Figure 5Rescue assays . Myc - Egg WT and Myc - SV40 - NLS - Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion ( siEgg and siWde ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . Western blotting of Myc - SV40 - NLS - Egg WT in whole ( Whole ) , cytoplasm / nucleoplasmic ( Cyto / Np ) and chromatin ( Chromatin ) fractions before ( - ) and after Flag - Wde co - expression . Anti - Myc and anti - Flag antibodies were used . Histone H3 was detected as a marker of chromatin fraction . Immunofluorescence analysis of H3K9me3 ( green ) , Myc - Egg WT ( red ) , and Myc - SV40 - NLS - Egg WT ( red ) in OSCs before and after Flag - Wde ( yellow ) co - expression . Anti - H3K9me3 , anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B ( p values based on unpaired Welch ' s t - test ) . Box plots show median ( line ) and 25th - 75th percentile ( box ) ± 1 . 5 interquartile range ( n > 25 cells for each box ) . Circles represent outliers .

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Figure 5Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction.Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.


Figure 6Heat map of Egg ChIP - seq within a 10kb window centered on Egg peaks . Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Heatmaps of Egg ChIP - seq signal within 10 kb windows centered on Egg peaks in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs . Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Metapolts showing Egg ChIP - seq signals for genomic regions around transposon ( TE ) insertions . ChIP - qPCR was performed before ( siEGFP ) and after ( siWde ) Wde depletion . qPCR was performed for Piwi target transposons ( Stalker2 , mdg1 , and gypsy ) and normalized to % input of a PIwi non - target transposon ( roo ) . Error bars represent SD from three independent experiments . Density plots for Egg ChIP - seq signal on Stalker2 , mdg1 , and gypsy in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs . Input : input DNA was used as a control . RPM : reads per million mapped reads .

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Figure 6Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.


Figure 1G . Immunostain for Ki67 in wildtype and lymphoma - bearing spleen . Representative images are shown at left . The proportion of nuclei positive for Ki67 ( proliferation index ) is quantified at right for n = 3 spleens ( T - test p = 0 . 00002331 ) .

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Figure 1G. Immunostain for Ki67 in wildtype and lymphoma-bearing spleen. Representative images are shown at left. The proportion of nuclei positive for Ki67 (proliferation index) is quantified at right for n=3 spleens(T-test p=0.00002331).


Figure 2F , G ( F ) PCR for Cγ1 - Cre and wildtype Cγ1 alleles and ( G ) , wildtype , floxed or excised Blimp1 alleles in DNA from wildtype ( wt ) control C57bl / 6J cells and purified T - cells or CD45 . 2 + cells from mouse with lymphoma ( T1 , T2 ) . PCR data were confirmed in at least 3 independent tests . Lad . = ladder .

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Figure 2F, G (F) PCR for Cγ1-Cre and wildtype Cγ1 alleles and (G), wildtype, floxed or excised Blimp1 alleles in DNA from wildtype (wt) control C57bl/6J cells and purified T-cells or CD45.2+ cells from mouse with lymphoma (T1, T2). PCR data were confirmed in at least 3 independent tests. Lad. = ladder.


Figure 5F . Western blot for c - MYC in whole spleen lysates from control C57bl / 6J and mPTCL mice . Quantification of c - MYC expression normalized to vinculin housekeeper gene levels ( n = 6 ) . T - test p = 0 . 0007 . Bars represent SEM .

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Figure 5F. Western blot for c-MYC in whole spleen lysates from control C57bl/6J and mPTCL mice. Quantification of c-MYC expression normalized to vinculin housekeeper gene levels (n=6). T-test p=0.0007. Bars represent SEM.


Figure 6B . Western blot for phosphorylated and total ZAP70 , AKT , S6 and GSK3β in lysates from three control ( C ) C57bl / 6J and three mPTCL ( T ) spleens . Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling . H . Western blot for γH2AX , total H2AX , CHK1 and RPA32 in control C57bl / 6J and mPTCL spleens . GAPDH is a loading control . Three representative samples per group are shown in the blot ( left ) and quantification was performed on n = 6 at right . T - test p = 0 . 0798 ( CHK1 ; not statistically significant , ns ) , p = 8 . 69E - 05 ( RPA32 ) , p = 2 . 11E - 03 ( γH2AX ) . * * * p < 0 . 001 .

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Figure 6B. Western blot for phosphorylated and total ZAP70, AKT, S6 and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.H. Western blot for γH2AX, total H2AX, CHK1 and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left) and quantification was performed on n=6 at right. T-test p=0.0798 (CHK1; not statistically significant, ns), p=8.69E-05 (RPA32), p=2.11E-03 (γH2AX). ***p<0.001.


Figure 7A . Sanger screen IC50 drug sensitivity data ( IC50 values ) for DDR inhibitors targeting ATR , CHK1 / 2 and Wee1 in T - ALL , PTCL ( DEL , KARPAS - 299 , SUP - M2 ) and pan - cancer cell lines . ANOVA p = 0 . 413 ( AZD6738 , ATR inhibitor ) , p = 0 . 750 ( AZD772 , CHK1 / 2 inhibitor ) , p = 0 . 119 ( MK - 8776 , ATR inhibitor ) and 0 . 282 ( AZD1775 , Wee1 inhibitor ) . Bars represent SEM of biological replicates . B . PTCL ( red ) and T - ALL ( yellow ) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo . Average IC50 values from three experiments are plotted . Bars represent SEM . F . Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 ( ATR ; n = 4 ) relative to untreated mPTCL spleens taken at endpoint ( n = 6 ) . T - test p = 2 . 70E - 06 . PC9 cells dosed in vitro 24h with 1 µM aphidicolin ( Aph ; an inhibitor of DNA replication ) or with AZD6738 ( ATR ) are positive controls for DNA damage . Bars represent SEM . * * * p < 0 .

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Figure 7A. Sanger screen IC50 drug sensitivity data (IC50 values) for DDR inhibitors targeting ATR, CHK1/2 and Wee1 in T-ALL, PTCL (DEL, KARPAS-299, SUP-M2) and pan-cancer cell lines. ANOVA p=0.413 (AZD6738, ATR inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR inhibitor) and 0.282 (AZD1775, Wee1 inhibitor). Bars represent SEM of biological replicates.B. PTCL (red) and T-ALL (yellow) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo. Average IC50 values from three experiments are plotted. Bars represent SEM.F. Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 (ATR; n=4) relative to untreated mPTCL spleens taken at endpoint (n=6). T-test p=2.70E-06. PC9 cells dosed in vitro 24h with 1 µM aphidicolin (Aph; an inhibitor of DNA replication) or with AZD6738 (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001


Figure 1 ( C ) Figures show the presence of differentially expressed genes within the analyzed single cells . Black color indicates cells with RPKM values above 40 for Pax3 , 300 for Stmn2 , 200 for Slc1a3 and 100 for

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Figure 1(C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp


Figure 2 ( B ) Expression ( RPKM ) of Sox3 and Sox9 in NPC and GPC cultures from RNA - seq experiments . Results are represented as mean ± SEM from 3 - 4 experiments ( n = 3 for NPC and n = 4 for GPC , * * * equals p = 4x10 - 5 , t . test ) ( C ) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures . Analysis of TUJ1 , GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes . Scale bar represents 40µm1 ] . ( F ) H3K27Ac read density in NPCs or GPCs around DNA - regions bound by SOX3 in NPCs . SOX3 DNA - regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci . Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci ( G ) Expression pattern of genes associated with group I and II loci ( from Fig . 2E ) within differentially expressed gene sets . Significance calculated by prop . test R , ( * * * ) P < 0 . 001 . ( H ) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs . Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs . ( I ) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs . Bar graph shows expression pattern of genes co - bound by SOX3 and SOX9 in GPCs . ( J ) ChIP - seq peak graphics around the astrocyte gene Fgfbp3 . ChIP - seq peaks are derived from three different experiments ; SOX3 ChIPs in NPCs , SOX3 ChIPs in GPCs , SOX9 ChIPs in GPCs . Both ChIP - seq reads and called peak regions ( underlying black lines ) are shown for all data sets . Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors . P - values ( phyper , R ) were calculated from the total number of protein coding genes in mm10 assembly ( 23 ´ 389 )

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Figure 2(B) Expression (RPKM) of Sox3 and Sox9 in NPC and GPC cultures from RNA-seq experiments. Results are represented as mean ± SEM from 3-4 experiments (n=3 for NPC and n=4 for GPC, *** equals p=4x10-5, t.test)(C) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures. Analysis of TUJ1, GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes. Scale bar represents 40µm1]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci(G) Expression pattern of genes associated with group I and II loci (from Fig. 2E) within differentially expressed gene sets. Significance calculated by prop.test R, (***) P<0.001. (H) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs. Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs. (I) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs. Bar graph shows expression pattern of genes co-bound by SOX3 and SOX9 in GPCs. (J) ChIP-seq peak graphics around the astrocyte gene Fgfbp3. ChIP-seq peaks are derived from three different experiments; SOX3 ChIPs in NPCs, SOX3 ChIPs in GPCs, SOX9 ChIPs in GPCs. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets. Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors. P-values (phyper, R) were calculated from the total number of protein coding genes in mm10 assembly (23´389)


Figure 4 ( A ) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA - seq ( B ) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets . Yellow bar represents enrichment within NPC / GPC genes , green bar within neuronal genes , blue bar within astrocytic genes and red bar within oligodendrocytic genes . SOX10 ChIP - seq data from [ 37 ( F ) Expression of Nfia , Ehf and Zbtb3 among spinal cord cells analyzed with scRNA - seq . Red color indicates cells with RPKM values above 55 for Nfia , 5 for Zbtb3 and 5 for Ehf . n = 3 for SOX9 and 2 for SOX3 . P - values ( phyper , R ) were calculated from the total number of genes for mm10 ( 23 ´ 389 )

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Figure 4(A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq(B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37(F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)


Figure 5 ( A ) Luc - reporter assays in P19 cells . Luc - reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA ; an activation that could be efficiently blocked by SOX3 . No activation of these reporters could be detected with SOX10 or SOX11 . ( B ) Luc - reporters containing SOX9 and SOX10 bound DNA - regions around the astrocyte genes Aldh1l1 and Atp1a2 , which are not expressed in SOX10 + oligodendrocytes , could not be activated by SOX9 or SOX10 ( C ) Luc - reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10 . No activation could be detected with SOX11 . ( D ) Luc - reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2 , which are sequentially bound by SOX3 and SOX11 , could be activated by SOX11 and efficiently repressed by SOX3 . No activation could be detected with SOX10 ( E ) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes . Scale bar , 25µm . ( F ) Statistical view of Fig . 5E . Bars represents mean values ( of 3 experiments ) of cells expressing GFAP or SOX10 in GFP or Sox3 - transduced cells . Error bars in figure 5 indicate standard deviation of 3 - 7 individual samples and three - star significance > 0 . 001 ( t - test ) . ( * ) 0 , 05 < P < 0 , 01 ; ( * * ) 0 , 01 < P < 0 , 001 ; ( * * * ) P < 0 ,

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Figure 5 (A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10 (C) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10(E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001


Figure 6 ( A ) SOX3 over - expression experiments in chicken spinal cord at E4 . 48 hrs post electroporation , transfected side showed decreased expression of FGFR2 , NFIA and Fgfr3 . Bar : 50µm . ( B ) 24 hrs after miss - expression of a dominant negative version of SOXB1 ( dnSOXB1 ) , premature expression of FGFR2 , NFIA and Fgfr3 were found in the transfected side . Bar : 40µm . ( C ) 24 hrs after SOX9 over - expression in chicken spinal cord ( at E4 ) , increased expression of FGFR2 , NFIA and Fgfr3 was shown compared to the un - transfected control side . Bar : 40µm . ( D ) Sox3 and Sox9 co - electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2 , NFIA and Fgfr3 levels were comparable to the un - transfected control side . Embryos were analyzed 24 hrs post electroporation . Bar : 40µm . ( E

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Figure 6 (A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E


Figure 1 ( B ) Immunoblotting of whole cell extracts obtained from parental and RAP80 - / - clones in the RPE - 1 hTERT p53 - / - Cas9 , RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 , U2OS , U2OS - 2 - 6 - 3 and U2OS Flp - In / TREx cell lines with RAP80 antibodies . Tubulin is used as loading control . Representative of at least two independent immunoblots . See Figure EV1 for further details on the gene editing strategy used to knockout RAP80 . ( C , D ) The indicated parental and RAP80 - / - cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against ABRAXAS1 and γH2AX . Shown in ( C ) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs of RPE1 - hTERT p53 - / - Cas9 wild type ( WT ) and RAP90 - / - cells are shown in ( D ) . ( E , The indicated cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against BRCA1 and γH2AX . Shown in ( E ) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates for all conditions except n = 5 for U2OS WT , n = 2 for U2OS 2 - 6 - 3 cells , n = 7 , 5 , 4 for U2OS Flp - In / T - REx cells ) . ( G ) RPE - 1 hTERT p53 - / - Cas9 parental ( WT ) and RAP80 - / - cells were untreated ( t = 0 h ) or irradiated with a 10 Gy dose . Samples were collected at the indicated time points , processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX . Shown is the percentage of cells with > 5 BRCA1 foci . A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S . D . ( n = 3 biological replicates ) . All scale bars are 5 μm .

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

Figure 1(B) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80.(C, D) The indicated parental and RAP80-/- cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against ABRAXAS1 and γH2AX. Shown in (C) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs of RPE1-hTERT p53-/- Cas9 wild type (WT) and RAP90-/- cells are shown in (D).(E, The indicated cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against BRCA1 and γH2AX. Shown in (E) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates for all conditions except n=5 for U2OS WT, n=2 for U2OS 2-6-3 cells, n=7, 5, 4 for U2OS Flp-In/T-REx cells).(G) RPE-1 hTERT p53-/- Cas9 parental (WT) and RAP80-/- cells were untreated (t=0 h) or irradiated with a 10 Gy dose. Samples were collected at the indicated time points, processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX. Shown is the percentage of cells with > 5 BRCA1 foci. A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S.D. (n=3 biological replicates). All scale bars are 5 μm.


Figure 2 ( A , B ) U2OS Flp - In / T - Rex parental ( WT ) and RAP80 - / - cells were transfected with siRNA pools targeting BRCA1 , RNF8 , RNF168 or BARD1 or with a non - targeting control siRNA ( CTRL ) . 48 h post - transfection cells were irradiated ( 2 Gy ) and processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 and γH2AX . Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in ( A ) . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs are shown in ( B ) . ( C , D ) Parental ( WT ) RPE1 - hTERT p53 - / - Cas9 , RNF8 - / - , and RNF168 - / - cells were treated with either a non - targeting siRNA pool ( CTRL ) or a pool targeting RAP80 . 48 h post - transfection cells were irradiated ( 2 Gy ) and processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 and γH2AX . Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in ( C ) . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs are shown in ( D ) . All scale bars are 5 μm .

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Figure 2(A, B) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B).(C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


Figure 3 ( A , B ) U2OS Flp - In / T - REx cells with integrated transgenes encoding GFP - BRCA1BRCT or - BRCA1BRCT S1655A were transfected with non - targeting siRNA ( CTRL ) or siRNA targeting RAP80 or BRCA1 . Following doxycycline treatment to induce transgene expression ( 5 μg / mL , 24 h ) , cells were irradiated ( 10 Gy ) and processed for immunofluorescence 1 h post - IR for GFP and antibodies against RAP80 and γH2AX . Shown in ( A ) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S . D ( n = 4 ) . Representative micrographs are shown in ( B ) . ( C , D ) U2OS Flp - In / T - REx cells stably integrated with the indicated transgenes were treated with doxycycline ( 5 μg / mL , 36 h ) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non - targeting siRNA ( CTRL ) or siRNAs targeting RAP80 . 1 h post - irradiation ( 10 Gy ) cells were processed for immunofluorescence using GFP ( to detect the BRCA1 fusions ) and antibodies against γH2AX . Shown in ( C ) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in ( D ) . All scale bars are 5 μm .

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Figure 3(A, B) U2OS Flp-In/T-REx cells with integrated transgenes encoding GFP-BRCA1BRCT or -BRCA1BRCT S1655A were transfected with non-targeting siRNA (CTRL) or siRNA targeting RAP80 or BRCA1. Following doxycycline treatment to induce transgene expression (5 μg/mL, 24 h), cells were irradiated (10 Gy) and processed for immunofluorescence 1 h post-IR for GFP and antibodies against RAP80 and γH2AX. Shown in (A) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=4). Representative micrographs are shown in (B).(C, D) U2OS Flp-In/T-REx cells stably integrated with the indicated transgenes were treated with doxycycline (5 μg/mL, 36 h) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non-targeting siRNA (CTRL) or siRNAs targeting RAP80. 1 h post-irradiation (10 Gy) cells were processed for immunofluorescence using GFP (to detect the BRCA1 fusions) and antibodies against γH2AX. Shown in (C) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


Figure 4 ( A , B ) U2OS 2 - 6 - 3 parental ( WT ) or RAP80 - / - cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins . 48 h post - nucleofection , mCherry - LacR - FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry . Shown in ( A ) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S . D ( n = 50 , 50 , 40 , 40 , 40 , 40 , 20 , 20 , 30 , 30 cells analyzed from 3 biological replicates ) . Representative micrographs for the BRCA1 and BRCA1 - I26A conditions are shown in ( B ) . Additional micrographs for the other conditions are in Figure EV2C . ( C ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 cells ( WT ) or their isogenic RAP80 - / - counterparts expressing the indicated GFP fusion proteins were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in Figure EV3B . ( D ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 cells or their isogenic RAP80 - / - counterparts expressing the indicated GFP fusion proteins were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in Figure EV3C . Scale bar is 5 μm in ( B ) .

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Figure 4(A, B) U2OS 2-6-3 parental (WT) or RAP80-/- cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins. 48 h post-nucleofection, mCherry-LacR-FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry. Shown in (A) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S.D (n= 50, 50, 40, 40, 40, 40, 20, 20, 30, 30 cells analyzed from 3 biological replicates). Representative micrographs for the BRCA1 and BRCA1-I26A conditions are shown in (B). Additional micrographs for the other conditions are in Figure EV2C.(C) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells (WT) or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3B. (D) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3C. Scale bar is 5 μm in (B).


Figure 5 ( A , B ) MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated ( 10 Gy ) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation . Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies . Shown in ( A ) is the quantitation of a minimum of 100 EdU + cells per replicate and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in ( B ) . MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated ( 10 Gy ) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation . Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies . Representative micrographs are shown in ( D ) . ( E ) Clonogenic survival assays using the indicated dose of olaparib and MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions . Data is shown as the mean ± S . D ( n = 3 biological replicates ) . Representative clonogenic images are shown in Figure EV5D .

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 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

Figure 5(A, B) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B).MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Representative micrographs are shown in (D).(E) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.


Figure 1C , D . Oxidative stress biochemical markers ( lipid peroxidation , ROS production , GSH levels ) ( C ) and p - ERK expression ( D ) in PD and control ( CNTR ) fibroblasts ( n = 6 ) . E , F . Oxidative stress biochemical markers ( lipid peroxidation , ROS production , GSH levels ) ( E ) and p - ERK expression ( F ) in PD and control ( CNTR ) myoblasts ( n = 4 ) . In all instances indicators of stress were increased in PD , compared to the respective control samples . Data information : In each experiment at least biological triplicates were analyzed for each cell line or tissue sample ; each assay was performed at least in duplicate . Data are presented as mean ± SD . Student ' s t - test was applied . Statistically significant comparison p - values are indicated . Fi

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Figure 1C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6). E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n=4). In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fi


Figure 2A , B . Western Blot analysis of the autophagy marker LC3 ( A ) and quantitative analyses ( B ) in untreated control fibroblasts ( CNTR ) and in 2 PD fibroblasts cell lines ( PD1 , PD2 ) . PD cells were untreated ( NT ) or subjected to different treatments to modulate autophagy ( starvation , STAR ; rapamycin , RAPA ; MK6 - 83 ; bafilomycin , BAFI ) . Data information : Starvation of cells was performed for 4 hours ; treatments with 20 µM rapamycin or 30 µM MK6 - 83 or 100nM bafilomycin was performed for 24 hours . Data presented as mean ± SD . P values were calculated with ANOVA Šídák ' s multiple comparisons test . Statistically significant comparison p - values are indicated . C . ROS production , lipid peroxidation , and GSH levels in PD fibroblasts ( n = 3 ) . CNTR ( white bars ) , CNTR after treatment ( dark grey bars ) , PD samples ( black bars ) and PD after treatment ( light grey bars ) . Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD cells , while bafilomycin treatment further increased stress in CNTR and PD cells . Data information : Starvation of cells was performed for 4 hours ; treatments with 20 µM rapamycin or 30 µM MK6 - 83 or 100nM bafilomycin was performed for 24 hours . Data presented as mean ± SD . P values were calculated with ANOVA Šídák ' s multiple comparisons test . Statistically significant comparison p - values are indicated .

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 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

Figure 2A, B. Western Blot analysis of the autophagy marker LC3 (A) and quantitative analyses (B) in untreated control fibroblasts (CNTR) and in 2 PD fibroblasts cell lines (PD1, PD2). PD cells were untreated (NT) or subjected to different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.C. ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (n=3). CNTR (white bars), CNTR after treatment (dark grey bars), PD samples (black bars) and PD after treatment (light grey bars). Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD cells, while bafilomycin treatment further increased stress in CNTR and PD cells. Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.


Figure 3A , B , C . Correlations between the levels of single stress indicators and correction of GAA activity by rhGAA in 6 different PD fibroblasts . ROS production and lipid peroxidation inversely correlate with the GAA levels attained in cells after incubation with rhGAA for 4 hours . The analysis of correlation was calculated , and the coefficient of Pearson is indicated . D

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Figure 3A, B, C. Correlations between the levels of single stress indicators and correction of GAA activity by rhGAA in 6 different PD fibroblasts. ROS production and lipid peroxidation inversely correlate with the GAA levels attained in cells after incubation with rhGAA for 4 hours. The analysis of correlation was calculated, and the coefficient of Pearson is indicated. D


Figure 4A . Cell viability in control and PD fibroblasts ( 3 different cell lines ) measured by MTT assay in the presence of increased concentrations ( 0 - 100 µM ) of either sodium arsenite ( ARS ) or tert - butyl - peroxide ( TBP ) at different time points . Data presented as a mean ± SD . B . ROS production , lipid peroxidation , and GSH levels in fibroblasts ( 3 different cell lines ) after 6 hours of treatment with 100 µM ARS and or 10 µM TBP . Both oxidative agents induced increases in oxidative stress in control ( CNTR ) and in PD cells . Data presented as a mean ± SD . Significance was calculated by one - way ANOVA followed by Šídák ' s multiple comparisons test . C Data information : statistically significant p - values are indicated .

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Figure 4A. Cell viability in control and PD fibroblasts (3 different cell lines) measured by MTT assay in the presence of increased concentrations (0-100 µM) of either sodium arsenite (ARS) or tert-butyl-peroxide (TBP) at different time points. Data presented as a mean ± SD. B. ROS production, lipid peroxidation, and GSH levels in fibroblasts (3 different cell lines) after 6 hours of treatment with 100 µM ARS and or 10 µM TBP. Both oxidative agents induced increases in oxidative stress in control (CNTR) and in PD cells. Data presented as a mean ± SD. Significance was calculated by one-way ANOVA followed by Šídák's multiple comparisons test. C Data information: statistically significant p-values are indicated.


Figure 5A . Effect of antioxidants on ROS production , lipid peroxidation , and GSH levels in PD fibroblasts ( 3 different cell lines for each treatment , each cell line assayed at least in ducplicate ) . Cells were incubated for 24 hours with : idebenone ( IDE ) 0 . 5 µM ; edaravone ( EDA ) 50 µM ; N - acetylcysteine ( NAC ) 0 . 5 mM ; resveratrol ( RESV ) 30 µM . Mean of control ( CNTR ) values are taken as equal to 100 . The results are shown as mean ± SD . Data information : To calculate statistical significance one - way ANOVA was applied for all experiments followed by Dunnett ' s test . Statistically significant p - values are indicated . E . Confocal immunofluorescence analysis of GAA and LAMP2 ; representative fibroblast cell lines ( PD1 ) . Confocal 63x images ; Scale bar 50 µm ; Brightness + 20 % . F . Percent of GAA / LAMP2 colocalization in PD1 . The results are expressed as means ± SD of 5 images for each condition . G . Percent of GAA / LAMP2 colocalization ; mean of the analyses performed in 3 PD fibroblast cell lines . For each PD patient , 5 images for each condition were quantified . The results are expressed as means ± SD of 5 images for each condition . Data information : To calculate statistical significance one - way ANOVA was applied for all experiments followed by Dunnett ' s test . Statistically significant p - values are indicated .

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Figure 5A. Effect of antioxidants on ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (3 different cell lines for each treatment, each cell line assayed at least in ducplicate). Cells were incubated for 24 hours with: idebenone (IDE) 0.5 µM; edaravone (EDA) 50 µM; N-acetylcysteine (NAC) 0.5 mM; resveratrol (RESV) 30 µM. Mean of control (CNTR) values are taken as equal to 100. The results are shown as mean ± SD. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.E. Confocal immunofluorescence analysis of GAA and LAMP2; representative fibroblast cell lines (PD1). Confocal 63x images; Scale bar 50 µm; Brightness +20%. F. Percent of GAA/LAMP2 colocalization in PD1. The results are expressed as means ± SD of 5 images for each condition. G. Percent of GAA/LAMP2 colocalization; mean of the analyses performed in 3 PD fibroblast cell lines. For each PD patient, 5 images for each condition were quantified. The results are expressed as means ± SD of 5 images for each condition. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.


Figure 6D . Mean of results obtained in control ( two cell lines in duplicate ) and PD fibroblast ( 3 different cell lines , each cell line tested in duplicate in three different experiments ) . For each cell line , the amount of M6PR positive cells was normalized , taking that observed in not - treated fibroblasts as 1 . Data presented as mean ± SD . Data information : For statistical analysis a one - way ANOVA followed by Šídák ' s post hoc test was applied .

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Figure 6D. Mean of results obtained in control (two cell lines in duplicate) and PD fibroblast (3 different cell lines, each cell line tested in duplicate in three different experiments). For each cell line, the amount of M6PR positive cells was normalized, taking that observed in not-treated fibroblasts as 1. Data presented as mean ± SD. Data information: For statistical analysis a one-way ANOVA followed by Šídák's post hoc test was applied.


Figure 7C . GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone ( black bars ) ( n of mice = 10 ) and with co - dosing of rhGAA and 2 gr / Kg / day NAC ( n of mice = 7 , dark grey bars ) . Data presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - test was applied to compare the results in each of the tissues . D . GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone ( black bars ) ( n of mice = 4 ) and with co - dosing of rhGAA and 100 mg / Kg / day IDE ( n of mice = 6 , light grey bars ) . Data presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - test was applied to compare the results in each of the tissues . E E , F . Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone ( n of mice = 2 ) or in combination with NAC ( n of mice = 3 ) ( E ) or IDE ( n of mice = 3 ) ( F ) . In F Data are presented as mean ± SD . G . Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone ( n of mice = 3 ) or in combination with NAC ( n of mice = 3 ) . H . Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone ( n of mice = 4 ) or in combination with IDE ( n of mice = 6 ) . Data information : i data are presented as mean ± SD . Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100 . A Student ' s t - test was applied to compare the results in each tissue .

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Figure 7C. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. D. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. E E, F. Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD.G. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice = 3). H. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with IDE (n of mice = 6). Data information: i data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.


Figure 1A Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data ( No . GSE14905 ) . B Expression of ptpn11 in human PBMCs of psoriatic patients ( n = 14 ) and normal controls ( n = 16 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . C Representative SHP2 staining in skin sections of psoriatic patients ( n = 13 ) and normal controls ( n = 5 ) . Scale bars : 200 μm . D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls . E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients ( n = 25 ) and normal controls ( n = 25 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . F Representative p - ERK staining of skin sections of psoriatic patients and normal controls . Scale bars : 200 μm . G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 ( n = 6 / group ) . Data were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . H Representative histological sections of IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 . Scale bar : 100 μm . I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients ( n = 20 ) and normal healthy controls ( n = 20 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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Figure 1A Expression of PTPN11 (gene encoding SHP2) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). B Expression of ptpn11 in human PBMCs of psoriatic patients (n=14) and normal controls (n=16). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.C Representative SHP2 staining in skin sections of psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm.D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls.E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.F Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 μm.G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5 (n=6/group). Data were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.H Representative histological sections of IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5. Scale bar: 100 μm.I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients (n=20) and normal healthy controls (n=20). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Figure 2C57BL / 6 mice ( n = 6 / group ) were treated with indicated dose of SHP099 or vehicle for 4 days . A Phenotypic presentation ( top ) , H & E staining ( middle ) and statistical data ( bottom ) ( mean ± SEM ) of dorsal skin . Scale bar : 100 μm . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . ELISA quantification of protein levels of cytokines in mouse serum . Quantitative PCR analysis of mRNA encoding psoriasis - related cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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Figure 2C57BL/6 mice (n=6/group) were treated with indicated dose of SHP099 or vehicle for 4 days. A Phenotypic presentation (top), H&E staining (middle) and statistical data (bottom) (mean ± SEM) of dorsal skin. Scale bar: 100 μm. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.ELISA quantification of protein levels of cytokines in mouse serum. Quantitative PCR analysis of mRNA encoding psoriasis-related cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Figure 3Violin plot showed the relative expression of 47 inflammation - associated genes based on sample groups and cell clusters .

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Figure 3Violin plot showed the relative expression of 47 inflammation-associated genes based on sample groups and cell clusters.


Figure 4D Gene set enrichment analysis ( GSEA ) of the NF - κB signaling pathway . E Volcano plots of DEGs that were upregulated ( red ) or downregulated ( blue ) in myeloid cells . Examples of DEGs were labeled . G Expression patterns of the top ten hub genes in ( F ) . Heatmap ( top ) displays the average expression in the three groups and in different myeloid cell subsets . Boxplots ( bottom ) show the corresponding expression distribution .

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Figure 4D Gene set enrichment analysis (GSEA) of the NF-κB signaling pathway.E Volcano plots of DEGs that were upregulated (red) or downregulated (blue) in myeloid cells. Examples of DEGs were labeled.G Expression patterns of the top ten hub genes in (F). Heatmap (top) displays the average expression in the three groups and in different myeloid cell subsets. Boxplots (bottom) show the corresponding expression distribution.


Figure 5Representative images ( A and E ) , histological sections of dorsal back ( B and F ) and clinical scores ( C and G ) and quantitative PCR analysis of mRNA levels in dorsal skin ( D and H ) from wild - type ( n = 6 ) and M - Shp2 - / - mice or DC - Shp2 - / - mice ( n = 6 ) treated with IMQ for 4 days . Scale bar : 100 μm . Left : H & E ( hematoxylin and eosin ) staining data ; right : statistical data ( mean ± SEM ) . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values were calculated by Tukey multiple comparison test ( B , D , F , H ) or Bonferroni multiple comparison test ( C , G ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant .

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Figure 5Representative images (A and E), histological sections of dorsal back (B and F) and clinical scores (C and G) and quantitative PCR analysis of mRNA levels in dorsal skin (D and H) from wild-type (n=6) and M-Shp2-/- mice or DC-Shp2-/- mice (n=6) treated with IMQ for 4 days. Scale bar: 100 μm. Left: H&E (hematoxylin and eosin) staining data; right: statistical data (mean ± SEM). Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values were calculated by Tukey multiple comparison test (B, D, F, H) or Bonferroni multiple comparison test (C, G). *P<0.05, **P<0.01, ns, not significant.


Figure 6A Heat map showing mRNA expression in peritoneal macrophages derived from wild - type and M - Shp2 - / - mice after IMQ treatment based on RNA sequencing ( n = 3 / group ) . Colors represent high ( red ) and low ( blue ) intensity . Genes labeled in green indicate they are in the NF - κB signaling . D BMDMs and PMs derived from wild - type and M - Shp2 - / - mice were stimulated by IMQ ( 10 µg / ml ) for indicated times . Whole cell lysates were subjected to Western blotting . E Representative p - p65 staining of skin sections from mouse . Scale bar : 100 μm .

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Figure 6A Heat map showing mRNA expression in peritoneal macrophages derived from wild-type and M-Shp2-/- mice after IMQ treatment based on RNA sequencing (n=3/group). Colors represent high (red) and low (blue) intensity. Genes labeled in green indicate they are in the NF-κB signaling.D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting.E Representative p-p65 staining of skin sections from mouse. Scale bar: 100 μm.


Figure 7E Immunoblotting of HEK293T cells co - transfected for 48 h with GFP - TLR7 , plus HA - SHP2 , HA - SHP2 mutant vectors , followed by immunoprecipitation with anti - GFP beads . F PMA - differentiated THP - 1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 ( 10 µg / ml ) . Subcellular fractionation was performed , and cytoplasmic and cell membrane proteins were probed by respective antibodies . G , H Immunoblot analysis of TLR7 and SHP2 in the isolated ER , Golgi and endosome from PMA - differentiated THP - 1 cells with either SHP2 overexpressed ( G ) and SHP2 deficiency ( H ) . I Skin sections from psoriatic patients and healthy controls were immune - stained for TLR7 together with a marker of the early endosome ( EEA1 ) or late endosome / lysosome ( LAMP1 ) prior to analysis by confocal microscopy , showing TLR7 localization in different organelles ( arrows ) . Scale bar : 50 μm .

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Figure 7E Immunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-SHP2, HA-SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP beads.F PMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies.G, H Immunoblot analysis of TLR7 and SHP2 in the isolated ER, Golgi and endosome from PMA-differentiated THP-1 cells with either SHP2 overexpressed (G) and SHP2 deficiency(H).I Skin sections from psoriatic patients and healthy controls were immune-stained for TLR7 together with a marker of the early endosome (EEA1) or late endosome/lysosome (LAMP1) prior to analysis by confocal microscopy, showing TLR7 localization in different organelles (arrows). Scale bar: 50 μm.


Figure 8B , Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP - TLR7 or GFP - TLR7 mutant vectors . G HEK293T cells transfected with NF - κB - luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ ( 10 µg / ml ) for 24 h were harvested for luciferase assay . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * * P < 0 . 01 , ns , not significant . I Representative p - TLR7 Y1024 staining of skin sections from human . Scale bar : 100 μm .

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Figure 8B, Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP-TLR7 or GFP-TLR7 mutant vectors.G HEK293T cells transfected with NF-κB-luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ (10 µg/ml) for 24 h were harvested for luciferase assay. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test **P<0.01, ns, not significant.I Representative p-TLR7 Y1024 staining of skin sections from human. Scale bar: 100 μm.


Figure 9Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . A Phenotypic presentation ( top ) and H & E staining ( bottom ) of dorsal skin . Scale bar : 100 μm . Left : H & E staining data ; right : statistical data ( mean ± SEM ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . D Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines and other psoriasis - related cytokines in the dorsal skin . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . E Representative p - p65 staining of the dorsal skin . Scale bars : 100 μm .

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Figure 9Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm.


Figure 1 ( A ) Remdesivir inhibition of SARS - CoV - 2 infections of Vero E6 cells . drugs , , were used at the indicated concentrations . Viral RNA level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) . ( B ) hrsACE2 inhibition of SARS - CoV - 2 infections of Vero E6 cells . murine recombinant soluble ACE2 ( mrsACE2 , control treatment ) , were used at the indicated concentrations . Viral RNA level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) .

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Figure 1(A) Remdesivir inhibition of SARS-CoV-2 infections of Vero E6 cells. drugs, , were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU). (B) hrsACE2 inhibition of SARS-CoV-2 infections of Vero E6 cells. murine recombinant soluble ACE2 (mrsACE2, control treatment), were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).


Figure 2 ( B ) Cell viability of isolated cell clones and WT AN3 - 12 cells following 72h remdesivir treatment with the indicated doses . Mean ± SEM of 2 - 4 biological replicates is displayed .

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Figure 2(B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed.


Figure 3 ( A ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 cells with Human recombinant soluble ACE 2 ( hrsACE2 ) ( 200µg / ml ) and remdesivir ( Remd . 4µM ) . Viral RNA level was determined at 15 hours after virus inoculation . ( B ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected human kidney organoids with hrsACE2 ( 200µg / ml ) and / or remdesivir ( Remd . 4µM ) ) . Viral RNA was determined by qRT - PCR 72 hours after the inoculation of 106 PFU of SARS - CoV - 2 . ( C ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 cells with clinical doses of hrsACE2 ( 5 and 10µg / ml ) and remdesivir ( Remd . 4µM ) ) . ( D ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected kidney organoids with hrsACE2 ( 10µg / ml ) and remdesivir ( 4µM ) . ( E , F ) Progeny virus released from untreated Vero - E6 cells or Vero - E6 cells treated with clinical doses of hrsACE2 ( 5 and 10µg / ml ) and remdesivir ( Remd . 4µM ) . Progeny was determined ( E ) 15 hours and ( F ) 48 hours post - infection ( hpi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 (A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation.  (B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2.  (C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)).  (D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM).  (E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).


Figure 1 ( A ) Quantitative analysis of phosphorylated S6 ( p - S6 ) ( mTORC1 activity read - out ) immunoreactivity . N = 11 - 15 pigmented or grey HFs from 4 different donors . ( B ) Representative images of p - S6 immunofluorescence . Only anagen VI HFs were investigated and analyzed immediately after surgery . Analyses were performed in defined reference areas ( dotted areas ) in the HFPU . Nuclei stained with DAPI . ( C ) Quantitative analysis of TSC2 protein levels . N = 11 - 14 pigmented or grey HFs from 4 different donors . ( D ) Representative images of TSC2 immunofluorescence . Only anagen VI HFs were investigated and analyzed immediately after surgery . Analyses were performed in defined reference areas ( dotted areas ) in the HFPU . Nuclei stained with DAPI .

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Figure 1(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 11-15 pigmented or grey HFs from 4 different donors. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(C) Quantitative analysis of TSC2 protein levels. N = 11-14 pigmented or grey HFs from 4 different donors. (D) Representative images of TSC2 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.


Figure 2 ( A ) Quantitative analysis of phosphorylated S6 ( p - S6 ) ( mTORC1 activity read - out ) immunoreactivity . N = 23 - 30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( B ) Representative images of p - S6 immunofluorescence . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Nuclei stained with DAPI . ( C ) Hair cycle staging was performed using Ki - 67 and Masson - Fontana histochemistry Mean ± SEM ; N = 36 - 39 HFs per group from 6 different donors treated with Rapamycin 20 ng / ml or vehicle ( control ) for 7 days . ( D ) Representative fluorescence images of Ki - 67 and bright field microscopic images of Masson - Fontana . for C - D where all HFs were analyzed ) were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test Nuclei stained with DAPI . ( E ) Quantitative histomorphometry of melanin production by Masson - Fontana histochemistry . N = 28 - 30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( F ) Representative bright field microscopic images of Masson - Fontana histochemistry . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test ( G ) Quantitative analysis of tyrosinase activity . N = 18 - 21 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( H ) Representative images of tyrosinase activity immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test Nuclei stained with DAPI . ( I ) Quantitative analysis of gp100 expression . N = 24 - 26 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( J ) Representative images of gp100 immunofluorescence . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test Nuclei stained with DAPI . ( K ) Quantitative analysis of melanocyte dendricity . N = 22 - 24 anagen VI HFs from 6 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( L ) Representative images of gp100 immunofluorescence . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test Nuclei stained with DAPI . ( M ) Quantitative analysis of α - MSH expression . N = 17 - 20 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( N ) Representative images of α - MSH immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . ± SEM , Unpaired Student ' s t - test Nuclei stained with DAPI . ( O ) Quantitative analysis of phosphorylated S6 ( p - S6 ) ( mTORC1 activity read - out ) immunoreactivity . N = 8 - 10 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng / ml , 2ug / ml Agouti , Rapamycin + Agouti or untreated ( vehicle ) for 7 days . ( P ) Representative images of p - S6 immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and One - Way ANOVA Nuclei stained with DAPI . ( Q ) Quantitative histomorphometry of melanin production by Masson - Fontana histochemistry . N = 8 - 11 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng / ml , 2 ug / ml Agouti , Rapamycin + Agouti or untreated ( vehicle ) for 7 days . ( R ) Representative bright field microscopic images of Masson - Fontana histochemistry . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and One - Way

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 23-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry Mean ± SEM; N = 36-39 HFs per group from 6 different donors treated with Rapamycin 20 ng/ml or vehicle (control) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 28-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test(G) Quantitative analysis of tyrosinase activity. N = 18-21 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(I) Quantitative analysis of gp100 expression. N = 24-26 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(K) Quantitative analysis of melanocyte dendricity. N = 22-24 anagen VI HFs from 6 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (L) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(M) Quantitative analysis of α-MSH expression. N = 17-20 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (N) Representative images of α-MSH immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(O) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 8-10 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (P) Representative images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA Nuclei stained with DAPI.(Q) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 8-11 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2 ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (R) Representative bright field microscopic images of Masson-Fontana histochemistry. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA