J Representative IHC images , and statistical analysis of immunoreactive score ( IRS ) of METTL14 expression in p53 - WT ( n = 63 ) and p53 - MT ( n = 41 ) CRC samples . The horizontal lines represent the median ; the bottom and top of the boxes represent the 25 % and 75 % percentiles , respectively ; and the vertical bars represent the range of the data . The insets show enlarged images of indicated p53 - WT and p53 - MT CRC tissues , respectively . Scale bars = 20 μm and 2 μm ( inset ) .

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J Representative IHC images, and statistical analysis of immunoreactive score (IRS) of METTL14 expression in p53-WT (n = 63) and p53-MT (n = 41) CRC samples. The horizontal lines represent the median; the bottom and top of the boxes represent the 25% and 75% percentiles, respectively; and the vertical bars represent the range of the data. The insets show enlarged images of indicated p53-WT and p53-MT CRC tissues, respectively. Scale bars = 20 μm and 2 μm (inset).


K ChIP assay verified the potential p53 binding site in the METTL14 promoter region in HCT116 and Lovo cell lines . Input fractions and IgG were used as controls .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

K ChIP assay verified the potential p53 binding site in the METTL14 promoter region in HCT116 and Lovo cell lines. Input fractions and IgG were used as controls.


A Colony formation assay of p53 - WT ( HCT116 , Lovo and RKO ) and p53 - MT ( HT29 , SW620 , SW1116 and SW480 ) cells stably infected with shNC or shMETTL14 . Data are presented as mean ± SD ( biological replicates , n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , ns = no significance ) .

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A Colony formation assay of p53-WT (HCT116, Lovo and RKO) and p53-MT (HT29, SW620, SW1116 and SW480) cells stably infected with shNC or shMETTL14. Data are presented as mean ± SD (biological replicates, n = 3; *P < 0.05, **P < 0.01, ns = no significance).


B , C Representative images and analysis of tumors in nude mice bearing stably transfected shNC or shMETTL14 p53 - WT ( HCT116 and Lovo ) and p53 - MT ( HT29 and SW620 ) cells . Data are presented as mean ± SD ( biological replicates , n = 7 , ns = no significance ) .

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B, C Representative images and analysis of tumors in nude mice bearing stably transfected shNC or shMETTL14 p53-WT (HCT116 and Lovo) and p53-MT (HT29 and SW620) cells. Data are presented as mean ± SD (biological replicates, n = 7, ns = no significance).


G Comparison of colorectum length between METTL14WT ( n = 14 , n = 24 ) and METTL14ΔIEC ( n = 13 , n = 21 ) mice from AOM / DSS - induced and AOM - induced CRC models , respectively . Data are expressed as mean ± SD .

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G Comparison of colorectum length between METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models, respectively. Data are expressed as mean ± SD.


I Representative HE staining images of colorectum in METTL14WT ( n = 14 , n = 24 ) and METTL14ΔIEC ( n = 13 , n = 21 ) mice from AOM / DSS - induced and AOM - induced CRC models . Lower panels show enlarged images of indicated normal or CRC tissues . Scale bars = 2 mm ( upper ) and 40 μm ( lower ) . Black dashed line refers to the border of tumor ( T ) and normal ( N ) tissues . Tumors are classified as adenomas with low to focal high - grade dysplasia . The percentages of mice with dysplasia are shown ( right ) .

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I Representative HE staining images of colorectum in METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models. Lower panels show enlarged images of indicated normal or CRC tissues. Scale bars = 2 mm (upper) and 40 μm (lower). Black dashed line refers to the border of tumor (T) and normal (N) tissues. Tumors are classified as adenomas with low to focal high-grade dysplasia. The percentages of mice with dysplasia are shown (right).


C Lactate production , ATP level , glucose uptake and pyruvate level in stably transfected Lv - vector and Lv - METTL14 or shNC and shMETTL14 HCT116 ( p53 - WT ) cells . Data are presented as mean ± SD ( biological replicates , n = 3 ; * * P < 0 . 01 ) .

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C Lactate production, ATP level, glucose uptake and pyruvate level in stably transfected Lv-vector and Lv-METTL14 or shNC and shMETTL14 HCT116 (p53-WT) cells. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01).


H Western blot analysis of METTL14 , SLC2A3 and PGAM1 protein levels of intestinal epithelial cells from AOM / DSS - induced Mettl14WT ( n = 4 ) and Mettl14ΔIEC ( n = 4 ) and mice .

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H Western blot analysis of METTL14, SLC2A3 and PGAM1 protein levels of intestinal epithelial cells from AOM/DSS-induced Mettl14WT (n = 4) and Mettl14ΔIEC (n = 4) and mice.


I Representative IHC staining images and quantitative analysis of SLC2A3 and PGAM1 in tumor tissues and non - tumor tissues from AOM / DSS - induced Mettl14ΔIEC and Mettl14WT mice CRC models . The insets show enlarged images of tumor tissues and non - tumor tissues , respectively . Scale bars = 40 μm and 4 μm ( inset ) . Data are presented as mean ± SD ( biological replicates , n = 6 ; * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I Representative IHC staining images and quantitative analysis of SLC2A3 and PGAM1 in tumor tissues and non-tumor tissues from AOM/DSS-induced Mettl14ΔIEC and Mettl14WT mice CRC models. The insets show enlarged images of tumor tissues and non-tumor tissues, respectively. Scale bars = 40 μm and 4 μm (inset). Data are presented as mean ± SD (biological replicates, n = 6; **P < 0.01, ***P < 0.001).


E Western blot analysis of SLC2A3 and PGAM1 protein levels in HCT116 and Lovo ( p53 - WT ) cells transfected with control or miRNA mimics .

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E Western blot analysis of SLC2A3 and PGAM1 protein levels in HCT116 and Lovo (p53-WT) cells transfected with control or miRNA mimics.


F Representative IF staining and quantitative analysis of the SLC2A3 ( green ) and PGAM1 ( green ) proteins in HCT116 ( p53 - WT ) cells transfected with control or miRNA mimics . Nuclei were stained with DAPI ( blue ) . Scale bars = 20 μm . Data are presented as mean ± SD ( biological replicates , n = 4 ; * * P < 0 . 01 ) . G Representative IF staining and quantitative analysis of the SLC2A3 ( green ) and PGAM1 ( green ) proteins in HT29 ( p53 - MT ) cells transfected with control or miRNA mimics . Nuclei were stained with DAPI ( blue ) . Scalebars = 20 μm . Data are presented as mean ± SD ( biological replicates , n = 4 ; ns = no significance ) .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HCT116 (p53-WT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scale bars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; **P < 0.01). G Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HT29 (p53-MT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scalebars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; ns = no significance).


J qRT - PCR analysis of the pri - miR - 6769b and pri - miR - 499a levels in stably transfected Lv - vector and Lv - METTL14 HCT116 cells with control , YTHDF1 - 3 , YTHDC1 - 2 or IGF2BP1 - 3 siRNAs . Data are presented as mean ± SD ( biological replicates , n = 3 ; * * P < 0 . 01 ) . The dotted line represents basal mRNA expression of pri - miR - 6769b and pri - miR - 499a in HCT116 cells , acting as a control .

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J qRT-PCR analysis of the pri-miR-6769b and pri-miR-499a levels in stably transfected Lv-vector and Lv-METTL14 HCT116 cells with control, YTHDF1-3, YTHDC1-2 or IGF2BP1-3 siRNAs. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01). The dotted line represents basal mRNA expression of pri-miR-6769b and pri-miR-499a in HCT116 cells, acting as a control.


N Representative images of tumors and analysis in nude mice intervened with control , miR - 6769b - 3p or miR - 499a - 3p expression p53 - WT ( HCT116 and Lovo ) cells . Data are presented as mean ± SD ( biological replicates , n = 7 ) . O Representative images of tumors and analysis in nude mice intervened with control , miR - 6769b - 3p or miR - 499a - 3p expression p53 - MT ( HT29 and SW620 ) cells . Data are presented as mean ± SD ( biological replicates , n = 7 , ns , no significance ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N Representative images of tumors and analysis in nude mice intervened with control, miR-6769b-3p or miR-499a-3p expression p53-WT (HCT116 and Lovo) cells. Data are presented as mean ± SD (biological replicates, n = 7). O Representative images of tumors and analysis in nude mice intervened with control, miR-6769b-3p or miR-499a-3p expression p53-MT (HT29 and SW620) cells. Data are presented as mean ± SD (biological replicates, n = 7, ns, no significance).


A Glucose uptake , Lactate , ATP , pyruvate levels were determined in HCT116 ( p53 - WT ) cells treated with control , miR - 6769b - 3p mimics or inhibitor for 48h . B Glucose uptake , Lactate , ATP , pyruvate levels were determined in HCT116 ( p53 - WT ) cells treated with control , miR - 499a - 3p mimics or inhibitor for 48h .

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A Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-6769b-3p mimics or inhibitor for 48h. B Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-499a-3p mimics or inhibitor for 48h.


E Glucose uptake , Lactate , ATP and pyruvate levels were determined in stably transfected Lv - vector and Lv - METTL14 HCT116 ( p53 - WT ) cells treated with control or mixture inhibitors for 48h . Western blot analysis of the corresponding METTL14 , SLC2A3 and PGAM1 protein levels in indicated treatment . F Glucose uptake , Lactate , ATP and pyruvate levels were determined in stably transfected Lv - vector and Lv - METTL14 HCT116 ( p53 - WT ) cells treated with control or mixture mimics for 48h . Western blot analysis of the corresponding METTL14 , SLC2A3 and PGAM1 protein levels in indicated treatment .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

E Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture inhibitors for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment. F Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture mimics for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment.


I , J Representative images and analysis of tumors in nude mice generated by stably transfected shNC and shMETTL14 or miR - 6769b - 3p and miR - 499a - 3p overexpressing HCT116 ( p53 - WT ) cells with or without METTL14 knockdown . Data are presented as mean ± SD ( biological replicates , n = 7 , * * * P < 0 . 001 ) .

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I, J Representative images and analysis of tumors in nude mice generated by stably transfected shNC and shMETTL14 or miR-6769b-3p and miR-499a-3p overexpressing HCT116 (p53-WT) cells with or without METTL14 knockdown. Data are presented as mean ± SD (biological replicates, n = 7, ***P < 0.001).


A Representative IHC and ISH staining images and corresponding quantitative analysis of miR - 6769b - 3p / SLC2A3 IRS and miR - 499a - 3p / PGAM1 IRS in p53 - WT ( n = 63 ) and p53 - MT ( n = 41 ) samples from Cohort 3 . The insets show enlarged images of indicated p53 - WT and p53 - MT CRC tissues , respectively . Scale bars = 200 μm and 20 μm ( inset ) . The horizontal lines represent the median ; the bottom and top of the boxes represent the 25 % and 75 % percentiles , respectively ; and the vertical bars represent the range of the data .

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A Representative IHC and ISH staining images and corresponding quantitative analysis of miR-6769b-3p/SLC2A3 IRS and miR-499a-3p/PGAM1 IRS in p53-WT (n = 63) and p53-MT (n = 41) samples from Cohort 3. The insets show enlarged images of indicated p53-WT and p53-MT CRC tissues, respectively. Scale bars = 200 μm and 20 μm (inset). The horizontal lines represent the median; the bottom and top of the boxes represent the 25% and 75% percentiles, respectively; and the vertical bars represent the range of the data.


B Representative IHC and ISH images of METTL14 , SLC2A3 , PGAM1 , miR - 6769b - 3p and miR - 499a - 3p in CRC tissues with higher or lower METTL14 expression in p53 - WT ( n = 63 ) samples from Cohort 3 . Right panels show enlarged images of indicated p53 - WT CRC tissues . Scale bars = 200 μm ( left ) and 20 μm ( right ) .

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B Representative IHC and ISH images of METTL14, SLC2A3, PGAM1, miR-6769b-3p and miR-499a-3p in CRC tissues with higher or lower METTL14 expression in p53-WT (n = 63) samples from Cohort 3. Right panels show enlarged images of indicated p53-WT CRC tissues. Scale bars = 200 μm (left) and 20 μm (right).


F Kaplan - Meier survival curves of OS in CRC patients with wild type p53 ( n = 63 ) and mutant p53 ( n = 41 ) from Cohort 3 database based on expression levels of METTL14 .

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F Kaplan-Meier survival curves of OS in CRC patients with wild type p53 (n = 63) and mutant p53 (n = 41) from Cohort 3 database based on expression levels of METTL14.


( a ) HEK293 cells stably expressing GFP - DFCP1 were transfected with or without shRNA against Atg14L . The cell lysates were subjected to immunoblotting with the indicated antibodies .

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(a) HEK293 cells stably expressing GFP-DFCP1 were transfected with or without shRNA against Atg14L. The cell lysates were subjected to immunoblotting with the indicated antibodies.


( b ) GFP signals were observed in these cells before or after starvation . ( c ) The number of GFP - positive puncta per cell was counted ; mean ± SD values are presented . N , nutrient rich ; S , starvation .

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(b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation.


( d ) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods . Equivalent amounts of total , cytosolic , and ER fractions were subjected to immunoblotting with each organelle marker antibody . The percentage of recovery in each fraction is presented as mean ± SD values .

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(d) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods. Equivalent amounts of total, cytosolic, and ER fractions were subjected to immunoblotting with each organelle marker antibody. The percentage of recovery in each fraction is presented as mean ± SD values.


( e ) HEK293 cells stably expressing GFP - DFCP1 were transiently transformed with Flag - tagged Atg14L and subjected to starvation . The cells were fixed and stained with the indicated antibodies . Insets show higher magnification of the boxed areas .

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(e) HEK293 cells stably expressing GFP-DFCP1 were transiently transformed with Flag-tagged Atg14L and subjected to starvation. The cells were fixed and stained with the indicated antibodies. Insets show higher magnification of the boxed areas.


( f ) HEK293 cells were transfected with GFP - Atg14L with or without mCherry - linked dominant - negative ULK1 , and these cells were cultured in complete or starvation medium for 60 min . Bars , 10 µm .

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(f) HEK293 cells were transfected with GFP-Atg14L with or without mCherry-linked dominant-negative ULK1, and these cells were cultured in complete or starvation medium for 60 min. Bars, 10 µm.


( a ) HEK293 cells were simultaneously transfected with four plasmids harboring One - Strep - Flag ( OSF ) - tagged Atg14L , Beclin - 1 , hVps34 , and hVps15 , and Atg14L was pulled down using Strep - Tactin Sepharose beads . Empty plasmid was used as control instead of Atg14L . The precipitates were subjected to SDS - PAGE and Coomassie brilliant blue staining .

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(a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)-tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining.


( c ) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti - GFP antibody . Each precipitant was subjected to immunoblotting with the indicated antibodies . IP , immunoprecipitation .

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(c) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti-GFP antibody. Each precipitant was subjected to immunoblotting with the indicated antibodies. IP, immunoprecipitation.


( e ) NRK cells were transiently transfected with adenovirus expressing GFP - Atg14L or GFP - Atg14L4C4A and fixed under nutrient - rich conditions . The cells were stained with anticalnexin antibody as an ER membrane marker . Higher magnification of the boxed areas is shown below . Bars , 10 µm .

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(e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.


( a ) A549 cells were transiently transfected with adenovirus expressing GFP - Atg14L or GFP - Atg14LCAAX and subjected to starvation . The cells were fixed and stained with anti - Beclin - 1 antibody . Higher magnification of the boxed areas is shown below .

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(a) A549 cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14LCAAX and subjected to starvation. The cells were fixed and stained with anti-Beclin-1 antibody. Higher magnification of the boxed areas is shown below.


( b ) HEK293 cells were cotransfected with MEF - hVps34 and GFP or GFP - Atg14LCAAX and stained with anti - Flag antibody . Bars , 10 µm .

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(b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.


Autophagy induction by overexpressed Atg14L . A549 cells were transiently transfected with adenovirus harboring GFP - Atg14L or GFP - Atg14L4C4A . ( a and b ) The cells before starvation ( a ) or after starvation ( b ) were fixed and stained with anti - LC3 antibody . ( c ) The number of LC3 - positive puncta per cell was counted , and mean ± SD values are presented . Bars , 10 µm .

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Autophagy induction by overexpressed Atg14L. A549 cells were transiently transfected with adenovirus harboring GFP-Atg14L or GFP-Atg14L4C4A. (a and b) The cells before starvation (a) or after starvation (b) were fixed and stained with anti-LC3 antibody. (c) The number of LC3-positive puncta per cell was counted, and mean ± SD values are presented. Bars, 10 µm.


The effect of Atg14L localization on its function . ( a ) Atg14L knockout mouse ES cells were transfected with GFP , GFP - Atg14L , GFP - Atg14L4C4A , or GFP - ER - Atg14L4C4A of the pCAG vector and subjected to starvation . The cells were fixed and immunostained with anti - Atg16L or anti - LC3 antibodies . ( b and c ) The number of Atg16L - positive puncta ( b ) and LC3 - positive puncta ( c ) per cell was counted in at least 10 GFP - positive cells , and mean ± SD values are presented . Bars , 10 µm .

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The effect of Atg14L localization on its function. (a) Atg14L knockout mouse ES cells were transfected with GFP, GFP-Atg14L, GFP-Atg14L4C4A, or GFP-ER-Atg14L4C4A of the pCAG vector and subjected to starvation. The cells were fixed and immunostained with anti-Atg16L or anti-LC3 antibodies. (b and c) The number of Atg16L-positive puncta (b) and LC3-positive puncta (c) per cell was counted in at least 10 GFP-positive cells, and mean ± SD values are presented. Bars, 10 µm.


( 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 .

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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 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

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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 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

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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 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 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

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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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) . 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 ) .

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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 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 .

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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 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 .

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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 DNA . The DNA binding inhibitor EtBr was added to the MDA - MB - 231 cell lyses followed by immunoprecipitation of KLF4 complex .

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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 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 .

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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 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 .

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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 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 .

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

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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 ) 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 .

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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 + / + 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 .

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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 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 .

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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 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell 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 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 .

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

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 amino acid stretch 411 - 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells . .

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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 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 .

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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 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 .

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

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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 ) 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 DNA damage - induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells . ORC2 is the 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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 the DNA damage - induced p21 expression , resulting in the failure of cell cycle arrest in MDA - MB - 231 cells .

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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 DNA damage response in MDA - MB - 231 cells .

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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 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 .

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

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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 DNA as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA assay . ( 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 cells . n = 3 , p values were labeled in figure , one - way ANOVA assay .

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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 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 .

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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.


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 .

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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.


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-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 .

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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the expression of PARP1 , p21 and Bax . Loss of PARylation on KLF4 impairs KLF4 - mediated inhibition of apoptosis in the 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 ) 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 .

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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 temperature - 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 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 .

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

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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 the KLF4 - promoted cellular transformation in U2OS 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 .

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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 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 .

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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 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 .

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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 ) cells , 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 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 .

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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 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 .

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

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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 cellular response of MCF7 to Olaparib in clonogenic assay .

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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 ± 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 .

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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 ± 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 .

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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 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 .

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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 ) 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 .

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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.


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

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

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 ( F ) . The image of MDA - MB - 231 xenograft

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


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 .

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

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 .

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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.


Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 ( indicated by asterisk ) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate . The predicted molecular weight sizes of putative , unanchored , poly - NEDD8 chains are denoted by N2 through to N5 . Unconjugated NEDD8 is denoted by N1 .

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Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 (indicated by asterisk) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate. The predicted molecular weight sizes of putative, unanchored, poly-NEDD8 chains are denoted by N2 through to N5. Unconjugated NEDD8 is denoted by N1.


NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells . Recombinant HALO - NEDP1 C163A ( CA ) conjugated to HALO - Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells . Enriched proteins were resolved by SDS - PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies . HALO - NEDP1 CA specifically enriches for NEDD8 - reactive proteins in both WT and NEDP1 KO cells , but does not enrich for Ubiquitin - modified proteins in either cell line .

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NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.


Components of the NEDD8 conjugation machinery are enriched in HALO - NEDP1 pulldowns from NEDP1 KO lysates . Neddylated proteins from HEK293 KO cells were enriched by HALO - NEDP1 CA pulldown , as in ( B ) but not by the NEDD8 nonbinder mutant , HALO - NEDP1 DAGC ( D29W A98K G99K C163A ) . The NEDD8 E1s , UBA3 and ULA1 , are modified in NEDP1 KO cells , as well as E2 UBE2M , and co - E3s DCNL1 and DCNL2 . Cul2 and Cul3 are hyper - neddylated in NEDP1 KO cells . CSN components , CSN5 and CSN8 , also co - precipitate in HALO - NEDP1 CA pulldowns .

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Components of the NEDD8 conjugation machinery are enriched in HALO-NEDP1 pulldowns from NEDP1 KO lysates. Neddylated proteins from HEK293 KO cells were enriched by HALO-NEDP1 CA pulldown, as in (B) but not by the NEDD8 nonbinder mutant, HALO-NEDP1 DAGC (D29W A98K G99K C163A). The NEDD8 E1s, UBA3 and ULA1, are modified in NEDP1 KO cells, as well as E2 UBE2M, and co-E3s DCNL1 and DCNL2. Cul2 and Cul3 are hyper-neddylated in NEDP1 KO cells. CSN components, CSN5 and CSN8, also co-precipitate in HALO-NEDP1 CA pulldowns.


Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation / de - conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells . Apart from UBA3 , there is no detectable amount of NEDD8 - modified enzymes in whole - cell lysates from NEDP1 KO cells .

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Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation/de-conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells. Apart from UBA3, there is no detectable amount of NEDD8-modified enzymes in whole-cell lysates from NEDP1 KO cells.


Poly - NEDD8 chains can be generated by in vitro reactions ( Rxn ) . NAE ( 0 . 15 μM ) , UBE2M , and NEDD8 ( 20 μM ) were incubated on ice or incubated at 30 ° C for 3 hours and reactions were stopped by addition of LDS loading buffer . Reactions were resolved by SDS - PAGE and stained with colloidal Coomaisse . Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis . The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2 - N4 . Unconjugated NEDD8 is indicated by N1 . UBE2M modified by NEDD8 is indicated with an asterisk .

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Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.


Neddylated species are NEDD8 E1 dependent . WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time . Lysed cells were then processed for Western blot analysis . NEDD8 E1 inhibition results in a time - dependent decrease in the amount of Cullin and non - Cullin NEDD8 reactive bands .

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Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.


Neddylated species are UBE2M dependent . WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours . Lysed cells were then processed for Western blot analysis . Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment ( si2M ) , but not with control siRNA ( siCTRL ) or when NEDD8 E2 UBE2F was depleted ( si2F ) . The double knockdown ( si2M / 2F ) does not further reduce NEDD8 modified proteins .

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Neddylated species are UBE2M dependent. WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours. Lysed cells were then processed for Western blot analysis. Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment (si2M), but not with control siRNA (siCTRL) or when NEDD8 E2 UBE2F was depleted (si2F). The double knockdown (si2M/2F) does not further reduce NEDD8 modified proteins.


( Left ) Western blot analysis of large scale HALO - NEDP1 pulldowns from HEK 293 NEDP1 KO lysates . ( Right ) Diagram of mass spectrometry sample preparation .

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(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.


Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1 - CA pulldown . iBAQ analysis of proteins identified in A ) are plotted as the log2 value of the enrichment ratio ( mass spectrometry intensity of the HALO - NEDP1 CA pulldown over HALO - NEDP1 DAGC pulldown ) versus the log10 value of the iBAQ intensity from the HALO - NEDP1 CA pulldown . Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP - 1 in the database of ADP - ribosylated proteins , ADPriboDB ( Vivelo et al . , 2017 ) .

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Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).


Non - cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa ( as indicated by the arrow ) . WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis .

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Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.