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Structured Review

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PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, <t>RPA2</t> and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.
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1) Product Images from "The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability"

Article Title: The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkae1249

PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.
Figure Legend Snippet: PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.

Techniques Used: Expressing, Construct, Western Blot, Transfection, Two Tailed Test



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PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, <t>RPA2</t> and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.
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PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, <t>RPA2</t> and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.
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PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, <t>RPA2</t> and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.
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Proteins known to co-localize with DBP in infected cells are identified by TMT-MS analysis of RC-enriched subnuclear fractions. ( A ) Hierarchical heatmap of proteins expected to be in RCs and identified in the TMT-MS analysis. ( B ) HFFs infected with HAdV-C5 were fixed at 24 hpi, and <t>RPA2,</t> Usp7, TopBP1, and IVa2 (green) were co-stained with DBP (red) and DNA (DAPI, blue). The samples were visualized by confocal microscopy as z-stacks, and the images are presented as maximum-intensity projections. The scale bars correspond to 10 µm.
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Cells derived from individuals with the CHAMP1 PTC mutations exhibit HR deficiency. ( A ) <t>pRPA2</t> foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 4 h after release, and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots. The bottom and top of the box show the lower and upper quartile values, respectively. The median is indicated with a bar in the box, and the whiskers denote the range within 1.5× the size of the box. The data represent 30 cells for each category. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in B. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant.
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Functional investigation of Top Hits from CRISPR screen to identify necessary genes for CHAF1A KO 293T cell survival. ( A ) 53BP1 immunofluorescence showing that depletion of selected top hits in CHAF1A KO 293T cells result in an increase in 53BP1 foci induction compared to 293T WT cells in the absence of exogenous DNA damage. At least 129 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance ( t -test two-tailed, unpaired). ( B ) DNA fiber assay to evaluate fork degradation showing differing impacts between the selected top hits when depleted in CHAF1A KO 293T cells. Depletion of CHK1 results in a significant increase in fork degradation, while depletion of FEN1 leads to an incomplete rescue of FP in CHAF1A KO cells. Depletion of RAD1 and <t>RPA2</t> demonstrated no change in degradation in CHAF1A KO cells despite a loss of FP in WT cells, while depletion of RPA3 and DNA2 did not alter FP status in either cell line. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). Schematic representations of the DNA fiber combing assay conditions are presented. ( C ) BrdU alkaline comet assay showing that depletion of selected top hits besides RAD1 result in an increase in ssDNA gap accumulation in CHAF1A KO 293T cells compared to 293T WT cells upon treatment with 150 μM cisplatin. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is displayed.
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PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.

Journal: Nucleic Acids Research

Article Title: The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability

doi: 10.1093/nar/gkae1249

Figure Lengend Snippet: PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.

Article Snippet: Proteins were detected by western blotting or immunofluorescence using the following primary antibodies: rabbit anti-GFP (1:5000, Abcam, #ab290), mouse anti-GFP (1:1000, Roche, #11814460001), rabbit anti-PICH (1:1000, Cell Signaling, #8886), mouse anti-PICH (1:1000, Millipore, #04–1540), rabbit anti-OsTIR1 (1:1000, MBL, #PD048), rabbit anti-53BP1 (1:2000, Abcam, #ab36823), mouse anti-cyclin A (1:200, Santa Cruz, #sc-271682), rabbit anti-Rb phosphor-S807/811 (1:1000, Cell Signaling, #8516), mouse anti-p53 (1:1000, Santa Cruz, #sc-126), rabbit anti-p53 phospho-S15 (1:1000, Cell Signaling, #9284), mouse anti-p21 (1:1000, Santa Cruz, #sc-6246), rabbit anti-cGAS (1:1000, Cell Signaling, #15102), rabbit anti-IRF3 (1:1000, Abcam, #ab68481), rabbit anti-IRF3 phospho-S386 (1:1000, Abcam, #ab76493), rabbit anti-BLM (1:1000, Abcam, #ab2179), rabbit anti-TOP3A (1:1000, Proteintech, #14525–1-AP), rabbit anti-RMI1 (1:1000, Abcam, #ab70525), mouse anti-PLK1 (1:1000, Santa Cruz, #sc-17783), mouse anti-histone H3 phospho-S10 (1:5000, Abcam, #ab14955), mouse anti-α-tubulin (1:1000, Sigma–Aldrich, #00020911), mouse anti-CHK1 (1:1000, Sigma–Aldrich, #C9358), rabbit-anti-CHK1 phospho-S317 (1:1000, Cell Signaling, #2344), mouse anti-CHK2 (1:1000, Millipore, #05–649), rabbit anti-CHK2 phospho-T68 (1:1000, Cell Signaling, #2661), rabbit anti-KAP1 phospho-S824 (1:1000, Abcam, #ab70369), mouse anti-RPA2 (1:1000, Abcam, #ab2175), rabbit anti-RPA2 phospho-S4/S8 (1:1000, Bethyl, #A300-245A), rabbit anti-SMC2 (1:1000, Bethyl, #A300-058A), rabbit anti-STING phospho-S366 (1:1000, Cell Signaling, #19781), rabbit anti-TBK1 phospho-S172 (1:1000, Cell Signaling, #5483) and rabbit anti-RIF1 (1:1000, Bethyl, #A300-568A).

Techniques: Expressing, Construct, Western Blot, Transfection, Two Tailed Test

PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.

Journal: Nucleic Acids Research

Article Title: The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability

doi: 10.1093/nar/gkae1249

Figure Lengend Snippet: PICH plays both catalytic and protein recruitment function in UFB resolution. ( A ) RPE1 PICH-AG cells expressing GFP-PICH constructs were treated with Dox and IAA for 72 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( B ) Clonogenic cell survival assays were carried out on RPE1 PICH-AG cells expressing different GFP-PICH constructs treated with Dox and IAA. ( C ) RPE1 PICH-AG expressing GFP-PICH WT or GFP-PICH K128A were treated with Dox and IAA for 24 h. GFP, histone H3 pS10 and DNA were visualized using anti-GFP (green), anti-histone H3 pS10 (red) antibodies and DAPI (blue), respectively. ( D ) Quantification of the number of PICH-coated UFBs and chromatin bridges per cells (90 cells per condition) as visualized in panel (C). ( E ) RPE1 PICH-AG expressing GFP-PICH WT , GFP-PICH K128A or GFP- PICH Δ791–1000 were treated with Dox and IAA for 24 h. Cells were then treated with ICRF-193 (25 nM, 2 h) before fixed. GFP, RPA2 and DNA were visualized using anti-GFP (green), anti-RPA2 (red) antibodies and DAPI (blue), respectively. Scale bars, 10 μm. ( F ) Quantification of the number of PICH-coated and RPA2-coated UFBs per cells (90 cells per condition) as visualized in panel (E). ( G ) HeLa TOP3A-GFP cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by western blotting with the indicated antibodies. ( H ) Cells in panel (G) were treated with ICRF-193 (50 nM) for 2 h. GFP, PICH and DNA were visualized using anti-GFP (green), anti-PICH (red) antibodies and DAPI (blue), respectively. ( I ) Quantification of the number of PICH-coated and TOP3A-coated UFBs per cells (>50 cells per condition) as visualized in panel (H). In all quantified data, bars represent the mean ± SD of n = 3 independent experiments. ** P < 0.01; **** P < 0.0001; ns (not significant) P > 0.05; statistical significance values were determined with unpaired two-tailed t -tests.

Article Snippet: Proteins were detected by western blotting or immunofluorescence using the following primary antibodies: rabbit anti-GFP (1:5000, Abcam, #ab290), mouse anti-GFP (1:1000, Roche, #11814460001), rabbit anti-PICH (1:1000, Cell Signaling, #8886), mouse anti-PICH (1:1000, Millipore, #04–1540), rabbit anti-OsTIR1 (1:1000, MBL, #PD048), rabbit anti-53BP1 (1:2000, Abcam, #ab36823), mouse anti-cyclin A (1:200, Santa Cruz, #sc-271682), rabbit anti-Rb phosphor-S807/811 (1:1000, Cell Signaling, #8516), mouse anti-p53 (1:1000, Santa Cruz, #sc-126), rabbit anti-p53 phospho-S15 (1:1000, Cell Signaling, #9284), mouse anti-p21 (1:1000, Santa Cruz, #sc-6246), rabbit anti-cGAS (1:1000, Cell Signaling, #15102), rabbit anti-IRF3 (1:1000, Abcam, #ab68481), rabbit anti-IRF3 phospho-S386 (1:1000, Abcam, #ab76493), rabbit anti-BLM (1:1000, Abcam, #ab2179), rabbit anti-TOP3A (1:1000, Proteintech, #14525–1-AP), rabbit anti-RMI1 (1:1000, Abcam, #ab70525), mouse anti-PLK1 (1:1000, Santa Cruz, #sc-17783), mouse anti-histone H3 phospho-S10 (1:5000, Abcam, #ab14955), mouse anti-α-tubulin (1:1000, Sigma–Aldrich, #00020911), mouse anti-CHK1 (1:1000, Sigma–Aldrich, #C9358), rabbit-anti-CHK1 phospho-S317 (1:1000, Cell Signaling, #2344), mouse anti-CHK2 (1:1000, Millipore, #05–649), rabbit anti-CHK2 phospho-T68 (1:1000, Cell Signaling, #2661), rabbit anti-KAP1 phospho-S824 (1:1000, Abcam, #ab70369), mouse anti-RPA2 (1:1000, Abcam, #ab2175), rabbit anti-RPA2 phospho-S4/S8 (1:1000, Bethyl, #A300-245A), rabbit anti-SMC2 (1:1000, Bethyl, #A300-058A), rabbit anti-STING phospho-S366 (1:1000, Cell Signaling, #19781), rabbit anti-TBK1 phospho-S172 (1:1000, Cell Signaling, #5483) and rabbit anti-RIF1 (1:1000, Bethyl, #A300-568A).

Techniques: Expressing, Construct, Western Blot, Transfection, Two Tailed Test

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/eLife.104718

Figure Lengend Snippet:

Article Snippet: Antibody , Goat polyclonal Anti-RPA2 , A303-874 , Bethyl , 1/500.

Techniques:

Proteins known to co-localize with DBP in infected cells are identified by TMT-MS analysis of RC-enriched subnuclear fractions. ( A ) Hierarchical heatmap of proteins expected to be in RCs and identified in the TMT-MS analysis. ( B ) HFFs infected with HAdV-C5 were fixed at 24 hpi, and RPA2, Usp7, TopBP1, and IVa2 (green) were co-stained with DBP (red) and DNA (DAPI, blue). The samples were visualized by confocal microscopy as z-stacks, and the images are presented as maximum-intensity projections. The scale bars correspond to 10 µm.

Journal: mBio

Article Title: The protein composition of human adenovirus replication compartments

doi: 10.1128/mbio.02144-24

Figure Lengend Snippet: Proteins known to co-localize with DBP in infected cells are identified by TMT-MS analysis of RC-enriched subnuclear fractions. ( A ) Hierarchical heatmap of proteins expected to be in RCs and identified in the TMT-MS analysis. ( B ) HFFs infected with HAdV-C5 were fixed at 24 hpi, and RPA2, Usp7, TopBP1, and IVa2 (green) were co-stained with DBP (red) and DNA (DAPI, blue). The samples were visualized by confocal microscopy as z-stacks, and the images are presented as maximum-intensity projections. The scale bars correspond to 10 µm.

Article Snippet: , Anti-RPA2 mouse mAb 9H8 , IF , 1:200 , Santa Cruz Biotechnology.

Techniques: Infection, Staining, Confocal Microscopy

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Journal: mBio

Article Title: The protein composition of human adenovirus replication compartments

doi: 10.1128/mbio.02144-24

Figure Lengend Snippet: Antibodies a

Article Snippet: , Anti-RPA2 mouse mAb 9H8 , IF , 1:200 , Santa Cruz Biotechnology.

Techniques: Proximity Ligation Assay

Cells derived from individuals with the CHAMP1 PTC mutations exhibit HR deficiency. ( A ) pRPA2 foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 4 h after release, and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots. The bottom and top of the box show the lower and upper quartile values, respectively. The median is indicated with a bar in the box, and the whiskers denote the range within 1.5× the size of the box. The data represent 30 cells for each category. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in B. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant.

Journal: Scientific Reports

Article Title: CHAMP1 premature termination codon mutations found in individuals with intellectual disability cause a homologous recombination defect through haploinsufficiency

doi: 10.1038/s41598-024-83435-y

Figure Lengend Snippet: Cells derived from individuals with the CHAMP1 PTC mutations exhibit HR deficiency. ( A ) pRPA2 foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 4 h after release, and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots. The bottom and top of the box show the lower and upper quartile values, respectively. The median is indicated with a bar in the box, and the whiskers denote the range within 1.5× the size of the box. The data represent 30 cells for each category. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in patient-derived fibroblasts treated with CPT. Patient-derived fibroblasts were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in B. P values were obtained using the Steel–Dwass multiple comparisons test. n.s.: not statistically significant.

Article Snippet: The following rabbit polyclonal antibodies were used at the following dilutions for immunoprecipitation (IP), immunoblotting (IB) and immunofluorescence (IF): CHAMP1 (N-terminus, Thermo Fisher Scientific, PA5-52313, IP 1:500, IB 1:1,000), CHAMP1 (C-terminus, Thermo Fisher Scientific, A304-217 A, IB 1:1,000), 53BP1 (NOVUS Biologicals, NB100-304, IF 1:1,000), phospho-antibody against serine 4 and serine 8 residues of RPA2 (pRPA2) (BETHYL, A300-245 A, IF 1:500), and GFP (Thermo Fisher Scientific, A-11122, 1:1,000).

Techniques: Derivative Assay, Staining

The CHAMP1 PTC and missense mutants do not disrupt the HR function of CHAMP1 WT. ( A ) pRPA2 foci in U2OS cells expressing CHAMP1 mutants treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA, or cells expressing WT or mutant CHAMP1 tagged with EGFP were treated with CPT for 1 h, fixed 4 h after release and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in U2OS cells expressing CHAMP1 mutants treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA, or cells expressing WT or mutant CHAMP1 tagged with EGFP were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Journal: Scientific Reports

Article Title: CHAMP1 premature termination codon mutations found in individuals with intellectual disability cause a homologous recombination defect through haploinsufficiency

doi: 10.1038/s41598-024-83435-y

Figure Lengend Snippet: The CHAMP1 PTC and missense mutants do not disrupt the HR function of CHAMP1 WT. ( A ) pRPA2 foci in U2OS cells expressing CHAMP1 mutants treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA, or cells expressing WT or mutant CHAMP1 tagged with EGFP were treated with CPT for 1 h, fixed 4 h after release and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in U2OS cells expressing CHAMP1 mutants treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA, or cells expressing WT or mutant CHAMP1 tagged with EGFP were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Article Snippet: The following rabbit polyclonal antibodies were used at the following dilutions for immunoprecipitation (IP), immunoblotting (IB) and immunofluorescence (IF): CHAMP1 (N-terminus, Thermo Fisher Scientific, PA5-52313, IP 1:500, IB 1:1,000), CHAMP1 (C-terminus, Thermo Fisher Scientific, A304-217 A, IB 1:1,000), 53BP1 (NOVUS Biologicals, NB100-304, IF 1:1,000), phospho-antibody against serine 4 and serine 8 residues of RPA2 (pRPA2) (BETHYL, A300-245 A, IF 1:500), and GFP (Thermo Fisher Scientific, A-11122, 1:1,000).

Techniques: Expressing, Transfection, Mutagenesis, Staining

CHAMP1 PTC mutants, but not missense mutants, exhibit impaired HR activity. ( A ) pRPA2 foci in U2OS cells expressing CHAMP1 mutants depleted of endogenous CHAMP1 treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA or cells expressing WT or mutant CHAMP1 tagged with EGFP transfected with CHAMP1 siRNA were treated with CPT for 1 h, fixed 4 h after release and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in U2OS cells expressing CHAMP1 mutants depleted of endogenous CHAMP1 treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA or cells expressing WT or mutant CHAMP1 tagged with EGFP transfected with CHAMP1 siRNA were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Journal: Scientific Reports

Article Title: CHAMP1 premature termination codon mutations found in individuals with intellectual disability cause a homologous recombination defect through haploinsufficiency

doi: 10.1038/s41598-024-83435-y

Figure Lengend Snippet: CHAMP1 PTC mutants, but not missense mutants, exhibit impaired HR activity. ( A ) pRPA2 foci in U2OS cells expressing CHAMP1 mutants depleted of endogenous CHAMP1 treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA or cells expressing WT or mutant CHAMP1 tagged with EGFP transfected with CHAMP1 siRNA were treated with CPT for 1 h, fixed 4 h after release and immunostained with an antibody against pRPA2. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in U2OS cells expressing CHAMP1 mutants depleted of endogenous CHAMP1 treated with CPT. U2OS cells expressing EGFP transfected with or without CHAMP1 siRNA or cells expressing WT or mutant CHAMP1 tagged with EGFP transfected with CHAMP1 siRNA were treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. Cells were also stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Article Snippet: The following rabbit polyclonal antibodies were used at the following dilutions for immunoprecipitation (IP), immunoblotting (IB) and immunofluorescence (IF): CHAMP1 (N-terminus, Thermo Fisher Scientific, PA5-52313, IP 1:500, IB 1:1,000), CHAMP1 (C-terminus, Thermo Fisher Scientific, A304-217 A, IB 1:1,000), 53BP1 (NOVUS Biologicals, NB100-304, IF 1:1,000), phospho-antibody against serine 4 and serine 8 residues of RPA2 (pRPA2) (BETHYL, A300-245 A, IF 1:500), and GFP (Thermo Fisher Scientific, A-11122, 1:1,000).

Techniques: Activity Assay, Expressing, Transfection, Mutagenesis, Staining

Heterozygous CHAMP1 depletion impairs HR activity. ( A ) pRPA2 foci in DLD-1 cells expressing mAID-mClover-CHAMP1 treated with CPT. DLD-1 cells in which the mAID-mClover tag was integrated into one (WT/AID) or both (AID/AID) alleles of CHAMP1 were treated with 5-Ph-IAA for 2 h before treated with CPT for 1 h, fixed 4 h after release, and immunostained with an antibody against pRPA2. DLD-1 cells without the mAID-mClover tag (WT/WT) were also treated as a control. Cells were simultaneously stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in DLD-1 cells expressing mAID-mClover-CHAMP1 treated with CPT. DLD-1 cells in which the mAID-mClover tag was integrated into one (WT/AID) or both (AID/AID) alleles of CHAMP1 were treated with 5-Ph-IAA for 2 h before treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. DLD-1 cells without the mAID-mClover tag (WT/WT) were also treated as a control. Cells were simultaneously stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Journal: Scientific Reports

Article Title: CHAMP1 premature termination codon mutations found in individuals with intellectual disability cause a homologous recombination defect through haploinsufficiency

doi: 10.1038/s41598-024-83435-y

Figure Lengend Snippet: Heterozygous CHAMP1 depletion impairs HR activity. ( A ) pRPA2 foci in DLD-1 cells expressing mAID-mClover-CHAMP1 treated with CPT. DLD-1 cells in which the mAID-mClover tag was integrated into one (WT/AID) or both (AID/AID) alleles of CHAMP1 were treated with 5-Ph-IAA for 2 h before treated with CPT for 1 h, fixed 4 h after release, and immunostained with an antibody against pRPA2. DLD-1 cells without the mAID-mClover tag (WT/WT) were also treated as a control. Cells were simultaneously stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( B ) The number of pRPA2 foci in cells treated as in A is displayed as box and dot plots as in Fig. B. The data represent 30 cells per category. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant. ( C ) 53BP1 foci in DLD-1 cells expressing mAID-mClover-CHAMP1 treated with CPT. DLD-1 cells in which the mAID-mClover tag was integrated into one (WT/AID) or both (AID/AID) alleles of CHAMP1 were treated with 5-Ph-IAA for 2 h before treated with CPT for 1 h, fixed 1 h after release, and immunostained with an antibody against 53BP1. DLD-1 cells without the mAID-mClover tag (WT/WT) were also treated as a control. Cells were simultaneously stained with an antibody against Cyclin A to discriminate cells in the S/G2 phase. DNA was stained with DAPI. Scale bar: 5 μm. ( D ) The number of 53BP1 foci in cells treated as in C is displayed as box and dot plots as in Fig. B. P values were obtained using the Steel multiple comparisons test. n.s.: not statistically significant.

Article Snippet: The following rabbit polyclonal antibodies were used at the following dilutions for immunoprecipitation (IP), immunoblotting (IB) and immunofluorescence (IF): CHAMP1 (N-terminus, Thermo Fisher Scientific, PA5-52313, IP 1:500, IB 1:1,000), CHAMP1 (C-terminus, Thermo Fisher Scientific, A304-217 A, IB 1:1,000), 53BP1 (NOVUS Biologicals, NB100-304, IF 1:1,000), phospho-antibody against serine 4 and serine 8 residues of RPA2 (pRPA2) (BETHYL, A300-245 A, IF 1:500), and GFP (Thermo Fisher Scientific, A-11122, 1:1,000).

Techniques: Activity Assay, Expressing, Control, Staining

Functional investigation of Top Hits from CRISPR screen to identify necessary genes for CHAF1A KO 293T cell survival. ( A ) 53BP1 immunofluorescence showing that depletion of selected top hits in CHAF1A KO 293T cells result in an increase in 53BP1 foci induction compared to 293T WT cells in the absence of exogenous DNA damage. At least 129 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance ( t -test two-tailed, unpaired). ( B ) DNA fiber assay to evaluate fork degradation showing differing impacts between the selected top hits when depleted in CHAF1A KO 293T cells. Depletion of CHK1 results in a significant increase in fork degradation, while depletion of FEN1 leads to an incomplete rescue of FP in CHAF1A KO cells. Depletion of RAD1 and RPA2 demonstrated no change in degradation in CHAF1A KO cells despite a loss of FP in WT cells, while depletion of RPA3 and DNA2 did not alter FP status in either cell line. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). Schematic representations of the DNA fiber combing assay conditions are presented. ( C ) BrdU alkaline comet assay showing that depletion of selected top hits besides RAD1 result in an increase in ssDNA gap accumulation in CHAF1A KO 293T cells compared to 293T WT cells upon treatment with 150 μM cisplatin. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is displayed.

Journal: Nucleic Acids Research

Article Title: CAF-1 promotes efficient PrimPol recruitment to nascent DNA for single-stranded DNA gap formation

doi: 10.1093/nar/gkae1068

Figure Lengend Snippet: Functional investigation of Top Hits from CRISPR screen to identify necessary genes for CHAF1A KO 293T cell survival. ( A ) 53BP1 immunofluorescence showing that depletion of selected top hits in CHAF1A KO 293T cells result in an increase in 53BP1 foci induction compared to 293T WT cells in the absence of exogenous DNA damage. At least 129 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance ( t -test two-tailed, unpaired). ( B ) DNA fiber assay to evaluate fork degradation showing differing impacts between the selected top hits when depleted in CHAF1A KO 293T cells. Depletion of CHK1 results in a significant increase in fork degradation, while depletion of FEN1 leads to an incomplete rescue of FP in CHAF1A KO cells. Depletion of RAD1 and RPA2 demonstrated no change in degradation in CHAF1A KO cells despite a loss of FP in WT cells, while depletion of RPA3 and DNA2 did not alter FP status in either cell line. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). Schematic representations of the DNA fiber combing assay conditions are presented. ( C ) BrdU alkaline comet assay showing that depletion of selected top hits besides RAD1 result in an increase in ssDNA gap accumulation in CHAF1A KO 293T cells compared to 293T WT cells upon treatment with 150 μM cisplatin. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is displayed.

Article Snippet: Antibodies used for western blot at 1:500 (unless otherwise noted) were: CHAF1A (Cell Signaling Technology 5480s); CHK1 (Cell Signaling Technology 2360s); FEN1 (Santa Cruz Biotechnology sc-28355); RPA2 (Abcam AB2175); RPA3 (Santa Cruz Biotechnology sc-271564); DNA2 (Abcam AB96488); ASF1A (Santa Cruz Biotechnology sc-53171); BRCA1 (Santa Cruz Biotechnology sc-6954); BRCA2 (Calbiochem OP95); GAPDH (Santa Cruz Biotechnology sc-47724); PrimPol (Proteintech 29824–1-AP) (1:250); Vinculin (Santa Cruz Biotechnology sc-25336).

Techniques: Functional Assay, CRISPR, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Alkaline Single Cell Gel Electrophoresis