Figure 2 Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the SVZ of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone . Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the hippocampus of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone . ( E ) qRT - PCR for ZIKV RNA expression in brain , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by Student ' s t test . ( F - G ) Western blot for autophagy protein LC3 - II , p62 and quantification normalized with GAPDH , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by Student ' s t test .

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

Figure 2 Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone.  Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the hippocampus of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. (E) qRT-PCR for ZIKV RNA expression in brain, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.(F-G) Western blot for autophagy protein LC3-II, p62 and quantification normalized with GAPDH, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.


Figure 3 ( B ) RT - qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs . Mean ± SEM of biological triplicates . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . ( C - D ) RT - qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC ( C ) or FANCC - specific siRNA ( D ) . Mean ± SEM of biological triplicates . * * p < 0 . 01 by Student ' s t test . ( E ) Flow cytometry analysis of ZIKVE expression in mock - infected ( red ) , ZIKV - infected ( blue ) , ZIKV - infected siFANCC - transfected ( orange ) , and ZIKV - infected siE2F4 - transfected ( green ) hNSCs at 48 h post - infection . ( F ) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs . Scale bar = 100 µm . ( G ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA ( NTC ) or siFANCC - targeting siRNA . Scale bar = 100 µm . ( H - I ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control ( NTC ) or siE2F4 - targeting siRNA . Mean ± SEM of six imaging fields of biological triplicates , * * p < 0 . 01 by Student ' s t test , Scale bar = 100 µm , ( J ) Immunoblot of macroautophagy ( p62 , LC3I / LC3II ) and selective autophagy ( FANCC , TOMM20 ) proteins 24 h after overexpression of FLAG - tagged ZIKV NS1 , NS2B , NS3 , NS4A , NS4B and NS5 in 293FT cells . ( K ) Analysis of E2F4 ChIP - seq datasets ( Encode ) showing direct chromatin - binding interaction between E2F4 and FA pathway genes in HeLa ( GEO : GSM935365 , Farnham lab ) , K562 ( GEO : GSM935600 , Farnham lab ) , and MCF10A ( GEO : GSM : 935400 , Struhl lab ) cell lines . H3K9ac and H3K4me3 datasets identify promoter regions . The genomic region is displayed in log scale with the scale range identified in each panel ( 0 - 30 ) . The summed interaction ( Sum ) across all three cell types is represented as a heatmap . ( L - M ) Heatmaps of RT - qPCR analysis of FA pathway genes ( K ) or essential selective autophagy genes ( L ) in hNSCs 48 h after transfection with control ( NTC ) or E2F4 - targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains . Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes .

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 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 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 B-PANEL_START 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 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 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

Figure 3(B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test.(C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test.(E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection.(F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm.(G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm. (H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm, (J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells.(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.(L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.


Figure 4 ( A ) Representative images of mock - or ZIKV - infected human neurospheres expressing control ( NTC ) or FANCC - targeting siRNAs at 1 - 4 days post - infection . Scale bar = 250 µm . ( B - C ) Violin plot ( B ) and box plot ( C ) of relative size of neurospheres treated as described in ( A ) and measured on day 4 ( B ) or days 1 - 4 ( C ) . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * * * p < 0 . 001 by Student ' s t test .

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Figure 4(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm. (B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.


Figure 5Immunostaining of neural progenitor marker SOX2 ( green ) and ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in the hippocampus of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Right - most column in ( A ) shows enlargements of the regions . CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , STR = striatum , SVZ = subventricular zone . Scale bars , 100 μm . Immunostaining of neural progenitor marker SOX2 ( green ) and ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in the SVZ of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Right - most column in ( A ) shows enlargements of the regions . CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , STR = striatum , SVZ = subventricular zone . Scale bars , 100 μm . ( C ) RT - qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar - / - mice . mean ± SEM of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t test . ( D ) RT - qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock - and ZIKV Paraiba infected Ifnar - / - mice six days post - infection . Mean ± SEM of n = 3 biological replicates , * p < 0 . 05 and ns by Student ' s t test . ( E ) RT - qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock - and ZIKV Paraiba infected Ifnar - / - newborn mice . Mean ± SEM of n = 3 biological replicates , * * * p < 0 . 001 by Student ' s t test . ( F ) Western blot analysis of Fancc protein in brain regions from mock - and ZIKV Paraiba infected Ifnar - / - mice six days post - infection . HP = hippocampus , CT = cortex , ST = striatum and OB = Olfactory Bulb . ( G ) Bar graph showing the quantification of Fancc proteins , mean ± SEM of n = 3 biological replicates , * p < 0 . 05 and ns by Student ' s t test . ( H ) RT - qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post - infection . mean ± SEM of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t test . ( I ) qRT - PCR for Ifnab gene expression in wild type and Fancc KO mice , Mean ± SEM of n = 3 biological replicates , ns by Student ' s 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 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 O O O O O O 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 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 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 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 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

Figure 5Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the SVZ of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.(C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(D) RT-qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. Mean ± SEM of n=3 biological replicates, *p < 0.05 and ns by Student's t test.(E) RT-qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock- and ZIKV Paraiba infected Ifnar-/- newborn mice. Mean ± SEM of n=3 biological replicates, ***p < 0.001 by Student's t test.(F) Western blot analysis of Fancc protein in brain regions from mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(G) Bar graph showing the quantification of Fancc proteins, mean ± SEM of n=3 biological replicates, *p<0.05 and ns by Student's t test.(H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(I) qRT-PCR for Ifnab gene expression in wild type and Fancc KO mice, Mean ± SEM of n=3 biological replicates, ns by Student's t test.


Figure 6 ( A ) Immunostaining of immature neuronal marker DCX ( green ) and ZIKV envelope ( ZIKVE , red ) in the hippocampus of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Bottom row shows enlargements of the hippocampus region . GCL = granular cell layer , SGZ = subgranular zone , DG = dentate gyrus , InGr = internal granular layer , Me = medulla , OB = olfactory bulb . Scale bars , 50 and 100 μm . Immunostaining of immature neuronal marker DCX ( green ) and ZIKV envelope ( ZIKVE , red ) in the olfactory bulb of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . InGr = internal granular layer , Me = medulla , OB = olfactory bulb . Scale bars , 50 and 100 μm . ( C - E ) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post - infection of ZIKV ( MR766 ) . Mean ± SEM of n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 005 , by Student ' s t test . HP = hippocampus , CT = cortex , ST = striatum and OB = Olfactory Bulb . ( F ) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker ( Sox2 ) with apoptosis marker ( Cleaved - caspase - 3 , CC3 ) in hippocampus region of Fancc KO mice . Scale bar = 50µm . ( G ) Quantitative analysis of number of Sox2 + cells and Sox2 / CC3 + co - labelled cells in Fancc KO mice hippocampal brain region . Mean ± SEM , n = 3 biological replicates , * p < 0 . 05 by Student ' s t test .

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Figure 6(A) Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Bottom row shows enlargements of the hippocampus region. GCL = granular cell layer, SGZ = subgranular zone, DG = dentate gyrus, InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the olfactory bulb of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.(C-E) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post-infection of ZIKV (MR766). Mean ± SEM of n=3 biological replicates, *p<0.05, **p<0.005, by Student's t test. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(F) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker (Sox2) with apoptosis marker (Cleaved-caspase-3, CC3) in hippocampus region of Fancc KO mice. Scale bar=50µm. (G) Quantitative analysis of number of Sox2+ cells and Sox2/CC3+ co-labelled cells in Fancc KO mice hippocampal brain region. Mean ± SEM, n=3 biological replicates, *p < 0.05 by Student's t test.


Figure 1Micrographs of S2 cells stained with PicoGreen , following 5 days of treatment with the dsRNA indicated . GFP and tamas ( Polg , CG8987 ) were used as negative and positive controls , respectively . In rescreening , CG5924 was also used as a positive control . Both were detected in the blinded screen as positives . Other images show a typical negative ( CG31380 ) , a typical positive ( CG6413 ) and a typical case of a target classed as abnormal , in this case CG2028 ( CkIα ) , which showed a decreased number of nucleoid signals per cell . Images are optimized for brightness and contrast but with no other manipulations . Scale bar indicates 50 μm .

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Figure 1Micrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIα), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 μm.


Figure 3A - C Relative mtDNA level ( A ) , TMRM ( B ) and MitoSox ( C ) following treatment for 5 days with dsRNA against the genes indicated . D MitoSox fluorescence following treatment for 1 - 3 days with dsRNA against the genes indicated . E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated . F , G Relative mtDNA level ( F ) and MitoSox fluorescence ( G ) after 5 days of treatment with oligomycin .

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Figure 3A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.D MitoSox fluorescence following treatment for 1-3 days with dsRNA against the genes indicated.E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated.F, G Relative mtDNA level (F) and MitoSox fluorescence (G) after 5 days of treatment with oligomycin.


Figure 4A ‐ D Relative mtDNA level ( A , B ) and MitoSox fluorescence ( C , D ) following 5 days of treatment with dsRNA against the genes indicated . E ‐ G Relative mtDNA level ( E ) , MitoSox fluorescence ( F ) and TMRM fluorescence ( G ) of cells stably expressing AOX or empty vector , as indicated , after 5 days of treatment with dsRNA against CG2968 , as shown . Note that these cells were cultured in the presence of hygromycin to maintain the AOX ‐ expressing or control plasmid . H Indicated parameters , following 5 days of treatment with dsRNA against CG2968 , in cells grown in galactose ‐ containing ( Gal ) medium .

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Figure 4A‐D Relative mtDNA level (A, B) and MitoSox fluorescence (C, D) following 5 days of treatment with dsRNA against the genes indicated.E‐G Relative mtDNA level (E), MitoSox fluorescence (F) and TMRM fluorescence (G) of cells stably expressing AOX or empty vector, as indicated, after 5 days of treatment with dsRNA against CG2968, as shown. Note that these cells were cultured in the presence of hygromycin to maintain the AOX‐expressing or control plasmid.H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.


Figure 5A . LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes , normalized against the values for untreated cells . Mean ± SD for four independent experiments ; asterisks indicate significant differences from untreated , P < 0 . 05 . B . Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red . The scale bar is 10 μm . C . Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes , or with chloroquine . Mean ± SD for three independent experiments , in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane . Analysis of z − stacks in ≥ 30 cells gave similar results . Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes . Asterisks indicate significant differences from untreated , P < 0 . 01 . D . Western blot of total protein extracts from control S2 cells and cells treated with dsRNAs against GFP or CG2968 ( cV ) , probed for NDUFS3 ( cI ) , ATP synthase subunit α ( cV ) and GAPDH ( loading control ) . E . Relative mtDNA level after 5 days of treatment with dsRNAs against the indicated genes . Mean ± SD for five independent experiments ; other data classes were significantly different from untreated , P < 0 . 05 .

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

Figure 5A. LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes, normalized against the values for untreated cells. Mean ± SD for four independent experiments; asterisks indicate significant differences from untreated, P < 0.05.B. Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red. The scale bar is 10 μm.C. Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes, or with chloroquine. Mean ± SD for three independent experiments, in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane. Analysis of z−stacks in ≥ 30 cells gave similar results. Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes. Asterisks indicate significant differences from untreated, P < 0.01.D. Western blot of total protein extracts from control S2 cells and cells treated with dsRNAs against GFP or CG2968 (cV), probed for NDUFS3 (cI), ATP synthase subunit α (cV) and GAPDH (loading control).E. Relative mtDNA level after 5 days of treatment with dsRNAs against the indicated genes. Mean ± SD for five independent experiments; other data classes were significantly different from untreated, P < 0.05.


Figure 1 ( B ) Genotyping of KLF4loxp / loxp coupled AAV - Cre inducible mouse . 2x1011 particles of AAV7 - Cre - mCherry or AAV7 - mCherry were intraperitoneally injected into KLF4 loxp / loxp mice ( 6 ~ 8 weeks ) . Five weeks later , mice tails were cut and collected for DNA extraction and PCR analysis . PCR results were analyzed by 1 % agarose gel . Validation of inducible knockout of KLF4 in mouse intestine . Five weeks after injection of AAV7 - Cre - mCherry , mouse intestine was removed and followed by the preparation of tissue section . The KLF4 expression in the intestine was then detected by Western blot Validation of inducible knockout of KLF4 in mouse intestine . Five weeks after injection of AAV7 - Cre - mCherry , mouse intestine was removed and followed by the preparation of tissue section . The KLF4 expression in the intestine was then detected by immunohistochemistry ( E ) Kaplan - Meier survival curves of KLF4 loxp / loxp mice with intraperitoneal injection of AAV7 - Cre - mCherry or AAV7 - mCherry followed by the treatment with 8 - Gy ( total - body ) γ - irradiation 5 - weeks after then . AAV7 - mCherry , n = 10 per group ; AAV7 - Cre - mCherry , n = 12 per group . p = 0 . 0023 , log - rank test . ( F ) Histological analysis of intestinal epithelium of KLF4 loxp / loxp mice with AAV7 - mCherry or AAV7 - Cre - mCherry intraperitoneal injection followed by 8 - Gy ( total - body ) γ - irradiation 5 - weeks after then . Tissues were collected from the sham mice and mice at different time after exposure to γ - irradiation . Scale bars , 100 μm . ( G - H ) Immunofluorescent staining of γ - H2AX , 53BP1 and Cleaved Caspase - 3 in the intestinal epithelium of KLF4 loxp / loxp mice with injection of AAV7 - mCherry or AAV7 - Cre - mCherry followed by treatment of γ - irradiation . Tissues were collected from sham mice and mice at different time after exposure to γ - irradiation and then staining with indicated antibodies . ( G ) Quantification of γ - H2AX , 53BP1 and active Caspase - 3 - positive cells based on the Immunofluorescent staining results presented in H . Data are mean ± SEM ; n = 4 per group ; p = 0 . 0023 ( r - H2AX ) , p = 7 . 9x10 - 4 ( 53BP1 ) p = 5 . 5x10 - 3 ( Active Caspase - 3 ) . one - way ANOVA was used for the statistical analysis . Scale bars , 60 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genotyping of KLF4loxp/loxp coupled AAV-Cre inducible mouse. 2x1011 particles of AAV7-Cre-mCherry or AAV7-mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6~8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel.  Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot  Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by immunohistochemistry  (E) Kaplan-Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7-Cre-mCherry or AAV7-mCherry followed by the treatment with 8-Gy (total-body) γ-irradiation 5-weeks after then. AAV7-mCherry, n=10 per group; AAV7-Cre-mCherry, n=12 per group. p = 0.0023, log-rank test.  (F) Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7-mCherry or AAV7-Cre-mCherry intraperitoneal injection followed by 8-Gy (total-body) γ-irradiation 5-weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ-irradiation. Scale bars, 100 μm.  (G-H) Immunofluorescent staining of γ-H2AX, 53BP1 and Cleaved Caspase-3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7-mCherry or AAV7-Cre-mCherry followed by treatment of γ-irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ-irradiation and then staining with indicated antibodies. (G) Quantification of γ-H2AX, 53BP1 and active Caspase-3 -positive cells based on the Immunofluorescent staining results presented in H. Data are mean ± SEM; n=4 per group; p = 0.0023 (r-H2AX), p = 7.9x10-4 (53BP1) p = 5.5x10-3 (Active Caspase-3). one-way ANOVA was used for the statistical analysis. Scale bars, 60 μm.


Figure 2 ( A ) . Purification of KLF4 protein complex in the presence and absence of DNA damage based on TAP - KLF4 stable expression cells ( U2OS ) . Proteins that interacted with KLF4 were purified from U2OS cells expressing FLAG and HA - tagged KLF4 in the absence and presence of 5 Gy radiation at 4h after the treatment . The accumulated bind induced in response to radiation was isolated for mass spectrometry analysis . PARP1 was identified as a binding partner for KLF4 . ( C ) . The purified complex was further confirmed by Western blot detected by FLAG - KLF4 , PARP1 ( lane 1 ) . The interaction between KLF4 and PARP1 was significantly increased in response to γ - radiation detected by pulldown experiment ( lane 3 and 4 ) . ( D ) Validation of interaction between ectopically expressed KLF4 and PARP1 . 293T cells were transfected with FLAG - KLF4 and Myc - PARP . Whole cell lysates or IP complex pulled down by anti - FLAG antibody were analyzed by Western blotting . ( E ) Validation of interaction between endogenous KLF4 and PARP1 in cytosolic lysate and nuclear lysate using immunoprecipitation and Western blotting in MDA - MB - 231 cells . The Histone 3 ( H3 ) is the control for nuclear portion , and actin is the control for cytosol portion . ( F ) Co - immunoprecipitation of PARP1 with endogenous KLF4 is independent on DNA . The DNA binding inhibitor EtBr was added to the MDA - MB - 231 cell lyses followed by immunoprecipitation of KLF4 complex . Co - localization analysis for endogenous PARP1 and KLF4 in MDA - MB - 231 cells . PARP1 and KLF4 are co - localized in the nucleus , and this co - localization is increased by in response to radiation . Scale bars , 5 μm . ectopic expressed GFP - PARP1 and DsRed - KLF4 in MDA - MB - 231 cells . PARP1 and KLF4 are co - localized in the nucleus , and this co - localization is increased by in response to radiation . Scale bars , 5 μm . ( I ) . Validation of the interaction between endogenous KLF4 and PARP1 by in situ proximity ligation assay ( PLA ) . No positive staining in KLF4 / Rabbit IgG antibody PLA assay . Scale bar , 100 µm . The right panel show the blow - up . ( J ) KLF4 was poly ( ADP - ribosyl ) ated . KLF4 was immunoprecipitated and detected by anti - PAR and anti - KLF4 antibodies in MDA - MB - 231 cells . ( K ) The PARylation of KLF4 was increased after exposure to DNA damage . KLF4 complex was purified using immunoprecipitation from MDA - MB - 231 cells that were treated with 5 µM doxorubicin and were then collected at different times ( 1h , 2h , 4h and 8h ) . PARylation was detected by using antibody against PAR . ( L ) Loss of PARP1 attenuates KLF4 PARylation . PARP1 + / + and PARP1 - / - MEFs cells were transfected with FLAG - KLF4 followed by 4h after 5 Gy radiation , and then the PARylation of KLF4 was detected by pulldown . ( M ) PARP1 inhibitors decrease KLF4 PARylation . U2OS cells were pretreated with 10 µM various PARP1 inhibitor niraparib , Olaparib and rucaparib for 1hr followed by exposure to10 Gy radiation for 4 hours . KLF4 was pulled down and the PARylation was detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Purification of KLF4 protein complex in the presence and absence of DNA damage based on TAP-KLF4 stable expression cells (U2OS). Proteins that interacted with KLF4 were purified from U2OS cells expressing FLAG and HA-tagged KLF4 in the absence and presence of 5 Gy radiation at 4h after the treatment. The accumulated bind induced in response to radiation was isolated for mass spectrometry analysis. PARP1 was identified as a binding partner for KLF4.  (C). The purified complex was further confirmed by Western blot detected by FLAG-KLF4, PARP1(lane 1). The interaction between KLF4 and PARP1 was significantly increased in response to γ-radiation detected by pulldown experiment (lane 3 and 4).  (D) Validation of interaction between ectopically expressed KLF4 and PARP1. 293T cells were transfected with FLAG-KLF4 and Myc-PARP. Whole cell lysates or IP complex pulled down by anti-FLAG antibody were analyzed by Western blotting.  (E) Validation of interaction between endogenous KLF4 and PARP1 in cytosolic lysate and nuclear lysate using immunoprecipitation and Western blotting in MDA-MB-231 cells. The Histone 3 (H3) is the control for nuclear portion, and actin is the control for cytosol portion.  (F) Co-immunoprecipitation of PARP1 with endogenous KLF4 is independent on DNA. The DNA binding inhibitor EtBr was added to the MDA-MB-231 cell lyses followed by immunoprecipitation of KLF4 complex. Co-localization analysis for endogenous PARP1 and KLF4 in MDA-MB-231 cells. PARP1 and KLF4 are co-localized in the nucleus, and this co-localization is increased by in response to radiation. Scale bars, 5 μm. ectopic expressed GFP-PARP1 and DsRed-KLF4 in MDA-MB-231 cells. PARP1 and KLF4 are co-localized in the nucleus, and this co-localization is increased by in response to radiation. Scale bars, 5 μm.  (I). Validation of the interaction between endogenous KLF4 and PARP1 by in situ proximity ligation assay (PLA). No positive staining in KLF4/Rabbit IgG antibody PLA assay. Scale bar, 100 µm. The right panel show the blow-up.  (J) KLF4 was poly(ADP-ribosyl)ated. KLF4 was immunoprecipitated and detected by anti-PAR and anti-KLF4 antibodies in MDA-MB-231 cells.  (K) The PARylation of KLF4 was increased after exposure to DNA damage. KLF4 complex was purified using immunoprecipitation from MDA-MB-231 cells that were treated with 5 µM doxorubicin and were then collected at different times (1h, 2h, 4h and 8h). PARylation was detected by using antibody against PAR.  (L) Loss of PARP1 attenuates KLF4 PARylation. PARP1+/+ and PARP1-/- MEFs cells were transfected with FLAG-KLF4 followed by 4h after 5 Gy radiation, and then the PARylation of KLF4 was detected by pulldown.  (M) PARP1 inhibitors decrease KLF4 PARylation. U2OS cells were pretreated with 10 µM various PARP1 inhibitor niraparib, Olaparib and rucaparib for 1hr followed by exposure to10 Gy radiation for 4 hours. KLF4 was pulled down and the PARylation was detected.


Figure 3 ( A ) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines . ( B ) . Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues . Scale bars , 100 μm . Statistic results of KLF4 IHC staining . Normal ( n = 10 ) , tumor ( n = 183 ) ; p = 4 . 36x10 - 7 ( KLF4 ) one - way ANOVA was used for the statistical analysis . Statistic results of PARP1 IHC staining . Normal ( n = 10 ) , tumor ( n = 183 ) p = 0 . 0473 ( PARP1 ) . one - way ANOVA was used for the statistical analysis . ( E ) Statistical analysis of PARP1 staining among normal ( n = 10 ) , ER / PR positive ( ER / PR ) ( n = 68 ) , HER2 positive ( n = 62 ) , and triple negative breast cancer ( TNBC ) ( n = 48 ) . The p values were labeled in figure , one - way ANOVA was used for the statistical analysis . ( F ) Statistical analysis of KLF4 protein staining among normal ( n = 10 ) , ER / PR positive ( n = 68 ) , HER2 positive ( n = 62 ) and TNBC ( n = 47 ) . The p values were labeled in figure , one - way ANOVA was used for the statistical analysis . Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens ( same batch of staining as ( B ) ) . Represents of paired IHC staining of PARP1 and KLF4 ( case 1 - 3 ) are shown ( G ) . Scale bars , 100 μm . ( H ) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer . n = 183 , r = 0 . 434 , p = 8 . 68x10 - 10 , Pearson correlation coefficients . ( I ) Survival analysis of KLF4 protein expression in 117 breast cancer patients . Compared to patients with low KLF4 protein expression , patients with high KLF4 levels had an inferior cumulative survival rate ( Kaplan - Meier assay , LogRank P = 0 . 012 ) ; Low , staining weak and moderate ; High , staining strong . ( J ) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients . Compared to patients with both low KLF4 and PARP1 protein expression levels , patients with both high KLF4 and PARP1 levels had lower cumulative survival rate ( Kaplan - Meier assay , LogRank P = 0 . 022 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines.  (B). Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues. Scale bars, 100 μm. Statistic results of KLF4 IHC staining. Normal (n=10), tumor (n=183); p = 4.36x10-7 (KLF4) one-way ANOVA was used for the statistical analysis.Statistic results of PARP1 IHC staining. Normal (n=10), tumor (n=183) p = 0.0473 (PARP1). one-way ANOVA was used for the statistical analysis.(E) Statistical analysis of PARP1 staining among normal (n=10), ER/PR positive (ER/PR) (n=68), HER2 positive (n=62), and triple negative breast cancer (TNBC) (n=48). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. (F) Statistical analysis of KLF4 protein staining among normal (n=10), ER/PR positive (n=68), HER2 positive (n=62) and TNBC (n=47). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis.  Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens (same batch of staining as (B)). Represents of paired IHC staining of PARP1 and KLF4 (case 1-3) are shown (G). Scale bars, 100 μm.  (H) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer. n=183, r=0.434, p=8.68x10-10, Pearson correlation coefficients.  (I) Survival analysis of KLF4 protein expression in 117 breast cancer patients. Compared to patients with low KLF4 protein expression, patients with high KLF4 levels had an inferior cumulative survival rate (Kaplan-Meier assay, LogRank P=0.012); Low, staining weak and moderate; High, staining strong.  (J) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients. Compared to patients with both low KLF4 and PARP1 protein expression levels, patients with both high KLF4 and PARP1 levels had lower cumulative survival rate (Kaplan-Meier assay, LogRank P=0.022).


Figure 4 ( B ) Identification of amino acid stretch 411 - 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells . . ( D ) . Identification of amino acid stretch 411 - 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells . . PARylation for Flag tagged KLF4 deletion mutants were analyzed , respectively , by pulldown using PAR antibody followed by Western blotting using antibody against Flag . ( E ) Dissection of the PARylation region of KLF4 into three zinc finger motif - containing domains , Zinc 1 , Zinc 2 and Zinc 3 . While no effect was observed on Zinc 1 and Zinc 3 domains , deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation . ( G ) Identification of amino acid stretch 829 - 1014 on the C - terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells .

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 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 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

Figure 4(B) Identification of amino acid stretch 411- 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells..(D). Identification of amino acid stretch 411- 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells.. PARylation for Flag tagged KLF4 deletion mutants were analyzed, respectively, by pulldown using PAR antibody followed by Western blotting using antibody against Flag.(E) Dissection of the PARylation region of KLF4 into three zinc finger motif-containing domains, Zinc 1, Zinc 2 and Zinc 3. While no effect was observed on Zinc 1 and Zinc 3 domains, deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation.(G) Identification of amino acid stretch 829-1014 on the C-terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells.


Figure 5 ( B ) The effect of YYR motif mutations on KLF4 PARylation . Constructs of KLF4 - Zinc 1 - YKH / AAA mutation , KLF4 - Zinc 2 - YYR / AAA mutation and KLF4 - Zinc - 2 - Y451A mutation were co - transfected with Myc - PARP1 into 293T cells , respectively , and then pulled - down using M2 agarose followed by measuring KLF4 PARylation with anti - PAR antibody . ( C ) KLF4 - Zinc 2 - YYR / AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation , ubiquitylation , acetylation and sumoylation in 293T cells . The FLAG - tagged wild - type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti - SYM10 ( methylation ) , anti - SUMO ( sumoylation ) , Anti - ac - lysine ( acetylation ) , anti - Ub ( ubiquitination ) and anti - PAR ( PARylation ) . Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol , soluble nucleus and chromatin in MDA - MB - 231 cells . While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction . Ponceau S were the control of nuclear soluble fraction . E ) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol , soluble nucleus and chromatin in MDA - MB - 231 cells . , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction . PARP1 were the control of nuclear soluble fraction . ( F ) KLF4 - Zinc 2 - YYR / AAA mutation remarkably reduces DNA damage - induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells . ORC2 is the loading control for chromatid fraction . ( G ) . Confocal analysis of PAR and FLAG - tagged KLF4 showing that KLF4 - Zinc 2 - YYR / AAA mutation significantly reduces KLF4 PARylation in U2OS cells . Scale bars , 10 μm . ( I ) The effect of PARP1 mutations ( H909A or T824A ) on KLF4 PARylation , confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells . Myc - PARP1 wildtype or mutations ( H909A or T824A ) and FLAG - KLF4 were co - transfected into 293T cells and then the FLAG - KLF4 was pulldown by anti - FLAG antibody , the PARylation modification of KLF4 was blotted with anti - PAR antibody , and the binding PARP1 was blotted with anti - myc antibody . ( J ) . Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation . In PARP1 - / - MEFs , wild - type or mutant PARP1 ( H909A ) were co - transfected with FLAG - tagged wild - type or KLF4 - Zinc 2 - YYR / AAA mutation followed by immunoprecipitation using M2 - agarose and Western blotting by anti - PAR antibody . ( K ) KLF4 PARylation enhances its transcriptional function demonstrated by p21 promoter luciferase assay . 293T cells were co - transfected with p21 luciferase reporter plasmid with wild - type or mutant KLF4 ( YYR / AAA ) and wild - type or mutant PARP1 ( H909A ) and then submitted to luciferase assay . n = 3 , p = 4 . 74x10 - 6 ( PARP1WT vs PARP1H909A ) , Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B) The effect of YYR motif mutations on KLF4 PARylation. Constructs of KLF4-Zinc 1-YKH/AAA mutation, KLF4-Zinc 2-YYR/AAA mutation and KLF4-Zinc-2-Y451A mutation were co-transfected with Myc-PARP1 into 293T cells, respectively, and then pulled-down using M2 agarose followed by measuring KLF4 PARylation with anti-PAR antibody. (C) KLF4-Zinc 2-YYR/AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation, ubiquitylation, acetylation and sumoylation in 293T cells. The FLAG-tagged wild-type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti-SYM10 (methylation), anti-SUMO (sumoylation), Anti-ac-lysine (acetylation), anti-Ub (ubiquitination) and anti-PAR (PARylation).  Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction. Ponceau S were the control of nuclear soluble fraction.  E) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction. PARP1 were the control of nuclear soluble fraction.  (F) KLF4-Zinc 2-YYR/AAA mutation remarkably reduces DNA damage-induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells. ORC2 is the loading control for chromatid fraction.  (G). Confocal analysis of PAR and FLAG-tagged KLF4 showing that KLF4-Zinc 2-YYR/AAA mutation significantly reduces KLF4 PARylation in U2OS cells. Scale bars, 10 μm.  (I) The effect of PARP1 mutations (H909A or T824A) on KLF4 PARylation, confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells. Myc-PARP1 wildtype or mutations (H909A or T824A) and FLAG-KLF4 were co-transfected into 293T cells and then the FLAG-KLF4 was pulldown by anti-FLAG antibody, the PARylation modification of KLF4 was blotted with anti-PAR antibody, and the binding PARP1 was blotted with anti-myc antibody.  (J). Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation. In PARP1-/- MEFs, wild-type or mutant PARP1 (H909A) were co-transfected with FLAG-tagged wild-type or KLF4-Zinc 2-YYR/AAA mutation followed by immunoprecipitation using M2-agarose and Western blotting by anti-PAR antibody.  (K) KLF4 PARylation enhances its transcriptional function demonstrated by p21 promoter luciferase assay. 293T cells were co-transfected with p21 luciferase reporter plasmid with wild-type or mutant KLF4 (YYR/AAA) and wild-type or mutant PARP1 (H909A) and then submitted to luciferase assay. n=3, p=4.74x10-6 (PARP1WT vs PARP1H909A), Data are mean ± SEM; one-way ANOVA was used for the statistical analysis.


Figure 6 ( A ) Depletion of KLF4 directly diminishes the DNA damage - induced p21 expression , resulting in the failure of cell cycle arrest in MDA - MB - 231 cells . ( B ) Abolishment of KLF4 PARylation disrupts KLF4 - mediated p21 expression that in turn impairs DNA damage response in MDA - MB - 231 cells . ( C ) p - H3 staining analysis of KLF4 + / + , KLF4 - / - MEFs , KLF4 - / - MEF with transfection of wild - type or KLF4 - Zinc 2 - YYR / AAA mutation . n = 4 , p values were labeled in figure , one - way ANOVA assay . ( D Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA assay . Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA assay . ( E ) Summary of D . ( F ) Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells . n = 3 , p values were labeled in figure , one - way ANOVA assay . ( G ) Effect of KLF4 PARylation on NHEJ and HR . Wild - type or KLF4 - Zinc 2 - YYR / AAA mutant KLF4 were co - transfected with I - SCE construction in U2Os - GFP - EJ5 cells ( for NHEJ assay ) or U2Os - DR - GFP ( for HR assay ) . n = 3 , p values were labeled in figure , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4 - / - MEF n = 3 , p values were labeled in figures , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA - MB - 468 n = 3 , p values were labeled in figures , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA - MB - 231 n = 3 , p values were labeled in figures , one - way ANOVA assay . ( K ) Heatmap of BRCA1 , P21 , Bax expression on U2OS cells with expression of wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant as well as KLF4 - Zinc 2 ( Zinc 2 domain deletion ) mutant . No difference of BRCA1 expression was measured between expression of wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant , while loss of Zinc domain causes drops of BRCA1 expression levels . ( L ) In early responsive window ( 1 - 2 hours after exposure to γ - radiation ) , while elevation of wild - type KLF4 enhances the expression levels of p21 , disruption of KLF4 PARylation diminishes KLF4 - mediated p21 accumulation . Upon DNA damage , no matter PARylation or not , elevation of both wild - type or KLF4 - PARylation - deficient mutant leads to BRCA1 accumulation suggesting the KLF4 - mediated regulation of BRCA1 is independent of PARP1 in MDA - MB - 231 cells . ( M ) KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP - PCR . ( N ) inset , top , schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study . ChIP analysis at the BRCA1 promoter using KLF4 - specific or nonspecific control IgG ( α - Gal4 ) in MDA - MB - 231 cells . Shown is the enrichment at positions of the BRCA1 locus relative to the TSS , presented as percent recovery of input . ( O ) inset , top , schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter . Shown is the wild - type ( BRCA1 - WT ) or mutant ( BRCA1 - AA ) BRCA1 promoter luciferase reporter activity when co - transfect with KLF4 plasmids . KLF4 co - transfection promotes BRCA1 - WT but not BRCA1 - AA promoter reporter transcription . n = 3 , p = 0 . 018 , one - way ANOVA assay . ( P ) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA - MB - 231 at - . 3K positions relative to the TSS in untreated and Olaparib ( 10µM for 8h ) or 5Gy radiation treat cells . No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells . n = 3 , p = 0 . 80 , one - way ANOVA assay . ( Q ) BRCA1 reporter assay in KLF4 - / - MEFs , KLF4 - / - MEF with transfection of wild - type ( KLF4WT ) or mutant KLF4 ( KLF4YYR / AAA , KLF4C403A and KLF4ΔZinc2 ) . The depleted the zinc domain or mutated zinc finger ( KLF4C403A and KLF4ΔZinc2 ) on KLF4 impairs the KLF4 - driven BRCA1 expression , while no effect was observed between wild - type and KLF4YYR / AAA mutant . n = 3 , p values were labeled in figure , one - way ANOVA assay . ( R ) HR analysis . U2OS - DR - GFP cells were transfected with I - SceI , BRCA1 and siBRCA1 in KLF4 - wild - type and depletion condition , respectively . GFP positive cells representing HR repair rate were measured by flow cytometry 48 - 72 hour after then . Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells . Data are mean ± SEM ; n = 3 , p values were labeled in figure , one - way ANOVA assay .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A) Depletion of KLF4 directly diminishes the DNA damage-induced p21 expression, resulting in the failure of cell cycle arrest in MDA-MB-231 cells. (B) Abolishment of KLF4 PARylation disrupts KLF4-mediated p21 expression that in turn impairs DNA damage response in MDA-MB-231 cells. (C) p-H3 staining analysis of KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with transfection of wild-type or KLF4-Zinc 2-YYR/AAA mutation. n=4, p values were labeled in figure, one-way ANOVA assay. (D Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n=4, p=4.74x10-6 (KLF4WT vs KLF4YYR/AAA), one-way ANOVA assay.  Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n=4, p=4.74x10-6 (KLF4WT vs KLF4YYR/AAA), one-way ANOVA assay. (E) Summary of D.  (F) Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells. n=3, p values were labeled in figure, one-way ANOVA assay. (G) Effect of KLF4 PARylation on NHEJ and HR. Wild-type or KLF4-Zinc 2-YYR/AAA mutant KLF4 were co-transfected with I-SCE construction in U2Os-GFP-EJ5 cells (for NHEJ assay) or U2Os-DR-GFP (for HR assay). n=3, p values were labeled in figure, one-way ANOVA assay.The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4-/- MEF n=3, p values were labeled in figures, one-way ANOVA assay. The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA-MB-468 n=3, p values were labeled in figures, one-way ANOVA assay.  The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA-MB-231 n=3, p values were labeled in figures, one-way ANOVA assay.  (K) Heatmap of BRCA1, P21, Bax expression on U2OS cells with expression of wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant as well as KLF4-Zinc 2 (Zinc 2 domain deletion) mutant. No difference of BRCA1 expression was measured between expression of wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant, while loss of Zinc domain causes drops of BRCA1 expression levels.  (L) In early responsive window (1-2 hours after exposure to γ-radiation), while elevation of wild-type KLF4 enhances the expression levels of p21, disruption of KLF4 PARylation diminishes KLF4-mediated p21 accumulation. Upon DNA damage, no matter PARylation or not, elevation of both wild-type or KLF4-PARylation-deficient mutant leads to BRCA1 accumulation suggesting the KLF4-mediated regulation of BRCA1 is independent of PARP1 in MDA-MB-231 cells. (M) KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP-PCR.(N) inset, top, schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study. ChIP analysis at the BRCA1 promoter using KLF4-specific or nonspecific control IgG (α-Gal4) in MDA-MB-231 cells. Shown is the enrichment at positions of the BRCA1 locus relative to the TSS, presented as percent recovery of input.(O) inset, top, schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter. Shown is the wild-type (BRCA1-WT) or mutant (BRCA1-AA) BRCA1 promoter luciferase reporter activity when co-transfect with KLF4 plasmids. KLF4 co-transfection promotes BRCA1-WT but not BRCA1-AA promoter reporter transcription. n=3, p=0.018, one-way ANOVA assay. (P) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA-MB-231 at -.3K positions relative to the TSS in untreated and Olaparib (10µM for 8h) or 5Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells. n=3, p=0.80, one-way ANOVA assay.  (Q) BRCA1 reporter assay in KLF4-/- MEFs, KLF4-/- MEF with transfection of wild-type (KLF4WT) or mutant KLF4 (KLF4YYR/AAA, KLF4C403A and KLF4ΔZinc2). The depleted the zinc domain or mutated zinc finger (KLF4C403A and KLF4ΔZinc2) on KLF4 impairs the KLF4-driven BRCA1 expression, while no effect was observed between wild-type and KLF4YYR/AAA mutant. n=3, p values were labeled in figure, one-way ANOVA assay.  (R) HR analysis. U2OS-DR-GFP cells were transfected with I-SceI, BRCA1 and siBRCA1 in KLF4-wild-type and depletion condition, respectively. GFP positive cells representing HR repair rate were measured by flow cytometry 48-72 hour after then. Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells. Data are mean ± SEM; n=3, p values were labeled in figure, one-way ANOVA assay.


Figure 7 ( A ) KLF4 + / + , KLF4 - / - MEFs , KLF4 - / - MEF with wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant were treated with 5 - 10 µM cisplatin for 48 hours followed by measuring the expression of PARP1 , p21 and Bax . Loss of PARylation on KLF4 impairs KLF4 - mediated inhibition of apoptosis in the presence of genotoxic stress . ( B ) The apoptotic response in U2OS cells with expression of wild - type and mutant KLF4 . U2Os cells with wildtype ( pLenti - tet - on - KLF4WT ) or mutant ( pLenti - tet - on - KLF4YYR / AAA ) KLF4 were incubated with 10 ng / ml doxycycline for 24h and then treated with 10uM doxorubicin ( Dox ) or cisplatin ( CDDP ) for additional 24h . While expression of wild - type KLF4 decreases Bax expression and inhibits drug - induced PARP1 cleavage , expression of PARylation - deficient KLF4 leads to failure in inhibiting genotoxic - induced Bax expression and PARP1 cleavage . ( C ) Disruption of KLF4 PARylation leads to failure in inhibiting temperature - induced senescence in BTR model ( RAS V12 - induced senescence ) . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in MDA - MB - 231 measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in MCF10A measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in U2OS measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . KLF4 sensitizes cell to Olaparib . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . ( G ) The representative of MEFs clonogenic assay . KLF4 sensitizes cell to Olaparib . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . ( G ) The representative of MEFs clonogenic assay . ( H ) is the summary of G . ( I ) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy . ( J - K ) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA - MB - 231 cells , disruption of KLF4 PARylation partially rescue the KLF4 - mediated resistance to Olaparib . J ) The representative of MEFs clonogenic assay . ( K ) is the summary of J . ( L ) Elevated KLF4 expression increases resistance to Olaparib in MDA - MB - 468 ( BRCA - proficient ) cells , while disruption of KLF4 PARylation attenuates KLF4 - driven resistance to Olaparib . Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines HCC1937 cells . No significant difference between expression of wild - type KLF4 and KLF4 PARylation - deficient mutant was observed in HCC1937 cells . Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA - MB - 436 cells . No significant difference between expression of wild - type KLF4 and KLF4 PARylation - deficient mutant was observed in MDA - MB - 436 cells . Modulating KLF4 by knockdown or overexpression affects cellular response of MDA - MB - 231 to Olaparib in clonogenic assay . Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay . Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Doxorubicin in MDA - MB - 231 ( Q ) Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . The exact p - Values were supplied in Appendix Table S4 . Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Cisplatin in MCF7 Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . The exact p - Values were supplied in Appendix Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 (A) KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant were treated with 5-10 µM cisplatin for 48 hours followed by measuring the expression of PARP1, p21 and Bax. Loss of PARylation on KLF4 impairs KLF4-mediated inhibition of apoptosis in the presence of genotoxic stress.  (B) The apoptotic response in U2OS cells with expression of wild-type and mutant KLF4. U2Os cells with wildtype (pLenti-tet-on-KLF4WT) or mutant (pLenti-tet-on-KLF4YYR/AAA) KLF4 were incubated with 10 ng/ml doxycycline for 24h and then treated with 10uM doxorubicin (Dox) or cisplatin (CDDP) for additional 24h. While expression of wild-type KLF4 decreases Bax expression and inhibits drug-induced PARP1 cleavage, expression of PARylation-deficient KLF4 leads to failure in inhibiting genotoxic-induced Bax expression and PARP1 cleavage.  (C) Disruption of KLF4 PARylation leads to failure in inhibiting temperature-induced senescence in BTR model (RAS V12 -induced senescence). Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in MDA-MB-231 measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay. Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in MCF10A measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay.  Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in U2OS measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay. KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay.KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay. (H) is the summary of G. (I) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy.  (J-K) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA-MB-231 cells, disruption of KLF4 PARylation partially rescue the KLF4-mediated resistance to Olaparib. J) The representative of MEFs clonogenic assay. (K) is the summary of J.  (L) Elevated KLF4 expression increases resistance to Olaparib in MDA-MB-468 (BRCA-proficient) cells, while disruption of KLF4 PARylation attenuates KLF4-driven resistance to Olaparib.  Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines HCC1937 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in HCC1937 cells.  Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA-MB-436 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in MDA-MB-436 cells.  Modulating KLF4 by knockdown or overexpression affects cellular response of MDA-MB-231 to Olaparib in clonogenic assay.  Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay.  Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Doxorubicin in MDA-MB-231(Q) Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.  Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Cisplatin in MCF7 Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.


Figure 8 ( A ) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells ( 4T1 ) . 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB / c Nude mice . Drug treatment was started at 10th day . Placebo or Olaparib at the dose of 100 mg / kg was administrated daily for twelve days . Tumor volumes were measured every other day . Tumor volumes were measured every other day . n = 9 per group , p values were labeled in figure , one - way ANOVA assay . ( B & C ) The image of 4T1 xenograft tumors ( B ) and tumors were weighed and summarized ( C ) . n = 9 per group , p values were labeled in figure , one - way ANOVA assay . The combination effect when combine of Olaparib with ABT - 263 and dasatinib in suppression MDA - MB - 231 xenograft tumor growth . 8 weeks SCID / Beige female mice were injected 8x106 MDA - MB - 231 cells in matrigel ( 1 : 1 volume ) . When tumor reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four weeks . Tumor volumes were measured every other day ( F ) . The image of MDA - MB - 231 xenograft tumors The combination effect when combine of Olaparib with ABT - 263 and dasatinib in suppression MDA - MB - 231 xenograft tumor growth . 8 weeks SCID / Beige female mice were injected 8x106 MDA - MB - 231 cells in matrigel ( 1 : 1 volume ) . When tumor reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four weeks . Tumor volumes were measured every other day ( G ) . The tumors were weighed and summarized ( H ) . Placebo ( n = 7 ) , ABT263 ( n = 9 ) , Dasatinib ( n = 8 ) , Olaparib ( n = 6 ) , Olaparib + ABT263 ( n = 9 ) , Olaparib + Dasatinib ( n = 10 ) , p values were labeled in figure , one - way ANOVA assay . ( I ) Staining of H & E , KLF4 , PARP1 , p21 and Bax in MDA - MB - 231 xenograft tumors . Scale bars , 100 μm . Combination of ABT - 263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression , but increase Bax expression in xenograft tumors .

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

Figure 8 (A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay.  (B & C) The image of 4T1 xenograft tumors (B) and tumors were weighed and summarized (C). n=9 per group, p values were labeled in figure, one-way ANOVA assay.  The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors  The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay. (I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.


Figure 1Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hSynI - mWwox [ total n = 18 , spontaneously dead n = 6 , mice taken out for electrophysiology / electron microscopy / analysis , are shown in yellow , n = 12 ] compared to mice injected with AAV9 - hSynI - GFP ( n = 6 ) or the non - injected ( n = 8 ( P < 0 . 0001 , Log - rank Mantel - Cox test ) . Immunofluorescence images showing the expression of WWOX ( red ) in brain tissue ( cortex ) at P17 from WT and Wwox null mice that were injected with AAV9 - hSynI - mWwox ( 2x1010 ) . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . White arrows show the transgene expression in NeuN positive cells . Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain ( Cortex , Hippocampus and Cerebellum ) from KO mice injected with AAV - mWwox at P17 . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - mWwox injected mice ( n = 3 ) brains .

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 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

Figure 1Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test).Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells. Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains.


Figure 2Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hWWOX [ total n = 16 , alive n = 6 , spontaneously dead n = 6 , 4 mice ( shown in yellow ) were taken out for analysis ) compared to the non - injected ( n = 8 ) ] ( P < 0 . 0001 , Log - rank Mantel - Cox test ) . D - F . Immunofluorescence images showing the expression of WWOX ( magenta ) in cortex ( D ) , Hippocampus ( E ) and Cerebellum ( F ) at P19 from WT and Wwox null mice that were injected with AAV - hWWOX ( 2x1010 ) or Wwox null alone . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . Graph represents the percentage of neurons showing the transgene ( NeuN and WWOX positive ) expression at different parts ( cortex , hippocampus and cerebellum ) of the KO brain at P19 after AAV injection . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - hWWOX injected mice ( n = 3 ) brains .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test).D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.   Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.


Figure 3Representative traces of cell - attached recordings performed in WT ( blue ) , KO ( red ) and KO injected with either with AAV9 - hSynI - mWwox ( KO + A - mW shown in purple ) or AAV9 - hSynI - hWWOX ( KO + A - hW shown in green ) pups at P18 - 21 days . Traces represent spontaneous neocortical activity ( action potentials shown ) . The panels represent 12 s recording with an inset presenting a zoom - in of an 0 . 5 s interval . The graph shows the recordings ( P18 - 21 ) averages over 30 neurons from WT ( n = 3 ) , 30 neurons from KO + A - mWwox ( n = 3 ) , 30 neurons from KO + A - hWWOX ( n = 3 ) , and 45 neurons from KO ( n = 3 ) . Immunofluorescence staining with anti - GFAP ( shown in green ) and anti - Iba1 ( shown in magenta ) antibodies showing the expression of astrocytes and microglia at P19 from WT , KO and KO + A - hWWOX . The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes . F , G . Graphs represents the quantification of GFAP ( F ) and Iba1 ( G ) positive cells from the images shown in E . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group ( n = 3 per each genotype ) . H . Immunocytochemical images showing astrocytes ( anti - GFAP ) and microglia ( anti - Iba1 ) in CA3 region of hippocampus from WT ( n = 3 ) and rescued ( KO + AAV - hWWOX ) ( n = 3 ) mice at 9 months . Magnified area is shown with square box . I , J . Graphs represents the quantification of GFAP ( I ) and Iba1 ( J ) positive cells from the images shown in H . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections ( paraffin embedded ) from CA3 region per group ( n = 3 per each genotype ) .

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 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

Figure 3Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.   The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).   Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes. F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype). H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box. I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).


Figure 4Images of whole brain sagittal sections that were immunolabelled with anti - MBP at P17 from indicated mice ( n = 3 for each group ) . MBP staining ( shown in gray ) in cortex ( green dotted line represents the border of the cortex ) , hippocampus and cerebellum is presented ( magnified from the top panel ) . Sagittal section of the brain ( P17 ) tissues immunostained for CC1 ( green ) and PDGFRα ( magenta ) . Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV - mWwox compared to Wwox null injected with AAV - GFP virus . Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum ( area 0 . 5 mm2 ) of WT ( n = 3 ) , KO ( n = 3 ) and KO + A - mWwox ( n = 3 ) counted from three similar brain sagittal sections per mouse in each genotype .

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 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

Figure 4Images of whole brain sagittal sections that were immunolabelled with anti-MBP at P17 from indicated mice (n=3 for each group). MBP staining (shown in gray) in cortex (green dotted line represents the border of the cortex), hippocampus and cerebellum is presented (magnified from the top panel).Sagittal section of the brain (P17) tissues immunostained for CC1 (green) and PDGFRα (magenta). Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV-mWwox compared to Wwox null injected with AAV-GFP virus.   Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum (area 0.5 mm2) of WT (n=3), KO (n=3) and KO+A-mWwox (n=3) counted from three similar brain sagittal sections per mouse in each genotype.


Figure 5Electron micrograph ( EM ) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9 - hSynI - mWwox - injected mice compared to Wwox - null mice ( KO ) at P17 . Graph showing average number of myelinated axons in corpus callosum from WT ( n = 3 ) , KO ( n = 3 ) or KO mice injected with AAV9 - hSynI - mWwox ( n = 3 ) per field of view ( FOV ) . g - ratio analysis showing reduced myelin thickness of axons in corpus callosum KO ( n = 3 ) compared to the KO + AAV - Wwox ( n = 3 ) . Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum ( total no of axons n = 350 per each group ) . EM images showing increased number of myelinated axons in optic nerve in AAV9 - hSynI - mWwox injected mice compared to KO mice at P17 . Graph showing the average number of myelinated axons ( counted per FOV ) in optic nerve from WT ( n = 3 ) , KO ( n = 3 ) and KO + AAV - mWwox ( n = 3 ) . g - ratio ' s of myelinated axons from optic nerves showing improved myelin thickness ( less g - ratio ) after WWOX restoration ( KO + AAV - mWwox ) compared to KO alone ( n = 3 per genotype , total no of axons n = 200 per each group ) .

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Figure 5Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.   Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).   g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).   EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.   Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).   g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).


Figure 6Representative images of open field test showing tracking pattern ( open arena ) of WT ( females , n = 7 , males n = 6 ) and AAV - mWwox - injected KO mice ( females n = 7 , males n = 5 ) at 8 - 10 weeks . Dot plot graphs represent the velocity ( B ) , total distance travelled ( C )

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Figure 6Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C)


Figure 1A . GAPDH smFISH and IF analysis using anti - G3BP antibody ( G3BP ) and anti - Dcp1b antibody ( DCP1 ) in A549 cells ( RL - WT ) or A549 RL - KO cells either mock transfected ( mock ) or transfected with poly ( I : C ) ( PIC ) for 5 hours . Scale bar 10 microns . B . Graph of the fraction of G3BP foci with different volumes in PIC treated RL - WT and RL - KO cells . C . Number of DCP1 foci per cell in PIC treated RL - WT and RL - KO cells relative to the number in mock treated cells . D . Average volume of individual Dcp1 foci in mock and PIC treated RL - WT and RL - KO cells .

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Figure 1A. GAPDH smFISH and IF analysis using anti-G3BP antibody (G3BP) and anti-Dcp1b antibody (DCP1) in A549 cells (RL-WT) or A549 RL-KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 hours. Scale bar 10 microns. B. Graph of the fraction of G3BP foci with different volumes in PIC treated RL-WT and RL-KO cells. C. Number of DCP1 foci per cell in PIC treated RL-WT and RL-KO cells relative to the number in mock treated cells. D. Average volume of individual Dcp1 foci in mock and PIC treated RL-WT and RL-KO cells.