Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot of RAD51 and γH2AX in HeLa cells untreated or treated with 20 μM ETO for 2 h and recovered at the indicated time points (left). Quantifications of RAD51 and γH2AX are shown on the right. Data represented as mean ± SD of three independent experiments. ( B ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in HeLa cells pretreated with DMSO and TSA (0.5 μM, 6 h), followed by ETO exposure (20 μM, 2 h), with cell lysates recovered at the indicated time points. DNA was stained by DAPI (blue). Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Immunofluorescence, Staining, Microscopy

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot of anti-RAD51 immunoprecipitates in HeLa cells untreated or treated with 20 μM ETO for 2 h and recovered at the indicated time points (left). Quantification of relative ac-RAD51 levels is shown (right). 0 h vs 1 h ( P = 0.0005), 0 h vs 2 h ( P < 0.0001), 0 h vs 4 h ( P < 0.0001), 0 h vs 8 h ( P = 0.1967), 0 h vs 24 h ( P = 0.5074). ( B ) Immunoblot of RAD51 in HeLa cells, fractionated as indicated and treated with or without 20 μM ETO for 2 h. Fractions include cytoplasmic, non-chromatin-bound (soluble), and chromatin-bound (chromatin). ( C ) Immunoblot (left) and quantification (right) of ac-RAD51 in anti-RAD51 immunoprecipitates from HeLa cells, fractionated as indicated after treatment with 20 μM ETO for 2 h and recovered for 1 h. Fractions include whole cell lysates (WCL), non-chromatin-bound (soluble, S), and chromatin-bound (C). P = 0.0064. ( D ) Immunoblot (left) of ac-RAD51 in anti-RAD51 immunoprecipitates from HeLa cells treated with DMSO, TSA (0.5 μM, 6 h), and NAM (1 mM, 6 h). Quantification of relative ac-RAD51 levels is shown (right). P = 0.0024. ( E ) Immunoblot (left) and quantification (right) of RAD51 from HeLa cells pretreated with DMSO and TSA (0.5 μM, 6 h), followed by 100 μg/mL CHX exposure, with cell lysates collected at the indicated time points. 3 h ( P = 0.9540), 6 h ( P = 0.009448), 9 h ( P = 0.009947). ( F ) Relative HR repair efficiency (right) in DMSO and TSA-treated (0.5 μM, 6 h) U2OS cells, as detected by HR reporter system (left). P = 0.0338. ( G ) Quantification of RAD51 (left) or γH2AX (right) foci from immunofluorescence in HeLa cells treated with or without 0.5 μM TSA for 6 h and recovered at the indicated points after ETO treatment (20 μM, 2 h) ( n = 50). RAD51, Utr ( P = 0.447789), 1 h ( P < 0.0001), 4 h ( P < 0.0001), 8 h ( P < 0.0001). γH2AX, Utr ( P = 0.389794), 1 h ( P = 0.429150), 4 h ( P < 0.0001), 8 h ( P < 0.0001). ( H ) Cell survival assay in TSA-treated (0.5 μM, 6 h) HeLa cells in response to different doses of ETO (left) and ADR (right). ETO, 3.3 μM ( P = 0.013734), 11 μM ( P = 0.015828), 33 μM ( P = 0.004219), 100 μM ( P = 0.003896). ADR, 0.3 μM ( P = 0.000937), 1.1 μM ( P = 0.002633), 3.3 μM ( P = 0.0006), 10 μM ( P = 0.009846). All data are represented as mean ± SD of three independent experiments. P values are from Student’s t tests ( A , C – F , H ) or Mann–Whitney U test ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Immunofluorescence, Clonogenic Cell Survival Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoprecipitation to detect the interaction between RAD51 and different Flag-tagged HATs in HEK293T cells. ( B ) Immunoblot of PCAF in anti-RAD51 immunoprecipitates from HEK293T cells. ( C ) Interaction between purified His-RAD51 and His-PCAF. ( D ) Immunoblot of in vitro acetylation assay, purified RAD51 and PCAF were incubated in acetyltransferase assay buffer with or without Ac-CoA. ( E ) Immunoblot (left) and quantification (right) of Flag-PCAF in anti-RAD51 immunoprecipitates from HeLa cells transfected with empty vector or Flag-PCAF and treated with or without ETO (20 μM, 2 h). Data represented as mean ± SD of three independent experiments. P values are from Student’s t tests. P < 0.0001. ( F ) Schematic of the RAD51 domains (upper). Immunoprecipitation was performed to determine the interaction and region between endogenous PCAF and the indicated Flag-RAD51 mutations (lower). ( G ) Schematic of the PCAF domains (upper). HAT Histone acetyltransferase. Immunoprecipitation was performed to determine the interaction and region between RAD51 and the indicated Flag-PCAF mutations. Asterisks indicate endogenous RAD51 (lower). .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Immunoprecipitation, Western Blot, Purification, In Vitro, Acetylation Assay, Incubation, Transfection, Plasmid Preparation

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoprecipitation to detect the interaction between RAD51 and p300 in HEK293T cells. ( B ) Immunoblot of RAD51 in anti-Flag immunoprecipitates from HEK293T cells transfected with empty vector or Flag-PCAF. ( C ) Immunoblot of Flag-PCAF in anti-His immunoprecipitates from HEK293T cells co-transfected with Flag-PCAF and His-RAD51 or empty vector. ( D ) Immunoblot of PCAF in anti-Flag-RAD51 immunoprecipitates from HEK293T cells transfected with empty vector or Flag-RAD51 and treated with or without DNase I. ( E ) Coomassie blue staining of purified His-RAD51 (upper) and His-PCAF (lower) protein. ( F ) Representative immunofluorescence images and quantification ( n = 30) of PCAF (red) and γH2AX (green) foci in HeLa cells treated with or without ETO (20 μM, 2 h) and recovered for 1 h. Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. P < 0.0001. ( G ) Representative immunofluorescence images and quantification ( n = 30) of RAD51 (red) and PCAF (green) foci in HeLa cells treated with or without ETO (20 μM, 2 h) and recovered for 1 h. Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. P < 0.0001. All data are represented as mean ± SD of three independent experiments. P values are from Mann–Whitney U test ( F , G ). *** P < 0.001. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Staining, Purification, Immunofluorescence, Microscopy, MANN-WHITNEY

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot (left) and quantification (right) of RAD51 in HeLa cells transfected with or without Flag-PCAF first, then treated with or without 10 μM MG132 for 6 h. EV vs Flag-PCAF ( P = 0.0002), Flag-PCAF vs Flag-PCAF + MG132 ( P = 0.0002). ( B , C ) Immunoblot (left) and quantification (right) of RAD51 in A549 ( B ) and MDA-MB-231 ( C ) cells transfected with or without Flag-PCAF. ( B ) ( P = 0.0006), ( C ) ( P = 0.00097). ( D ) Relative mRNA level of RAD51 in HEK293T (left) and HeLa (right) cells transfected with empty vector or Flag-PCAF for 48 h. HEK293T ( P = 0.7592), HeLa ( P = 0.5225). ( E ) Immunoblot (left) and quantification (right) of RAD51 in HeLa cells transfected with sgNC or sgPCAF. P = 0.0004. ( F ) Relative mRNA level of RAD51 in HEK293T (left) and HeLa (right) cells transfected with sgNC or sgPCAF. HEK293T ( P = 0.6320), HeLa ( P = 0.5095). All data are represented as mean ± SD of three independent experiments. P values are from Student’s t tests. *** P < 0.001; ns not significant ( A – F ).

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Transfection, Plasmid Preparation

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Diagram showing the sequence of acetylation sites of RAD51. ( B ) HR efficiency in U2OS cells transfected with scramble RNA or siRAD51 for 24 h, followed by transfection with the indicated Flag-RAD51 mutations for another 24 h, and treated with DMSO and TSA (0.5 μM, 6 h). Scr ( P = 0.001898), siRAD51 ( P = 0.066415), WT ( P = 0.000142), K40R ( P = 0.103938), K40Q ( P = 0.096139). ( C ) Schematic diagram of CRISPR/Cas9 targeting RAD51 (left) and immunoblot (right) of RAD51 in RAD51 KD HeLa cells. ( D ) Immunoblot of γH2AX in RAD51 KD HeLa cells transfected with indicated Flag-RAD51 mutations and treated with or without ETO (20 μM, 2 h). ( E ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in RAD51 KD HeLa cells transfected with different Flag-RAD51 mutations and treated with or without ETO (20 μM, 2 h). DNA was stained by DAPI (blue). Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( F ) Quantification of RAD51 (right) or γH2AX (left) foci per cell from ( E ) ( n = 50). RAD51, ETO − , NC vs RAD51 KD + EV ( P = 0.1333), NC vs RAD51 KD + WT ( P = 0.7142), NC vs RAD51 KD + 5KR ( P = 0.4249). RAD51, ETO + , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.2425), NC vs RAD51 KD + 5KR ( P = 0.0013). γH2AX, ETO − , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.3234), NC vs RAD51 KD + 5KR ( P = 0.165271). γH2AX, ETO + , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.8152), NC vs RAD51 KD + 5KR ( P < 0.0001). ( G , H ) Cell survival assay was performed in RAD51 KD HeLa cells transfected with indicated Flag-RAD51 mutations in response to different doses of ETO ( G ) and ADR ( H ). ETO, NC vs RAD51 KD + 5KR, 3.3 μM ( P = 0.237876), 11 μM ( P = 0.021469), 33 μM ( P = 0.001285), 100 μM ( P = 0.00063). NC vs RAD51 KD + EV, 3.3 μM ( P = 0.58183), 11 μM ( P = 0.005927), 33 μM ( P = 0.00068), 100 μM ( P = 0.000489). ADR, NC vs RAD51 KD + 5KR, 0.3 μM ( P = 0.020424), 1.1 μM ( P = 0.008277), 3.3 μM ( P = 0.060957), 10 μM ( P = 0.002279). NC vs RAD51 KD + EV, 0.3 μM ( P = 0.000134), 1.1 μM ( P = 0.019614), 3.3 μM ( P = 0.016448), 10 μM ( P = 0.000114). All data are represented as mean ± SD of three independent experiments. P values are from Mann–Whitney U test ( F ) or Student’s t tests ( B , G , H ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Sequencing, Transfection, CRISPR, Western Blot, Immunofluorescence, Staining, Microscopy, Clonogenic Cell Survival Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot (upper) and quantification (lower) of ac-RAD51 in anti-RAD51 immunoprecipitates from HeLa cells transfected with indicated Flag-RAD51 mutations after being treated with ETO (20 μM, 2 h) and recovered for 1 h. WT vs K40R ( P < 0.0001), WT vs 5KR ( P < 0.0001). ( B ) Immunoblot (upper) and quantification (lower) of ac-RAD51 immunoprecipitates from 293 T cells transfected with Flag-PCAF and His-RAD51 WT or K40R. P = 0.0012. ( C ) HR efficiency (upper) and immunoblot (lower) in U2OS cells transfected with scramble RNA or siRAD51 for 24 h, followed by transfection with the indicated Flag-RAD51 mutations for another 24 h. Scr vs siRAD51 ( P = 0.0149), siRAD51 vs WT ( P = 0.0028), siRAD51 vs K40R ( P < 0.0001), siRAD51 vs K40Q ( P = 0.0877). ( D ) Immunoblot of γH2AX in RAD51 KD HeLa cells transfected with indicated Flag-RAD51 mutations and treated with or without ETO (20 μM, 2 h) and recovered for 4 h. ( E ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in RAD51 KD HeLa cells transfected with different Flag-RAD51 mutations and treated with or without ETO (20 μM, 2 h) and recovered for 4 h. Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( F ) Quantification of RAD51 (right) or γH2AX (left) foci per cell from ( E ) ( n = 50). RAD51, ETO − , NC vs RAD51 KD + EV ( P = 0.0898), NC vs RAD51 KD + WT ( P = 0.3234), NC vs RAD51 KD + K40R ( P = 0.2311), NC vs RAD51 KD + K40Q ( P = 0.1450). RAD51, ETO + , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.0847), NC vs RAD51 KD + K40R ( P < 0.0001), NC vs RAD51 KD + K40Q ( P < 0.0001). γH2AX, ETO − , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.94), NC vs RAD51 KD + K40R ( P = 0.8332), NC vs RAD51 KD + K40Q ( P = 0.7641). γH2AX, ETO + , NC vs RAD51 KD + EV ( P < 0.0001), NC vs RAD51 KD + WT ( P = 0.4152), NC vs RAD51 KD + K40R ( P < 0.0001), NC vs RAD51 KD + K40Q ( P < 0.0001). ( G ) Cell survival assay was performed in RAD51 KD HeLa cells transfected with indicated Flag-RAD51 mutations in response to different doses of ETO (left) and ADR (right). ETO, NC vs RAD51 KD + K40R 7 μM ( P = 0.187613), 22 μM ( P = 0.001712), 66 μM ( P = 0.067985), 200 μM ( P = 0.003486). NC vs RAD51 KD + EV, 7 μM ( P < 0.0001), 22 μM ( P = 0.000255), 66 μM ( P < 0.0001), 200 μM ( P = 0.000195). ADR, NC vs RAD51 KD + K40R, 7 μM ( P = 0.030385), 22 μM ( P = 0.004562), 66 μM ( P = 0.555441), 200 μM ( P = 0.645064). NC vs RAD51 KD + EV, 7 μM ( P = 0.001492), 22 μM ( P < 0.0001), 66 μM ( P = 0.78915), 200 μM ( P = 0.882987). All data are represented as mean ± SD of three independent experiments. P values are from Student’s t tests ( A – C , G ) or Mann–Whitney U test ( F ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Transfection, Immunofluorescence, Microscopy, Clonogenic Cell Survival Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Differences in PCAF expression levels between tumor and normal tissues across various cancers in the UALCAN database ( https://ualcan.path.uab.edu/index.html ). TPM Transcripts per million. ( B ) UALCAN database ( https://ualcan.path.uab.edu/index.html ) analysis of PCAF expression levels to patient survival of Kidney Renal Cell Carcinoma (KIRC, upper) and Lung Adenocarcinoma (LUAD, lower). ( C ) Quantification of RAD51 (left) or γH2AX (right) foci per cell from immunofluorescence in HeLa cells transfected with empty vector or Flag-PCAF and recovered at indicated points after ETO treatment (20 μM, 2 h) ( n = 50). RAD51, Utr ( P = 0.81633), 0 h ( P < 0.0001), 1 h ( P < 0.0001), 2 h ( P = 0.049434), 4 h ( P = 0.129798), 8 h ( P < 0.0001), 24 h ( P < 0.0001). γH2AX, Utr ( P = 0.610136), 0 h ( P = 0.512304), 1 h ( P = 0.616873), 2 h ( P = 0.838133), 4 h ( P < 0.0001), 8 h ( P < 0.0001), 24 h ( P < 0.0001). ( D ) Quantification of RAD51 (left) or γH2AX (right) foci per cell from immunofluorescence in HeLa cells transfected with sgNC or sgPCAF and recovered at indicated points after ETO treatment (20 μM, 2 h) ( n = 50). RAD51, Utr ( P = 0.306286), 1 h ( P < 0.0001), 4 h ( P = 0.001603), 8 h ( P < 0.0001). γH2AX, Utr ( P = 0.539115), 1 h ( P = 0.946743), 4 h ( P < 0.0001), 8 h ( P < 0.0001). ( E ) Neutral comet assay in HeLa cells transfected with indicated vectors and recovered for 4 h after ETO treatment (20 μM, 2 h). Scale bars, 100 μm. ETO– ( P = 0.142071), ETO+ ( P < 0.0001). ( F ) Neutral comet assay in HeLa cells transfected with sgNC or sgPCAF and recovered for 4 h after ETO treatment (20 μM, 2 h). Scale bars, 100 μm. ETO– ( P = 0.223976), ETO+ ( P = 0.007230). ( G ) Relative HR repair efficiency in U2OS cells transfected with Flag-PCAF (left) or sgPCAF (right). EV vs Flag-PCAF ( P = 0.0006), sgNC vs sgPCAF ( P = 0.0201). ( H ) Quantification of RAD51 (left) or γH2AX (right) foci per cell from immunofluorescence in HeLa cells overexpressing PCAF or co-transfected with Flag-PCAF and His-RAD51 with or without ETO treatment (20 μM, 2 h) and recovered for 4 h ( n = 50). RAD51, ETO–, EV vs Flag-PCAF ( P = 0.7589), EV vs Flag-PCAF+His-RAD51 ( P = 0.8566). RAD51, ETO + , EV vs Flag-PCAF ( P < 0.0001), EV vs Flag-PCAF+His-RAD51 ( P = 0.5383). γH2AX, ETO–, EV vs Flag-PCAF ( P = 0.3643), EV vs Flag-PCAF+His-RAD51 ( P = 0.8373). γH2AX, ETO + , EV vs Flag-PCAF ( P < 0.0001), EV vs Flag-PCAF+His-RAD51 ( P = 0.9586). ( I ) The analysis of tail moment in HeLa cells overexpressing PCAF or co-transfected with Flag-PCAF and His-RAD51, with or without ETO treatment (20 μM, 2 h) and recovered for 4 h. ETO–, EV vs Flag-PCAF ( P = 0.4437), EV vs Flag-PCAF+His-RAD51 ( P = 0.2682). ETO + , EV vs Flag-PCAF ( P = 0.0021), EV vs Flag-PCAF+His-RAD51 ( P = 0.9869). ( J ) Cell survival assay was performed in HeLa cells transfected with empty vector or Flag-PCAF in response to different doses of ETO. 3.3 μM ( P = 0.078967), 11 μM ( P = 0.003003), 33 μM ( P = 0.010846), 100 μM ( P = 0.958042). ( K ) Cell survival assay was performed in HeLa cells transfected with sgNC or sgPCAF in response to different doses of ETO. 3.3 μM ( P = 0.016596), 11 μM ( P = 0.000242), 33 μM ( P = 0.031894), 100 μM ( P = 0.929595). ( L ) Cell survival assay in HeLa cells transfected with PCAF or co-transfected with Flag-PCAF and His-RAD51 in response to different doses of ETO. 3.3 μM ( P = 0.000127), 11 μM ( P = 0.003017), 33 μM ( P = 0.000569), 100 μM ( P = 0.006091). All data are represented as mean ± SD of three independent experiments. P values are from or Mann–Whitney U test ( C – F , H , I ) or Student’s t tests ( G , J – L ). * P < 0.05, ** P < 0.01 *** P < 0.001, ns not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Neutral Comet Assay, Clonogenic Cell Survival Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot of γH2AX and RAD51 in HeLa cells transfected with empty vector or Flag-PCAF, treated with 20 μM ETO for 2 h and recovered at the indicated time points. ( B ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in HeLa cells transfected with empty vector or Flag-PCAF, followed by ETO exposure (20 μM, 2 h), with cell lysates recovered at the indicated time points. DNA was stained by DAPI (blue). Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( C ) Immunoblot of γH2AX and RAD51 in HeLa cells transfected with sgNC or sgPCAF, treated with 20 μM ETO for 2 h and recovered at the indicated time points. ( D ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in HeLa cells transfected with sgNC or sgPCAF, followed by ETO exposure (20 μM, 2 h), with cell lysates recovered at the indicated time points. DNA was stained by DAPI (blue). Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( E ) Representative immunofluorescence images of RAD51 (red) and γH2AX (green) foci in HeLa cells transfected with Flag-PCAF or Flag-PCAF and His-RAD51, followed by ETO exposure (20 μM, 2 h), with cell lysates recovered at the indicated time points. DNA was stained by DAPI (blue). Scale bars, 10 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( F ) Neutral comet assay in HeLa cells overexpressing PCAF or co-transfected with Flag-PCAF and His-RAD51, with or without ETO treatment (20 μM, 2 h). Scale bars, 100 μm. X indicated that with the chosen microscopy settings, no signal was obtained. ( G ) Cell survival assay was performed in HeLa cells transfected with empty vector or Flag-PCAF in response to different doses of ADR. 0.3 μM ( P < 0.0001), 1.1 μM ( P = 0.00228), 3.3 μM ( P = 0.287791), 10 μM ( P = 0.522845). ( H ) Cell survival assay was performed in HeLa cells transfected with sgNC or sgPCAF in response to different doses of ADR. 0.3 μM ( P = 0.000153), 1.1 μM ( P = 0.000848), 3.3 μM ( P = 0.371428), 10 μM ( P = 0.24121). ( I ) Cell survival assay was performed in HeLa cells transfected with PCAF or co-transfected with Flag-PCAF and His-RAD51 in response to different doses of ADR. EV vs Flag-PCAF, 0.3 μM ( P = 0.000107), 1.1 μM ( P = 0.029975), 3.3 μM ( P = 0.192603), 10 μM ( P = 0.166067). All data are represented as mean ± SD of three independent experiments. P values are from Student’s t tests ( G – I ). ** P < 0.01, *** P < 0.001, ns: not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Microscopy, Neutral Comet Assay, Clonogenic Cell Survival Assay

Journal: EMBO Reports

Article Title: PCAF-mediated acetylation regulates RAD51 dynamic localization on chromatin during HR repair

doi: 10.1038/s44319-025-00513-6

Figure Lengend Snippet: ( A ) Immunoblot (left) and quantification (right) of RAD51 in HEK293T cells transfected with indicated Flag-PCAF variants. EV vs WT ( P = 0.0126), EV vs ΔHAT ( P = 0.2355). ( B ) Immunoblot (left) and quantification (right) of RAD51 in HeLa cells transfected with empty vector or Flag-PCAF-ΔHAT, treated with 100 μg/mL CHX, and collected cell lysates at indicated time points. 3 h ( P = 0.084659), 6 h ( P = 0.386378), 9 h ( P = 0.483797), 12 h ( P = 0.051411). ( C ) Immunoblot (left) and quantification (right) of ac-RAD51 in anti-RAD51 immunoprecipitates from HeLa cells transfected with indicated Flag-PCAF variants. EV vs WT ( P = 0.0006), EV vs ΔHAT ( P = 0.1937). ( D ) Immunoblot (left) and quantification (right) of RAD51 in non-chromatin-bound (soluble) and chromatin fractions of HeLa cells transfected with the indicated vectors and treated with ETO (20 μM, 2 h) and recovered for 1 h. EV vs WT ( P = 0.0006), EV vs ΔHAT ( P = 0.0075). ( E ) Representative immunofluorescence images (left) and quantifications (right) of RAD51 (red) and γH2AX (green) foci in HeLa cells transfected with indicated Flag-PCAF variants and treated with or without ETO (20 μM, 2 h). Scale bars, 10 μm, n = 50. X indicated that with the chosen microscopy settings, no signal was obtained. RAD51, ETO–, EV vs WT ( P = 0.6052), EV vs ΔHAT ( P = 0.6391). RAD51, ETO + , EV vs WT ( P < 0.0001), EV vs ΔHAT ( P = 0.2167). γH2AX, ETO–, EV vs WT ( P = 0.6194), EV vs ΔHAT ( P = 0.7694). γH2AX, ETO + , EV vs WT ( P < 0.0001), EV vs ΔHAT ( P = 0.9586). ( F ) Neutral comet assay (upper) and analysis of tail moment (lower) in HeLa cells transfected with indicated Flag-PCAF variants and treated with or without ETO (20 μM, 2 h). Scale bars, 100 μm. ETO–, EV vs WT ( P = 0.8539), EV vs ΔHAT ( P = 0.5024). ETO + , EV vs WT ( P < 0.0001), EV vs ΔHAT ( P = 0.7454). ( G ) Relative HR repair efficiency in U2OS cells transfected with indicated Flag-PCAF variants. EV vs WT ( P = 0.0004), EV vs ΔHAT ( P = 0.9078). ( H , I ) Cell survival assay in HeLa cells transfected with indicated Flag-PCAF variants in response to different doses of ETO ( H ) and ADR ( I ). ETO, 3.3 μM ( P = 000396), 11 μM ( P = 0.005928), 33 μM ( P = 0.013787), 100 μM ( P = 0.582992). ADR, 0.3 μM ( P < 0.0001), 1.1 μM ( P < 0.0001), 3.3 μM ( P < 0.0001), 10 μM ( P = 0.030867). All data are represented as mean ± SD of three independent experiments. P values are from Student’s t tests ( A – D , G – I ) or Mann–Whitney U test ( E , F ). *** P < 0.001, ns: not significant. .

Article Snippet: The human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa cells were purchased from ATCC, U2OS cells containing HRR reporter system and pCAGGS-I-SceI plasmid were gifts from Liu Songbai’s group at Suzhou Vocational Health College.

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Neutral Comet Assay, Clonogenic Cell Survival Assay, MANN-WHITNEY