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

Nucleic Acids Research

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.

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)."

Figure Legend

"Figure 6. 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. "