Actin is used as internal control for protein loading. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above).E Bar plot showing the percentage (%) of apoptotic cells as assessed by flow-cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX), and next treated with cisplatin for 72 hrs.F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients (N = 22). Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy (as evaluated in B), for each patient (values in Log2). Only patients who have experienced an incomplete response have been taken into account. After treatment H2AX staining was evaluated only on residual tumour cells. The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes.G Kaplan-Meier curves showing specific overall survival (Left) and progression-free survival (Right) of TN patients with respect to major (N=15) or minor (N=7) H2AX protein level decrease, observed upon chemotherapy. Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease (respectively 2/3 and 1/3 of patients).H Scatter plots showing the number of cleaved caspase 3-positive cells / mm2 in TN BC, categorized according to major- or minor-H2AX protein level decrease after chemotherapy.I Scatter plots showing variation of NRF2 histological score in patients with major-H2AX decrease before and after chemotherapy.J Box plots showing expression levels of representative NRF2-target genes in TN tumours with minor- or major-H2AX decrease, as indicated. Lines indicate Means and whiskers show 10-90 percentiles. ACOT7: Acyl-CoA Thioesterase 7; PGD: Phosphogluconate Dehydrogenase. AHR: Aryl Hydrocarbon Receptor; JAG1: Jagged1. P-values are from t-test.


Figure 1Immunoblot analysis of ADAR1 p110 , ADAR1 p150 and ADAR2 expression in various organs isolated from wild - type ( WT ) mice . The expression of GAPDH is shown as a reference . The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) were normalized to the level of expression in DN thymocytes ( DN values set to 1 ) . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 5 ; Mann - Whitney U - test , * p < 0 . 05 , * * p < 0 . 01 ) . The relative expression of type I interferon - stimulated genes ( ISGs ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The relative expression of ISGs was normalized against the level of expression in DN thymocytes ( DN values set to 1 ) .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).


Figure 2Total number of thymocytes in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( B ) . The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( C ) . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and were gated by the high expression of TCRβ ( TCRβhigh ) . Representative images are shown ( D ) . The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( E ) . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with antibodies against CD5 and analyzed by flow cytometry . Representative flow cytometric images for DP and 4SP thymocytes are shown ( upper panel ) . The percentages of CD5 - DP and 4SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C).Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D).The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E).Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Figure 3CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with HY - TCR + mice to generate Cd4cre A1fl / fl HY - TCR + mice . Thymocytes isolated from male ( A ) and female ( B ) HY - TCR + or Cd4cre A1fl / fl HY - TCR + mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( left panels ) . The absolute number of DP and 8SP thymocytes were compared between HY - TCR + and Cd4cre A1fl / fl HY - TCR + mice ( right panels ) . Thymocytes isolated from WT and Cd4cre A1fl / fl mice were labeled with Cell Proliferation Dye and then stimulated with plate - bound anti - CD3 and anti - CD28 antibodies for 4 days . Apoptotic ( Annexin V + ) and proliferating thymocytes ( reduced intensity of Proliferation Dye ) were determined by flow cytometry . Representative flow cytometric images are shown ( left panel ) . The percentages of either Annexin V + or Annexin V - proliferating thymocytes were compared between WT ( n = 5 ) and Cd4cre A1fl / fl ( n = 4 ) mice ( right panel ) . The relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 , N . S . ; not significant ) . The mean value for the relative expression of each anti - apoptotic gene in WT 4SP thymocytes was set as one . Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from WT and Cd4cre A1fl / fl mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels).Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel).The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively


Figure 4Incidence of spontaneous colitis observed in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice ( n = 11 for each group ; Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl / fl mouse . Scale bar , 200 μm . Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ , anti - IL17 or anti - Foxp3 antibodies together with anti - CD4 antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The absolute number of IFNγ - , IL17 - , or Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( right panel ) . Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) . Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - Foxp3 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) . Effector T ( Teff ) cells isolated from the spleen of a WT mouse were stimulated with soluble anti - CD3 ( 5 μg / ml ) in the presence of mitomycin C - treated splenocytes and were cultured with regulatory T ( Treg ) cells isolated from WT and Cd4cre A1fl / fl mice at the indicated ratios . After 72 h , the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , N . S . ; not significant ) . The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0 % .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group;Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm.Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel).Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.


Figure 5RNA editing in the 3 ′ UTR ( C ) and intronic region ( D ) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild - type ( WT ) DP , WT 4SP thymocytes , and 4SP thymocytes isolated from CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice . The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in WT DP thymocytes was set as one . Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl / fl mice ( n = 2 for each group ) . The expression of GAPDH is shown as a reference .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one.Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.


Figure 6CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with MDA5 knockout ( Ifih1 - / - ) mice to generate Cd4cre A1fl / fl Ifih1 - / - mice . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The percentage of DN , DP , 4SP and 8SP thymocytes were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl ( n = 7 ) mice ( right panel ) . The same data shown in Fig 2C was used for Cd4cre A1fl / fl mice . The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in Ifih1 - / - 4SP thymocytes was set as one . Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 , respectively and displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , N . S . ; not significant ) ( right panel ) by setting the mean value of stimulated Ifih1 - / - samples at 60 sec as 1 . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stimulated with plate - bound anti - CD3 ( 5 μg / ml ) and anti - CD28 ( 1 μg / ml ) antibodies for 72 h and the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the activity of Ifih1 - / - thymocytes in an unstimulated condition was set as one . For comparative analysis , the same data shown in Appendix Fig S1B was used for Cd4cre A1fl / fl mice . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared among Ifih1 - / - ( n = 5 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 3 ) and Cd4cre A1fl / fl ( n = 3 ) mice ( lower panel ) . The same data shown in Fig 2F was used for Cd4cre A1fl / fl mice . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared among Ifih1 - / - , Cd4cre A1fl / fl Ifih1 - / - and Cd4cre A1fl / fl mice ( lower panel ) . The same data shown in Fig 2H was used for Cd4cre A1fl / fl mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with MDA5 knockout (Ifih1-/-) mice to generate Cd4cre A1fl/fl Ifih1-/- mice. Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The percentage of DN, DP, 4SP and 8SP thymocytes were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl (n = 7) mice (right panel). The same data shown in Fig 2C was used for Cd4cre A1fl/fl mice.The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in Ifih1-/- 4SP thymocytes was set as one.Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, N.S.; not significant) (right panel) by setting the mean value of stimulated Ifih1-/- samples at 60 sec as 1.Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared among Ifih1-/- (n = 5), Cd4cre A1fl/fl Ifih1-/- (n = 3) and Cd4cre A1fl/fl (n = 3) mice (lower panel). The same data shown in Fig 2F was used for Cd4cre A1fl/fl mice.Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared among Ifih1-/-, Cd4cre A1fl/fl Ifih1-/- and Cd4cre A1fl/fl mice (lower panel). The same data shown in Fig 2H was used for Cd4cre A1fl/fl mice.


Figure 7Splenocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( left panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl mice ( n = 6 ) ( right panel ) . The same data shown in Fig 4E was used for Cd4cre A1fl / fl mice . No incidence of spontaneous colitis observed in Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl Ifih1 - / - ( n = 11 ) mice . For side - by - side comparison with Cd4cre A1fl / fl miceRepresentative images of HE staining of colon dissected from an Ifih1 - / - and a Cd4cre A1fl / fl Ifih1 - / - mouse . Scale bar , 200 μm .

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Figure 7Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice.No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl miceRepresentative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm.


Figure 1 ( B ) Whole mount low magnification images show marking in interscale ( K10 + ) vs scale regions , C ) interscale ( K10 + ) vs scale regions , quantified in ( C ) . Error bars are SDs . The data shown is average of 4 different timepoints ( 2 week , 3month , 6month and 1 year ) each consisting of 2 mice and 5 images per mice . p - value is calculated for difference between scale vs interscale using Student ' s t - test ( mixed Anova ) . ( D ) Tail skin sections immunofluorescence stained for interscale ( K10 ) or scale ( K31 ) supra - basal markers , and basal markers . Scale bar = 20 µm or 50 µm as indicated on the panels . Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer . Dashed lines indicate ROI in basement membrane . ( E ) Quantification of images like those in ( D ) with n = 3 - 4 mice and 6 - 7 images per mouse showing preferred - gene enrichment . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effect model )

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Figure 1(B) Whole mount low magnification images show marking in interscale (K10+) vs scale regions,C) interscale (K10+) vs scale regions, quantified in (C). Error bars are SDs. The data shown is average of 4 different timepoints (2 week, 3month, 6month and 1 year) each consisting of 2 mice and 5 images per mice. p-value is calculated for difference between scale vs interscale using Student's t-test (mixed Anova).(D) Tail skin sections immunofluorescence stained for interscale (K10) or scale (K31) supra-basal markers, and basal markers. Scale bar =20 µm or 50 µm as indicated on the panels. Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer. Dashed lines indicate ROI in basement membrane.(E) Quantification of images like those in (D) with n=3-4 mice and 6-7 images per mouse showing preferred-gene enrichment. Error bars are SDs. P-values calculated by Student's t-test (mixed effect model)


Figure 2 ( A - F ) Human skin immunofluorescence images of mouse basal non - LRC / LRC preferred - factor staining in rete ridges ( RR ) versus inter - ridges ( IR ) . Representative images from 30 samples of different body regions and ages Scale bars are indicated on the panels ( G ) LRC / non - LRC preferred gene expression in RR and IR samples from two 30 - year - old breast samples , quantified using background subtraction P - values from paired Student ' s t - test . Error bars are SDs . 8 - 10 images were quantified each from two females .

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Figure 2(A-F) Human skin immunofluorescence images of mouse basal non-LRC/LRC preferred-factor staining in rete ridges (RR) versus inter-ridges (IR). Representative images from 30 samples of different body regions and ages Scale bars are indicated on the panels(G) LRC/non-LRC preferred gene expression in RR and IR samples from two 30-year-old breast samples, quantified using background subtraction P-values from paired Student's t-test. Error bars are SDs. 8-10 images were quantified each from two females.


Figure 5 ( A ) Schematic showing the tamoxifen injection paradigm for tdTomato - labelling of all IFE lineages in tail and back . Co - expression of markers with the lineage - traced cells at 1 - month post - tamoxifen . Dlx1 - CrER is shown as example in tail ; Scale bar = 50 µm ( B ) Quantification of images like those in ( A ) showing % tdTomato + cells expressing indicated markers . Sox6 ( black bar ) was not analyzed in K14 - Cre / EYFP lineage . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effects model ) from n = 2 - 3 mice and 5 - 6 images per replicate . ( G ) Immunofluorescence images of Sox6 staining in noUVB ( 0hr ) or UVB - exposed ( 6hrs - or 24hrs - post ) mouse back skin . ( H ) Boxplot shows % Sox6 expressing basal cells quantified from images like those in ( G ) . Central band is the mean value , the top of box indicates 25th percentile while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

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Figure 5(A) Schematic showing the tamoxifen injection paradigm for tdTomato-labelling of all IFE lineages in tail and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER is shown as example in tail; Scale bar =50 µm (B) Quantification of images like those in (A) showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP lineage. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice and 5-6 images per replicate.(G) Immunofluorescence images of Sox6 staining in noUVB (0hr) or UVB-exposed (6hrs- or 24hrs-post) mouse back skin. (H) Boxplot shows % Sox6 expressing basal cells quantified from images like those in (G). Central band is the mean value, the top of box indicates 25th percentile while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


Figure 6 ( B - G ) ( B , D , F ) Images of tail skin immune - stained for markers indicated and ( C , E , G ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates . ( I - N ) ( I , K , M ) Images of skin immune - stained for markers indicated and ( J , L , N ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

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Figure 6(B-G) (B,D,F) Images of tail skin immune-stained for markers indicated and (C,E,G) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates.(I-N) (I,K,M) Images of skin immune-stained for markers indicated and (J,L,N) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


Figure 4BN - PAGE of 20 µg solubilized BHM with several of the distinct complexes annotated . The dashed box indicates the monomeric ATP synthase ( complex V ) band subjected to IGX - MS .

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Figure 4BN-PAGE of 20 µg solubilized BHM with several of the distinct complexes annotated. The dashed box indicates the monomeric ATP synthase (complex V) band subjected to IGX-MS.


Figure 3D . ( Top ) Relative enrichment of the three microRNAs across different cell types . Quantitative expression of microRNAs in primary hippocampal neurons , primary astrocytes and primary microglia . miR - 146a - 5p is significantly enriched in microglia , while miR - 148a - 3p and miR - 181a - 5p are significantly enriched in neurons . N = 5 / group , Two - way ANOVA , Tukey ' s multiple comparisons test , * P < 0 . 05 , * * P < 0 . 01 . Bars and error bar indicates mean ± sem . ( Bottom ) miRNA expression in different cell types of mouse brain stem . The data was retrieved from Hoye et al . 2017 . E . Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA - seq analysis . miR - 181a - 5p was overexpressed in primary hippocampal neurons , while miR - 146a - 5p was overexpressed in microglia culture . Immortalized microglial cell line was used for this purpose . Given that miR - 148a - 3p was highly enriched in neurons ( D ) , primary hippocampal neurons were treated with the corresponding miR - 148a - 3p mimic . PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another . Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples ( FDR < 0 . 05 ) . Red color indicates the up - regulated genes while the blue color represents the genes those were down - regulated . H . qPCR analysis confirms the over - expression of pro - inflammation related genes ( IL - 1ß , IL - 6 , TNF - alpha ) due to overexpression of miR - 146a - 5p . Expression of anti - inflammatory gene , IL - 10 was downregulated in mimic treated cells compared to the controls . Unpaired t - tests , two - tailed , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 . Bars and error bar indicates mean ± sem . Number of biological replicates : 5 - 6 / group .

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Figure 3D. (Top) Relative enrichment of the three microRNAs across different cell types. Quantitative expression of microRNAs in primary hippocampal neurons, primary astrocytes and primary microglia. miR-146a-5p is significantly enriched in microglia, while miR-148a-3p and miR-181a-5p are significantly enriched in neurons. N = 5/group, Two-way ANOVA, Tukey's multiple comparisons test, *P<0.05, **P<0.01. Bars and error bar indicates mean ± sem. (Bottom) miRNA expression in different cell types of mouse brain stem. The data was retrieved from Hoye et al. 2017.E. Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA-seq analysis. miR-181a-5p was overexpressed in primary hippocampal neurons, while miR-146a-5p was overexpressed in microglia culture. Immortalized microglial cell line was used for this purpose. Given that miR-148a-3p was highly enriched in neurons (D), primary hippocampal neurons were treated with the corresponding miR-148a-3p mimic. PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another. Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples (FDR <0.05). Red color indicates the up-regulated genes while the blue color represents the genes those were down-regulated.H. qPCR analysis confirms the over-expression of pro-inflammation related genes (IL-1ß, IL-6, TNF-alpha) due to overexpression of miR-146a-5p. Expression of anti-inflammatory gene, IL-10 was downregulated in mimic treated cells compared to the controls. Unpaired t-tests, two-tailed, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Bars and error bar indicates mean ± sem. Number of biological replicates: 5-6/group.


Figure 4B . Functional mature synapses were quantified via co - localizations of pre - ( Synatophysin 1 ) and the post - synaptic ( PSD - 95 ) markers and compared between 3 - miR - mix and control groups . Scale bar : 10 μm . Two independent methods ( SynQuant and Colocalization ) were used for quantification . 3 - miR - mix reduced the number of functional synapses compared to controls ( n = 24 - 30 images ) C . Dendrite labeling and quantification . Dendritic spines were stained with Dil . Scale bar : 10 μm . Spine density and total spine length are substantially reduced in 3 - miR mix treated primary neurons compared to those treated with scramble RNA ( n = 49 - 97 images )

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Figure 4B. Functional mature synapses were quantified via co-localizations of pre- (Synatophysin 1) and the post-synaptic (PSD-95) markers and compared between 3-miR-mix and control groups. Scale bar: 10 μm. Two independent methods (SynQuant and Colocalization) were used for quantification. 3-miR-mix reduced the number of functional synapses compared to controls (n = 24-30 images)C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images)


Figure 5All data have been adjusted for age , gender and other latent covariates for downstream eigenvalue calculation . A . Eigen value showing the increased expression of the 3 - microRNA signature in blood plasma samples of age matched MCI ( n = 23 ) patients compared to controls ( n = 27 ) . B . Expression of 3 - microRNA signature is increased in PAXgene blood samples of MCI patients ( n = 71 ) compared to controls ( n = 65 ) from the DELCODE cohort . E . Eigen value showing the expression the 3 - microRNA signature in MCI patients for those the follow up diagnostics data assessed 2 years after were available . 15 % of these MCI patients developed Alzheimer ´ s disease ( AD ) , while the rest 85 % patients remained with MCI ( stable MCI ) . The boxplot depicts the increased expression levels of 3 - miR signature in patients who converted to AD ( n = 8 ) compared to those that had stable MCI ( n = 47 ) . F . Increased expression of 3 - microRNA signature in cerebrospinal fluid ( CSF ) of MCI patients ( n = 9 ) compared to controls ( n = 26 ) . Wilcoxon - rank test , P value is given on the corresponding panel . In the boxplots in ( A , B , E , F ) , the center line indicates the median , while the upper and lower lines represent the 75th and 25th percentiles , respectively . The whiskers represent the smallest and largest values in the 1 . 5 × interquartile range .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5All data have been adjusted for age, gender and other latent covariates for downstream eigenvalue calculation. A. Eigen value showing the increased expression of the 3-microRNA signature in blood plasma samples of age matched MCI (n = 23) patients compared to controls (n = 27). B. Expression of 3-microRNA signature is increased in PAXgene blood samples of MCI patients (n=71) compared to controls (n=65) from the DELCODE cohort.E. Eigen value showing the expression the 3-microRNA signature in MCI patients for those the follow up diagnostics data assessed 2 years after were available. 15% of these MCI patients developed Alzheimer´s disease (AD), while the rest 85% patients remained with MCI (stable MCI). The boxplot depicts the increased expression levels of 3-miR signature in patients who converted to AD (n=8) compared to those that had stable MCI (n=47). F. Increased expression of 3-microRNA signature in cerebrospinal fluid (CSF) of MCI patients (n = 9) compared to controls (n = 26). Wilcoxon-rank test, P value is given on the corresponding panel. In the boxplots in (A, B, E, F), the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range.


Figure 6H . Escape latency during the water maze training comparing 7 months old wild type mice ( WT - control , n = 17 , male : 9 , female : 8 ) and APPPS - 21 mice ( APP - control , n = 8 , male : 6 , female : 2 ) injected with scramble control oligonucleotides and APPPS1 - 21 mice injected with microRNA inhibitors ( APP miR - inhibtor mix , n = 12 , male : 8 , female : 4 ) . Bars and error bars indicate mean ± sem . .

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Figure 6H. Escape latency during the water maze training comparing 7 months old wild type mice (WT-control, n = 17, male: 9, female: 8) and APPPS-21 mice (APP-control, n = 8, male: 6, female: 2) injected with scramble control oligonucleotides and APPPS1-21 mice injected with microRNA inhibitors (APP miR-inhibtor mix, n = 12, male: 8, female: 4). Bars and error bars indicate mean ± sem..


Figure 7D . qPCR assay for several synaptic genes ( LRKK2 , Cadm3 and Slc6a11 ) confirms reinstatement of gene expression with anti - miR - mix ( n = 5 - 7 , Kruskal - wallis test ) . G . qPCR data shows rescue of AFF2 / FMR2 and Hivep3 expression in APP / PS1 mice treated with inhibitor cocktail ( n = 6 - 7 , Kruskal - wallis test ) .

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Figure 7D. qPCR assay for several synaptic genes (LRKK2, Cadm3 and Slc6a11) confirms reinstatement of gene expression with anti-miR-mix (n = 5-7, Kruskal-wallis test).G. qPCR data shows rescue of AFF2/FMR2 and Hivep3 expression in APP/PS1 mice treated with inhibitor cocktail (n = 6-7, Kruskal-wallis test).