( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . The 293 cells ( 1 × 106 ) were transfected with the indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . The 293 - ATG12 - RNAi or control cells ( 1 × 106 ) were transfected with the indicated plasmids . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


( B ) Direct sequencing of PARN in a control , P1 and her parents .

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(B) Direct sequencing of PARN in a control, P1 and her parents.


( D ) Direct sequencing of PARN in a control , P2 and her parents .

(D) Direct sequencing of PARN in a control, P2 and her parents.


( E ) Aberrant splicing products detected in P2 and her mother but not in P2 ' s father nor in a healthy control . GAPDH RT - PCR was performed as control .

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(E) Aberrant splicing products detected in P2 and her mother but not in P2's father nor in a healthy control. GAPDH RT-PCR was performed as control.


( F ) Detection of the exon6 / 7 deletion by PCR with specific primers ( 1F / 2R ) .

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(F) Detection of the exon6/7 deletion by PCR with specific primers (1F/2R).


( G ) PARN detection in cell lysates from P1 and P2 and a healthy control . Actin was used as loading control .

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(G) PARN detection in cell lysates from P1 and P2 and a healthy control. Actin was used as loading control.


( H ) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T - transformed fibroblasts . Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect .

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(H) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T-transformed fibroblasts. Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect.


( A ) Mean telomere length ( kb ) of whole blood cells from patients and their parents estimated with the TRF method .

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(A) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method.


( B ) Representative picture of telomeric signals used for Q - FISH analysis on metaphase spreads from SV40T - transfomed fibroblasts and Muntjac cells used to normalize the signals . ( C ) Individual and mean values of Q - FISH analyses for control and patients . Mean fluorescence intensity of Muntjac cells was set to 1 . Error bars indicate s . d . . ( D ) Graphical representation of ( C ) showing the average of fluorescence ratios ( relative to Muntjac ) . Error bars indicate s . e . m . . The non - parametric Kruskal - Wallis test was applied to compare relative fluorescence values between Ctl and P1 , and Ctl and P2 .

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(B) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2.


( E ) Representative pictures of nuclei from two controls and patient ' s primary fibroblasts showing 53BP1 foci ( green ) and telomeres ( red , detected by Telo - FISH ) . Yellow arrows indicate telomere dysfunction - induced foci ( TIF ) . The scale bar corresponds to 5µm . ( F ) Quantification of ( E ) in primary fibroblasts from two healthy controls ( passages > 4 ) and from patient 1 ( passages < 4 ) . Control 1 : n = 224 ; Control 2 : n = 263 ; Patient 1 : n = 231 . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied when indicated .

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(E) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated.


( G ) Representative pictures of SA - β - galactosidase staining in control ( passage 15 ) and P1 ' s primary fibroblasts ( passage 6 ) . Results are expressed as the percentage of SA - β - galactosidase - positive cells ( averages , lower panel ) . Control : n = 436 ; P1 : n = 436 . A test to compare two population proportions was applied .

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(G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied.


( H ) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo - FISH . The scale bar corresponds to 1µm . ( I ) Quantification of terminal deletions and sister telomere losses ( J ) from three independent experiments ( counted chromatids : Ctl1 : n = 2224 ; Ctl2 : n = 5696 ; P1 : n = 4764 ; P2 : n = 7908 ) . Averages are shown and χ2 - tests were applied to compare Ctl1 / 2 with either P1 or P2 .

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(H) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.


( A ) Validation of PARN KO in HT1080 cells by Western blot . GAPDH is used as loading control .

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(A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control.


( B ) In vitro deadenylation activity assay using protein extracts from HT1080 ( WT ) and HT1080PARN KO ( PARN KO ) cells .

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(B) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells.


( C ) TRAP assay using successive dilutions ( 500 ng , 250 ng , 125 ng and 62 . 5 ng ) of cell extracts from WT or PARN KO cells . An internal control ( IC ) for PCR was used .

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(C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used.


( D ) qRT - PCR analysis of hTR expression in WT and PARN KO cells . Expression levels were normalized first to ACTB and then to WT . Three independent RNA extractions were performed for each cell line . Error bars indicate s . e . m . .

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(D) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m..


( E ) Representative pictures of FISH against hTR in WT and PARN KO cells . Arrows indicate hTR foci . Scale bar 5 μm .

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(E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm.


( F ) PARN detection by Western blot in the indicated conditions . Expression was induced by incubating cells with 10 ng / ml doxycycline for 72 h . Actin is used as loading control . PARN / actin ratios , normalized to doxycycline - treated WT / PARN cells , are shown below .

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(F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below.


( G ) In vitro deadenylation activity assay using protein extracts from doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells .

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(G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells.


( H ) qRT - PCR analysis of the indicated gene transcripts in doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells . Expression levels were normalized first to ACTB and then to WT / empty . Three independent doxycycline inductions were performed for each cell line . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied .

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(H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( I ) Representative western blot analysis of PARN , TRF2 and p53 . Actin was used as loading control . Mean TRF2 / Actin levels from two independent experiments are indicated below .

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(I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below.


( J ) Same as ( H ) for p53 and p21 transcripts . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied .

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(J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( K ) qRT - PCR analysis of TERRA from the indicated chromosome ends in doxycycline - treated KO / empty and KO / PARN cells . Expression levels were normalized first to ACTB and then to KO / empty . Three independent doxycycline inductions were performed for each cell line . Error bars indicate s . e . m . .

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(K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..


( A ) Analysis of telomeric aberrations by Telo - FISH in doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells . Two independent experiments were performed . Counted chromatids : WT / empty : n = 1824 ; WT / PARN : n = 1744 ; KO / empty : n = 1452 ; KO / PARN : n = 1920 . Averages are shown and χ2 - tests were applied to compare KO / empty with either WT / empty or KO / PARN .

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(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN.


( B ) TRF analysis of telomere length ( kb ) in KO / empty and KO / PARN cells treated with 10 ng / ml Dox for 72h . A control Western blot with PARN and Actin is shown below for the corresponding samples .

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(B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.


( A ) HT1080 ( WT ) and HT1080PARN KO ( KO ) cells stably transfected with GFP plasmid ( empty ) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT - PCR . siRNAs against luciferase ( siLuci ) were used as control . p53 expression levels were normalized first to ACTB and then to WT / empty / siLuci cells . Experiment was performed in triplicate . Error bars indicate s . e . m .

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(A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( B ) Cells were treated as in ( A ) and transcript levels of the indicated genes were measured by qRT - PCR . cDNA expression levels were normalized first to ACTB and then to WT / empty / siLuci cells . Experiment was performed in triplicate . Error bars indicate s . e . m .

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(B) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( C ) qRT - PCR analyses of the indicated genes were performed on RNA extracted from P1 ' s primary or SV40T - transformed fibroblasts . cDNA expression levels were normalized to primary fibroblasts .

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(C) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.


( C ) Northern blot analysis of pre - rRNAs from control and patient B - LCLs . ( D ) Log2 values of 18S - E / 21S and 18S - EFL / 21S for P1 and P2 were normalized to the values of the control and presented in a graphical format , as Log averages + standard deviation for two independent experiments .

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(C) Northern blot analysis of pre-rRNAs from control and patient B-LCLs. (D) Log2 values of 18S-E/21S and 18S-EFL/21S for P1 and P2 were normalized to the values of the control and presented in a graphical format, as Log averages + standard deviation for two independent experiments.


( E ) Northern blot analysis of pre - rRNAs from HT1080PARN WT and HT1080PARN KO cells , transduced with an empty vector . ( F ) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector . Western blot relative to actin assessed PARN levels . ( G ) Quantitative analyses of 18S - E / 21S and 18S - E FL / 21S ratios were as described in ( D ) for HT1080PARN KO cells relative to HT1080PARN WT ( left panel ; see ( E ) ) , and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector ( right panel ; see ( F ) ) , with Log averages + standard deviation for four independent experiments .

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(E) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.


( A ) Western blot analysis of Parn expression in control and Parn + / - MEFs .

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(A) Western blot analysis of Parn expression in control and Parn+/- MEFs.


( B ) Northern blot analysis of total RNAs from WT and Parn + / - MEFs .

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(B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs.


( D ) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analyzed . Statistical significance ( P = 1 . 5417E - 08 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test . ( E ) Frequencies of expected and observed E11 . 5 embryos of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analysed . Statistical significance ( P = 0 . 02535 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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(D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( F ) , ( G ) p53 knock - out does not rescue Parn - / - . No Parn - / - p53 - / - mouse was born from various combinations of Parn + / - and either p53 + / - or p53 - / - crosses ( 49 and 56 pups , respectively ) . Statistical significance ( P = 0 . 000886 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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(F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


C . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . Apc1 - loop500 WT or its derivatives ( ∆ 11 or 3A ) were incubated with the 35S - labelled Flag - B56γ in interphase extract ( Inter ) or anaphase extract ( Ana ) supplemented with CycB ∆ 167 at 23 ˚ C for 1 hr . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or coomassie brilliant blue ( CBB ) staining . The bar graph is quantification of bound B56γ . The intensities of MBP control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


D . Cdk - dependent in vitro kinase assay of Apc1 - loop500 . MBP fused WT or 3A Apc1 - loop500 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 23 ˚ C for 10 min , separated by SDS - PAGE and detected by autoradiography .

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D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.


E . Cdk can promote Apc1 - loop500 and B56 interaction . MBP fused WT or 3A Apc1 - loop500 fragment was incubated in the presence or absence of Cdk2 - cyclin A at 30 ˚ C for 60 min . MBP - fused peptides ( - / + kinase ) were isolated and incubated with purified Flag - B56γ at 23 ˚ C for 30 min . The bound proteins were recovered by amylose beads and analysed by SDS - PAGE and immunoblotting with indicated antibodies . F . Quantification of E . Bound B56γ to WT Apc1 - loop500 control ( - Cdk ) was arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies. F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant wild - type ( WT ) or B56 binding site mutant APC / C ( 1 - L557A / V560A ) was incubated with APC / C - depleted ( ∆ APC ) interphase extract ( Inter ) or ∆ APC anaphase extract supplemented with CycB ∆ 167 ( Ana ) at 23 ˚ C for indicated times . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . pApc1 ( pS314 / pS318 ) is a phospho - site specific antibody that binds only when both S314 and S318 are phosphorylated . B . Quantification of A . The intensities of WT control in interphase were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated. B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


C . B56 binding site mutant APC / C ( 1 - L557A / V560A ) is less active in ubiquitylation assay than WT APC / C . The purified recombinant WT APC / C or B56 binding motif mutant APC / C ( 1 - L557A / V560A ) was incubated with ∆ APC anaphase extract . The recovered APC / C - Cdc20 complex was subjected to ubiquitylation assay using 35S - labelled cyclin B as a substrate . Samples were taken at indicated time points and analysed by SDS - PAGE and autoradiography . D . Quantification of C . Error bars , s . e . m . from three independent experiments .

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

C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdc20 binding to APC / C loop domain mutants in interphase extract . The purified recombinant WT or Apc1 mutant APC / Cs ( 1 - ∆ L300 , 1 - ∆ L300 / ∆ 11 or 1 - ∆ L300 / L557A / V560A ) were incubated with ∆ APC interphase extract at 23 ˚ C for 1 hr . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments . B . ( left panel ) Cdc20 binding to APC / C loop domain mutants in anaphase extracts . Same as A , but ∆ APC anaphase extract was used and incubated at 23 ˚ C for 50 min in anaphase extract . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

A. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdk - dependent in vitro kinase assay of Apc1 - loop300 . 3xFlag - tagged WT or 7T Apc1 - loop300 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 30 ˚ C for 30 min , and separated by SDS - PAGE and detected by autoradiography . ( right panel ) 32P - phosphorylated WT or 7T Apc1 - loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS - PAGE followed by autoradiography . B . Quantification of removal of radioactivity in A . Error bars , s . e . m . from three independent experiments .

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

A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments.


C . Cdc20 - 5A efficiently binds to the B56 binding site mutant APC / C . The purified recombinant WT APC / C or Apc1 - loop500 mutant APC / C ( 1 - L557A / V560A ) was incubated with WT Cdc20 or non - phosphorylatable Cdc20 mutant ( 5A ) in ∆ APC ∆ Cdc20 anaphase extract at 23 ˚ C for 55 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . D . Quantification of C . The intensities of WT APC / C control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


E . The APC / C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A - B56γ at 23 ˚ C . Samples were taken at the indicated time points and analysed by SDS - PAGE and immunoblotting with antibodies , including phospho - site specific antibodies for pCdc20 ( pT79 ) and pApc1 ( pS314 / pS318 ) .

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E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).


D HCT116 , Lovo and RKO cells were transfected with control or p53 siRNAs . 48 h after transfection , total protein was then analyzed by western blot analysis . E HCT116 , Lovo and RKO cells were transfected with empty vector and p53 plasmid . 48 h after transfection , total protein was then analyzed by western blot analysis .

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D HCT116, Lovo and RKO cells were transfected with control or p53 siRNAs. 48 h after transfection, total protein was then analyzed by western blot analysis. E HCT116, Lovo and RKO cells were transfected with empty vector and p53 plasmid. 48 h after transfection, total protein was then analyzed by western blot analysis.


F Representative immunofluorescence staining of the p53 ( green ) and METTL14 ( red ) proteins in p53 - WT HCT116 , Lovo and RKO cells and p53 - MT HT29 ( p53R273H ) , SW480 ( p53R273H / P309S ) , SW620 ( p53R273H ) and SW1116 ( p53A159D ) cells . Nuclei were stained with DAPI ( blue ) . Scale bars = 10 μm . The relative mean fluorescence density was analyzed by ImageJ .

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F Representative immunofluorescence staining of the p53 (green) and METTL14 (red) proteins in p53-WT HCT116, Lovo and RKO cells and p53-MT HT29 (p53R273H), SW480 (p53R273H/P309S), SW620 (p53R273H) and SW1116 (p53A159D) cells. Nuclei were stained with DAPI (blue). Scale bars = 10 μm. The relative mean fluorescence density was analyzed by ImageJ.


I Western blot analysis of METTL14 levels after transfection with empty vector , wild - type or mutant p53 plasmids in HCT116 ( p53 - / - ) cells .

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I Western blot analysis of METTL14 levels after transfection with empty vector, wild-type or mutant p53 plasmids in HCT116 (p53-/-) cells.


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 .

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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 ) .

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

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 .

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

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 ) .

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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 .

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

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 B-MEASURED_VAR 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 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 B-MEASURED_VAR 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 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 ) .

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 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 O O O O O

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 .

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

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.