Immunostaining of neural progenitor marker SOX2 ( green ) ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in 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 regions . CC = cortical cortex , GCL = granular layer , LV = lateral ventricle , SGZ = subgranular zone , STR = striatum , SVZ = subventricular zone . Scale bars , 100 μm .

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Immunostaining 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 ) ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in 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 regions . CC = cortical cortex , GCL = granular 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 of relative ZIKV in different brain regions of Ifnar - / - mice . mean ± of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t .

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(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 of relative Fancc mRNA from whole brains or testes in mock - ZIKV Paraiba infected Ifnar - / - mice six days post - infection . Mean ± of n = 3 biological replicates , * p < 0 . 05 ns by Student ' s t .

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(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 of relative Fancc or Fancl mRNA from whole brains from mock - ZIKV Paraiba infected Ifnar - / - newborn mice . Mean ± of n = 3 biological replicates , * * * p < 0 . 001 by Student ' s t .

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(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 of Fancc in brain regions from mock - ZIKV Paraiba infected Ifnar - / - mice six days post - infection . HP = hippocampus , CT = cortex , ST = striatum OB = Olfactory Bulb .

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(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 of Fancc , mean ± of n = 3 biological replicates , * p < 0 . 05 ns by Student ' s t .

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(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 of relative ZIKV in different brain regions of Fancc KO neonatal mice six days post - infection . mean ± of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t .

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(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 Fancc KO mice , Mean ± of n = 3 biological replicates , ns by Student ' s t .

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(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.


( A ) Immunostaining of immature neuronal marker DCX ( green ) ZIKV envelope ( ZIKVE , red ) in hippocampus of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Bottom row shows enlargements of hippocampus region . GCL = granular layer , SGZ = subgranular zone , DG = dentate gyrus , InGr = internal granular layer , Me = medulla , OB = olfactory bulb . Scale bars , 50 100 μm .

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(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 ) ZIKV envelope ( ZIKVE , red ) in 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 , 50 100 μm .

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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 LC3 p62 in different brain region of Fancc KO neonatal mice six days post - infection of ZIKV ( MR766 ) . Mean ± of n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 005 , by Student ' s t . HP = hippocampus , CT = cortex , ST = striatum OB = Olfactory Bulb .

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(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 showing immunoreactivity of neural progenitor marker ( Sox2 ) with apoptosis marker ( - caspase - 3 , CC3 ) in hippocampus region of Fancc KO mice . Scale bar = 50µm . ( G ) Quantitative of number of Sox2 + Sox2 / CC3 + co - labelled in Fancc KO mice hippocampal brain region . Mean ± , n = 3 biological replicates , * p < 0 . 05 by Student ' s t .

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(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.


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

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Micrographs 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.


A - C Relative mtDNA level ( A ) , TMRM ( B ) MitoSox ( C ) following treatment for 5 days with dsRNA against genes indicated .

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A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.


D MitoSox following treatment for 1 - 3 days with dsRNA against genes indicated .

D MitoSox fluorescence following treatment for 1-3 days with dsRNA against the genes indicated.


E Whole respiration following treatment for 5 days with dsRNA against genes indicated .

E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated.


F , G Relative mtDNA level ( F ) MitoSox ( G ) after 5 days of treatment with oligomycin .

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F, G Relative mtDNA level (F) and MitoSox fluorescence (G) after 5 days of treatment with oligomycin.


A ‐ D Relative mtDNA level ( A , B ) MitoSox ( C , D ) following 5 days of treatment with dsRNA against genes indicated .

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A‐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 ( F ) TMRM ( G ) of stably expressing AOX or empty vector , as indicated , after 5 days of treatment with dsRNA against CG2968 , as shown . Note that these were cultured in presence of hygromycin to maintain AOX ‐ expressing or control plasmid .

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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 grown in galactose ‐ containing ( Gal ) medium .

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H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.


A . LysoTracker Red NAO of treated as indicated for 5 days with dsRNA against indicated genes , normalized against values for untreated . Mean ± SD for four independent experiments ; asterisks indicate significant differences from untreated , P < 0 . 05 .

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A. 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 of living treated for 5 days with dsRNA against indicated genes stained with both MitoTracker Green LysoTracker Red . scale bar is 10 μm .

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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 in treated for 5 days with dsRNA against indicated genes , or with chloroquine . Mean ± SD for three independent experiments , in each of which ≥ 50 were analyzed by confocal microscopy in a single plane . of z − stacks in ≥ 30 gave similar results . Note that increase in lysosome content was intermediate between that of untreated treated with chloroquine to block lysosomal turnover of autophagosomes . Asterisks indicate significant differences from untreated , P < 0 . 01 .

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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 extracts from control S2 treated with dsRNAs against GFP or CG2968 ( cV ) , probed for NDUFS3 ( cI ) , ATP synthase subunit α ( cV ) GAPDH ( loading control ) .

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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 indicated genes . Mean ± SD for five independent experiments ; other data classes were significantly different from untreated , P < 0 . 05 .

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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.


( 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 ) . Five later , mice tails were cut collected for extraction PCR . PCR results were analyzed by 1 % agarose gel .

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(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 after injection of AAV7 - Cre - mCherry , mouse intestine was removed followed by preparation of tissue section . KLF4 expression in intestine was then detected by Western blot

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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 after injection of AAV7 - Cre - mCherry , mouse intestine was removed followed by preparation of tissue section . KLF4 expression in intestine was then detected by Western

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Validation of inducible knockout of KLF4 in mouse intestine . Five after injection of AAV7 - Cre - mCherry , mouse intestine was removed followed by preparation of tissue section . KLF4 expression in intestine was then detected by immunohistochemistry

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


Validation of inducible knockout of KLF4 in mouse intestine . Five after injection of AAV7 - Cre - mCherry , mouse intestine was removed followed by preparation of tissue section . KLF4 expression in intestine was then detected by

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( E ) Kaplan - Meier survival curves of KLF4 loxp / loxp mice with intraperitoneal injection of AAV7 - Cre - mCherry or AAV7 - mCherry followed by treatment with 8 - Gy ( total - ) γ - irradiation 5 - after then . AAV7 - mCherry , n = 10 per group ; AAV7 - Cre - mCherry , n = 12 per group . p = 0 . 0023 , log - rank .

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(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 of intestinal epithelium of KLF4 loxp / loxp mice with AAV7 - mCherry or AAV7 - Cre - mCherry intraperitoneal injection followed by 8 - Gy ( total - ) γ - irradiation 5 - after then . Tissues were collected from sham mice mice at different time after exposure to γ - irradiation . Scale bars , 100 μm .

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(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 of γ - H2AX , 53BP1 Caspase - 3 in 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 mice at different time after exposure to γ - irradiation then with indicated antibodies . ( G ) of γ - H2AX , 53BP1 active Caspase - 3 - positive on Immunofluorescent results presented in H . Data are mean ± ; 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 statistical . Scale bars , 60 μm .

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(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.


( A ) . Purification of KLF4 in presence absence of damage on TAP - KLF4 stable expression ( U2OS ) . that interacted with KLF4 were purified from U2OS expressing FLAG HA - tagged KLF4 in absence presence of 5 Gy radiation at 4h after treatment . accumulated bind induced in response to radiation was isolated for mass spectrometry . PARP1 was identified as a binding partner for KLF4 .

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(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 ) . purified was further confirmed by Western blot detected by FLAG - KLF4 , PARP1 ( lane 1 ) . interaction between KLF4 PARP1 was significantly increased in response to γ - radiation detected by pulldown experiment ( lane 3 4 ) .

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(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 PARP1 . 293T were transfected with FLAG - KLF4 Myc - PARP . Whole lysates or IP pulled down by anti - FLAG antibody were analyzed by Western blotting .

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(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 PARP1 in lysate lysate using immunoprecipitation Western blotting in MDA - MB - 231 . Histone 3 ( H3 ) is control for portion , actin is control for cytosol portion .

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(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 . binding inhibitor EtBr was added to MDA - MB - 231 lyses followed by immunoprecipitation of KLF4 .

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(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 for endogenous PARP1 KLF4 in MDA - MB - 231 . PARP1 KLF4 are co - localized in nucleus , this co - localization is increased by in response to radiation . Scale bars , 5 μm .

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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 DsRed - KLF4 in MDA - MB - 231 . PARP1 KLF4 are co - localized in nucleus , this co - localization is increased by in response to radiation . Scale bars , 5 μm .

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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 interaction between endogenous KLF4 PARP1 by in situ proximity ligation ( PLA ) . No positive in KLF4 / Rabbit IgG antibody PLA . Scale bar , 100 µm . right panel show blow - up .

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(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 detected by anti - PAR anti - KLF4 antibodies in MDA - MB - 231 .

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(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 ) PARylation of KLF4 was increased after exposure to damage . KLF4 was purified using immunoprecipitation from MDA - MB - 231 that were treated with 5 µM doxorubicin were then collected at different times ( 1h , 2h , 4h 8h ) . PARylation was detected by using antibody against PAR .

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(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 + / + PARP1 - / - MEFs were transfected with FLAG - KLF4 followed by 4h after 5 Gy radiation , then PARylation of KLF4 was detected by pulldown .

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(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 were pretreated with 10 µM various PARP1 inhibitor niraparib , Olaparib rucaparib for 1hr followed by exposure to10 Gy radiation for 4 hours . KLF4 was pulled down PARylation was detected .

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(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.


( A ) Expression of PARP1 KLF4 in mammary epithelial various types of breast cancer lines .

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(A) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines.


( B ) . Accumulation of PARP1 KLF4 were detected in human breast cancer tissues with 183 breast invasive carcinoma 10 pairs of cancer adjacent normal tissue specimens by in comparison with adjacent normal breast tissues . Scale bars , 100 μm .

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(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 . Normal ( n = 10 ) , ( n = 183 ) ; p = 4 . 36x10 - 7 ( KLF4 ) one - way ANOVA was used for statistical .

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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 . Normal ( n = 10 ) , ( n = 183 ) p = 0 . 0473 ( PARP1 ) . one - way ANOVA was used for statistical .

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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 of PARP1 among normal ( n = 10 ) , ER / PR positive ( ER / PR ) ( n = 68 ) , HER2 positive ( n = 62 ) , triple negative breast cancer ( TNBC ) ( n = 48 ) . p values were labeled in figure , one - way ANOVA was used for statistical .

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(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 of KLF4 among normal ( n = 10 ) , ER / PR positive ( n = 68 ) , HER2 positive ( n = 62 ) TNBC ( n = 47 ) . p values were labeled in figure , one - way ANOVA was used for statistical .

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(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 KLF4 are significantly correlated in 183 breast invasive carcinoma human breast cancer tissue specimens ( same batch of as ( B ) ) . Represents of paired of PARP1 KLF4 ( case 1 - 3 ) are shown ( G ) . Scale bars , 100 μm .

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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 of 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 .

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(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 of KLF4 expression in 117 breast cancer . Compared to with low KLF4 expression , with high KLF4 had an inferior cumulative survival rate ( Kaplan - Meier , LogRank P = 0 . 012 ) ; Low , weak moderate ; High , strong .

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(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 of both KLF4 PARP1 expression in 66 breast cancer . Compared to with both low KLF4 PARP1 expression , with both high KLF4 PARP1 had lower cumulative survival rate ( Kaplan - Meier , LogRank P = 0 . 022 ) .

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(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).


( B ) Identification of acid stretch 411 - 441 on repression domain of KLF4 as a segment mediating interaction with PARP1 in 293T . .

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(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 acid stretch 411 - 441 on Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T . . PARylation for Flag tagged KLF4 deletion mutants were analyzed , respectively , by pulldown using PAR antibody followed by Western blotting using antibody against Flag .

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(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 PARylation region of KLF4 into three zinc finger motif - containing domains , Zinc 1 , Zinc 2 Zinc 3 . While no effect was observed on Zinc 1 Zinc 3 domains , deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation .

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(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 acid stretch 829 - 1014 on C - terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T .

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(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.


( B ) effect of YYR motif mutations on KLF4 PARylation . Constructs of KLF4 - Zinc 1 - YKH / AAA mutation , KLF4 - Zinc 2 - YYR / AAA mutation KLF4 - Zinc - 2 - Y451A mutation were co - transfected with Myc - PARP1 into 293T , respectively , then pulled - down using M2 agarose followed by measuring KLF4 PARylation with anti - PAR antibody .

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(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 methylation , ubiquitylation , acetylation sumoylation in 293T . FLAG - tagged wild - type or mutant KLF4 were immunoprecipitated with FLAG detected by anti - SYM10 ( methylation ) , anti - SUMO ( sumoylation ) , Anti - ac - lysine ( acetylation ) , anti - Ub ( ubiquitination ) anti - PAR ( PARylation ) .

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(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 distribution of KLF4 subcellular localization among cytosol , soluble nucleus in MDA - MB - 231 . While activation of PARP1 in response to genotoxic stress enhances recruitment of KLF4 ORC2 were loading control for chromatid fraction . Ponceau S were control of soluble fraction .

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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 distribution of KLF4 subcellular localization among cytosol , soluble nucleus in MDA - MB - 231 . , inhibition of PARP1 by Olaparib attenuates KLF4 accumulation in fraction H3 were loading control for chromatid fraction . PARP1 were control of soluble fraction .

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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 damage - induced recruitment of ectopic expressed KLF4 in U2OS . ORC2 is loading control for chromatid fraction .

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(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 of PAR FLAG - tagged KLF4 showing that KLF4 - Zinc 2 - YYR / AAA mutation significantly reduces KLF4 PARylation in U2OS . Scale bars , 10 μm .

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(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 ) effect of PARP1 mutations ( H909A or T824A ) on KLF4 PARylation , confirming critical role of H909 in mediating KLF4 PARylation in 293T . Myc - PARP1 wildtype or mutations ( H909A or T824A ) FLAG - KLF4 were co - transfected into 293T then FLAG - KLF4 was pulldown by anti - FLAG antibody , PARylation modification of KLF4 was blotted with anti - PAR antibody , binding PARP1 was blotted with anti - myc antibody .

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(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 YYR motif of KLF4 by triple alanines leads to 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 Western blotting by anti - PAR antibody .

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(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 . 293T were co - transfected with p21 luciferase reporter plasmid with wild - type or mutant KLF4 ( YYR / AAA ) wild - type or mutant PARP1 ( H909A ) then submitted to luciferase . n = 3 , p = 4 . 74x10 - 6 ( PARP1WT vs PARP1H909A ) , Data are mean ± ; one - way ANOVA was used for statistical .

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(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.


( A ) Depletion of KLF4 directly diminishes damage - induced p21 expression , resulting in failure of cycle arrest in MDA - MB - 231 .

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(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 damage response in MDA - MB - 231 .

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(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 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 .

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(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 as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA .

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(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 as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA . ( E ) Summary of D .

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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 . n = 3 , p values were labeled in figure , one - way ANOVA .

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(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 HR . Wild - type or KLF4 - Zinc 2 - YYR / AAA mutant KLF4 were co - transfected with I - SCE construction in U2Os - GFP - EJ5 ( for NHEJ ) or U2Os - DR - GFP ( for HR ) . n = 3 , p values were labeled in figure , one - way ANOVA .

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(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.


effect of overexpression or knockdown KLF4 on mRNA of BRCA1 in KLF4 - / - MEF n = 3 , p values were labeled in figures , one - way ANOVA .

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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.


effect of overexpression or knockdown KLF4 on mRNA of BRCA1 in MDA - MB - 468 n = 3 , p values were labeled in figures , one - way ANOVA .

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.


effect of overexpression or knockdown KLF4 on mRNA of BRCA1 in MDA - MB - 231 n = 3 , p values were labeled in figures , one - way ANOVA .

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 ) of BRCA1 , P21 , Bax expression on U2OS 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 .

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(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 expression of p21 , disruption of KLF4 PARylation diminishes KLF4 - mediated p21 accumulation . Upon damage , no matter PARylation or not , elevation of both wild - type or KLF4 - PARylation - deficient mutant leads to BRCA1 accumulation suggesting KLF4 - mediated regulation of BRCA1 is independent of PARP1 in MDA - MB - 231 .

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(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 .

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(M) KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP-PCR.


( N ) inset , top , schematic diagram of BRCA1 promoter relative positions of primer sets used in this study . ChIP at BRCA1 promoter using KLF4 - specific or nonspecific control IgG ( α - Gal4 ) in MDA - MB - 231 . Shown is enrichment at positions of BRCA1 locus relative to TSS , presented as recovery of input .

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(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 BRCA1 promoter cloning primer alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 SLC5A6 promoter . Shown is 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 .

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(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 of KLF4 binding to BRCA1 promoter in MDA - MB - 231 at - . 3K positions relative to TSS in untreated Olaparib ( 10µM for 8h ) or 5Gy radiation treat . No significant difference of KLF4 binds to BRCA1 promoter between untreated Olaparib or radiation treat . n = 3 , p = 0 . 80 , one - way ANOVA .

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(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 in KLF4 - / - MEFs , KLF4 - / - MEF with transfection of wild - type ( KLF4WT ) or mutant KLF4 ( KLF4YYR / AAA , KLF4C403A KLF4ΔZinc2 ) . depleted zinc domain or mutated zinc finger ( KLF4C403A KLF4ΔZinc2 ) on KLF4 impairs KLF4 - driven BRCA1 expression , while no effect was observed between wild - type KLF4YYR / AAA mutant . n = 3 , p values were labeled in figure , one - way ANOVA .

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(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 . U2OS - DR - GFP were transfected with I - SceI , BRCA1 siBRCA1 in KLF4 - wild - type depletion condition , respectively . GFP positive representing HR repair rate were measured by flow cytometry 48 - 72 hour after then . Overexpression of BRCA1 restores HR efficiency in KLF4 knockdown . Data are mean ± ; n = 3 , p values were labeled in figure , one - way ANOVA .

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(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.


( 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 expression of PARP1 , p21 Bax . Loss of PARylation on KLF4 impairs KLF4 - mediated inhibition of apoptosis in presence of genotoxic stress .

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(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 ) apoptotic response in U2OS with expression of wild - type mutant KLF4 . U2Os with wildtype ( pLenti - tet - on - KLF4WT ) or mutant ( pLenti - tet - on - KLF4YYR / AAA ) KLF4 were incubated with 10 ng / ml doxycycline for 24h then treated with 10uM doxorubicin ( Dox ) or cisplatin ( CDDP ) for additional 24h . While expression of wild - type KLF4 decreases Bax expression inhibits drug - induced PARP1 cleavage , expression of PARylation - deficient KLF4 leads to failure in inhibiting genotoxic - induced Bax expression PARP1 cleavage .

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(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 - induced senescence in BTR model ( RAS V12 - induced senescence ) .

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(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 KLF4 - promoted transformation in MDA - MB - 231 measured by soft agar . n = 3 , p values were labeled in figure , one - way ANOVA .

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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 KLF4 - promoted transformation in MCF10A measured by soft agar . n = 3 , p values were labeled in figure , one - way ANOVA .

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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 KLF4 - promoted transformation in U2OS measured by soft agar . n = 3 , p values were labeled in figure , one - way ANOVA .

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 to Olaparib . asterisk in panels represents significant difference ( p < 0 . 05 ) . ( G ) representative of MEFs clonogenic .

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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 to Olaparib . asterisk in panels represents significant difference ( p < 0 . 05 ) . ( G ) representative of MEFs clonogenic . ( H ) is summary of G .

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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 .

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(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 , disruption of KLF4 PARylation partially rescue KLF4 - mediated resistance to Olaparib . J ) representative of MEFs clonogenic . ( K ) is summary of J .

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(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 ) , while disruption of KLF4 PARylation attenuates KLF4 - driven resistance to Olaparib .

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(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 same effect as WT KLF4 to Olaparib in BRCA1 mutant lines HCC1937 . No significant difference between expression of wild - type KLF4 KLF4 PARylation - deficient mutant was observed in HCC1937 .

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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 same effect as WT KLF4 to Olaparib in BRCA1 mutant lines MDA - MB - 436 . No significant difference between expression of wild - type KLF4 KLF4 PARylation - deficient mutant was observed in MDA - MB - 436 .

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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 response of MDA - MB - 231 to Olaparib in clonogenic .

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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 response of MCF7 to Olaparib in clonogenic .

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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 ± ; one - way ANOVA was used for statistical . asterisk in panels represents significant difference ( p < 0 . 05 ) . exact p - Values were supplied in Appendix Table S4 .

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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 ± ; one - way ANOVA was used for statistical . asterisk in panels represents significant difference ( p < 0 . 05 ) . exact p - Values were supplied in Appendix Table S4 .

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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.


( A ) Depletion of KLF4 significantly enhances efficacy of Olaparib in killing TNBC ( 4T1 ) . 4T1 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 dose of 100 mg / kg was administrated daily for twelve days . volumes were measured every other day . volumes were measured every other day . n = 9 per group , p values were labeled in figure , one - way ANOVA .

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(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 ) image of 4T1 xenograft tumors ( B ) tumors were weighed summarized ( C ) . n = 9 per group , p values were labeled in figure , one - way ANOVA .

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(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.


combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When 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 . volumes were measured every other day ( F ) . image of MDA - MB - 231 xenograft tumors

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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


combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When 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 . volumes were measured every other day ( F ) . image of MDA - MB - 231 xenograft

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combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When 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 . volumes were measured every other day ( G ) . tumors were weighed 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 .

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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 ) of H & E , KLF4 , PARP1 , p21 Bax in MDA - MB - 231 xenograft tumors . Scale bars , 100 μm . Combination of ABT - 263 or dasatinib with Olaparib treatment decrease KLF4 p21expression , but increase Bax expression in xenograft tumors .

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(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.


Kaplan - 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 / , are shown in yellow , n = 12 ] compared to mice injected with AAV9 - hSynI - GFP ( n = 6 ) or non - injected ( n = 8 ( P < 0 . 0001 , Log - rank Mantel - Cox ) .

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Kaplan-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 showing expression of WWOX ( red ) in brain tissue ( cortex ) at P17 from WT Wwox null mice that were injected with AAV9 - hSynI - mWwox ( 2x1010 ) . Neurons are labelled with anti - NeuN antibody ( green ) . square box shows magnified . arrows show transgene expression in NeuN positive . Graph showing of NeuN WWOX double positive in different parts of brain ( Cortex , Hippocampus Cerebellum ) from KO mice injected with AAV - mWwox at P17 . NeuN WWOX double positive were calculated from 3 identical sagittal sections of AAV - mWwox injected mice ( n = 3 ) brains .

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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.


Kaplan - 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 ) compared to non - injected ( n = 8 ) ] ( P < 0 . 0001 , Log - rank Mantel - Cox ) .

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Kaplan-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 showing expression of WWOX ( magenta ) in cortex ( D ) , Hippocampus ( E ) Cerebellum ( F ) at P19 from WT Wwox null mice that were injected with AAV - hWWOX ( 2x1010 ) or Wwox null alone . Neurons are labelled with anti - NeuN antibody ( green ) . square box shows magnified . Graph represents of neurons showing transgene ( NeuN WWOX positive ) expression at different parts ( cortex , hippocampus cerebellum ) of KO brain at P19 after AAV injection . NeuN WWOX double positive were calculated from 3 identical sagittal sections of AAV - hWWOX injected mice ( n = 3 ) brains .

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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.


Representative traces of - attached recordings performed in WT ( blue ) , KO ( red ) 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 ) . panels represent 12 s recording with an inset presenting a zoom - in of an 0 . 5 s interval . graph shows 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 ) , 45 neurons from KO ( n = 3 ) .

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Representative 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 with anti - GFAP ( shown in green ) anti - Iba1 ( shown in magenta ) antibodies showing expression of astrocytes microglia at P19 from WT , KO KO + A - hWWOX . square box shows magnified from CA3 regions of hippocampus from genotypes . F , G . Graphs represents of GFAP ( F ) Iba1 ( G ) positive from shown in E . number of GFAP or Iba1 were counted from 3 identical sagittal sections from CA3 region per group ( n = 3 per each genotype ) .

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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 showing astrocytes ( anti - GFAP ) microglia ( anti - Iba1 ) in CA3 region of hippocampus from WT ( n = 3 ) rescued ( KO + AAV - hWWOX ) ( n = 3 ) mice at 9 months . Magnified is shown with square box . I , J . Graphs represents of GFAP ( I ) Iba1 ( J ) positive from shown in H . number of GFAP or Iba1 were counted from 3 identical sagittal sections ( paraffin embedded ) from CA3 region per group ( n = 3 per each genotype ) .

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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).


of whole brain sagittal sections that were immunolabelled with anti - MBP at P17 from indicated mice ( n = 3 for each group ) . MBP ( shown in gray ) in cortex ( green dotted represents border of cortex ) , hippocampus cerebellum is presented ( magnified from top panel ) .

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Images 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 brain ( P17 ) tissues immunostained for CC1 ( green ) PDGFRα ( magenta ) . showing increased number of matured oligodendrocytes in corpus callosum of Wwox null after treatment with AAV - mWwox compared to Wwox null injected with AAV - GFP . Graph represents of CC1 PDGFRα positive in corpus callosum ( 0 . 5 mm2 ) of WT ( n = 3 ) , KO ( n = 3 ) KO + A - mWwox ( n = 3 ) counted from three similar brain sagittal sections per mouse in each genotype .

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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.


Electron micrograph ( EM ) 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 - showing reduced myelin thickness of axons in corpus callosum KO ( n = 3 ) compared to KO + AAV - Wwox ( n = 3 ) . Axon diameter myelin thickness was calculated from electron micrograph of corpus callosum ( total no of axons n = 350 per each group ) .

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Electron 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 showing increased number of myelinated axons in optic nerve in AAV9 - hSynI - mWwox injected mice compared to KO mice at P17 . Graph showing average number of myelinated axons ( counted per FOV ) in optic nerve from WT ( n = 3 ) , KO ( n = 3 ) KO + AAV - mWwox ( n = 3 ) . g - ' s of myelinated axons from optic nerves showing improved myelin thickness ( less g - ) 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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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).


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

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Representative 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)


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

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A. 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.


A . Western of ( N ) or ( C ) fractions from whole lysates ( W ) from A549 ( RL WT ) , RNase L knock out A549 ( RL KO ) , or A549 with either wild - type RNase L ( NLS - RL - WT ) or RNase L - R667A ( NLS - RL - CM ) fused to a localization signal . Short long exposure with anti - RL antibody . GAPDH used as marker . Histone H3 used as marker . B . Graph depicting fraction of RNase L found in nucleus or cytoplasm . Mean of two experiments with experiment values plotted .

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A. Western analysis of nuclear (N) or cytoplasmic (C) fractions from whole cell lysates (W) from A549 cells (RL WT), RNase L knock out A549 cells (RL KO), or A549 cells with either wild-type RNase L (NLS-RL-WT) or RNase L-R667A (NLS-RL-CM) fused to a nuclear localization signal sequence. Short and long exposure with anti-RL antibody. GAPDH protein used as cytoplasmic marker. Histone H3 protein used as nuclear marker. B. Graph depicting the fraction of RNase L found in the nucleus or cytoplasm. Mean and SEM of two experiments with individual experiment values plotted.


C . FISH with probes to GAPDH mRNA or oligo ( dT ) to detect poly ( A ) + in A549 without or with either NLS - RL - WT or NLS - RL - CM either mock transfected or transfected with poly ( I : C ) for 4 hours . Scale bar 5 micron .

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C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron.


D . Graph depicting median value of of oligo ( dT ) in nuclei divided by volume in indicated types either mock transfected or transfected with poly ( I : C ) for 4 hours . At least 43 nuclei analyzed per condition .

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D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . A . FISH to detect poly ( A ) + nucleolar - localized snoRD3A . IF to detect nucleolar granular component nucleophosmin ( NPM1 ) . B . Graph of mean value of average volume of snoRD3A foci in nuclei . 17 nuclei mock treated , 31 nuclei PIC treated . C . Fraction of nuclei with NPM1 enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm . 17 nuclei mock treated , 31 nuclei PIC treated .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1). B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . D . IF to detect dense fibrillar component , ribosome processing factor 1 ( RPF1 ) . E . Graph of mean value of average volume of snoRD3A foci in nuclei . 43 nuclei mock treated , 69 nuclei PIC treated . F . Graph of mean value of average volume of RPF1 foci in 43 mock treated 69 nuclei PIC treated .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . G . FISH to detect poly ( A ) + speckle - localized MALAT1 IF with anti - sc35 antibody to detect speckle SRRM2 . H . Graph of mean value of of MALAT1 signal divided by volume of nuclei . 51 nuclei mock treated , 56 nuclei PIC treated . I . Graph of median value of volume of SRRM2 foci in nuclei . Mock 1526 SRRM2 foci in 51 nuclei . PIC 136 SRRM2 foci in 20 nuclei that contain < 10 SRRM2 foci .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.