Journal: bioRxiv
Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells
doi: 10.1101/2024.08.27.609980
Figure Lengend Snippet: a , Histogram ( right ) showing the impact of increasing IR doses on the induction of genomic amplifications in control U2OS cells or two independent clones with deletion of DNA-PKcs by CRISPR/Cas9. ( left ) Western blots illustrating the efficiency of deletion of DNA-PKcs in U2OS cells. b , Histogram showing the percentage of U2OS cells with genomic amplifications 72 h following the exposure to 9 Gy of control, DMSO-treated U2OS cells or U2OS cells treated with the DNA-PKcs inhibitor NU7441 for 24 h prior to IR. c, Immunoblot showing deletion of effectors of the c-NHEJ pathway (LIG4, XRCC4, XLF). d , Histogram showing the impact of deletion of the indicated effectors of c-NHEJ on the induction of genomic amplifications by IR. e , The impact of inhibiting DNA LIG4 on the induction of genomic amplifications by IR in U2OS cells. f , g , Deletion of KTM4B (coding for SUV4-20H1) but not deletion of KTM4C (coding for SUV4-20H2) inhibits the recruitment of 53BP1 to DSBs. f , Representative immunofluorescent images of 53BP1 showing formation of 53BP1 foci following exposure of control (pX330) U2OS cells or U2OS cells deleted of KTM4B or KTM4C to 5 Gy analysed 1 h post-exposure. g , Quantitation of the number of 53BP1 foci per cell in control (pX330) cells or U2OS deleted of KTM4B or KTM4C (two independent clones each). The results represent the average of a minimum of 100 nuclei in each of three independent experiments for each condition ± S.D. ns: non-significant, *** p < 0.001. h , i , Histograms showing the percentage of cells undergoing genomic amplifications following the exposure of control or ATM-deleted U2OS cells (inset: immunoblotting of ATM levels) to 9 Gy ( h ), or following pharmacological inhibition of ATM in parental U2OS cells by KU55933 24 h prior to IR ( i ). Genomic amplifications was monitored 72 h post-IR. j , Histogram showing the percentage of control (pX330) U2OS cells or U2OS cells deleted of RNF8 or RNF168 and exposed to 9 Gy. Genomic amplifications was monitored 72 h post-IR. k , l , Histograms showing the percentage of control (pX330) U2OS cells or U2OS cells deleted of 53BP1 (in two independent clones; immunoblots shown in inset ( k )) or RIF1 (immunoblots shown in inset ( l )) with genomic amplifications following the exposure to 9 Gy. Genomic amplifications was monitored 72 h post-IR. Data in all the histograms represent the average of three independent experiments ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Article Snippet: Primary antibodies recognizing the following proteins were used: ATM (1:1000, ab78; Abcam), tubulin (1:1000, sc-53646; Santa Cruz), SET8 (1:1000, C18B7; Cell Signaling), mono-methyl Histone H4 (K20) (1:500, #9724; Cell Signaling), di-methyl Histone H4 (K20) (1:500, #9759; Cell Signaling), Tri-methyl Histone H4 (K20) (1:500, #5737; Cell Signaling), DNA-PKcs (1:1000, ab44815; Abcam), 53BP1 (1:5000, N100-304; Novus Biologicals) POLD3 (1:1000, H00010714-M01; Abnova), POLQ (1:1000, H00010721-M09; Abnova), RIF1 (1:1000, A300-569A-M; Bethyl Laboratories), RNF8 (1:1000, sc-2711462; Santa Cruz), RNF168 (1:1000, ABE367; Millipore), CtIP (1:1000, sc-271339, Santa Cruz), EXO1 (1:1000, ab95012; Abcam), XRCC4 (1:1000, sc-271087; Santa Cruz), XLF (1:1000, A300-730A-M; Bethyl Laboratories), NBS1 (1:1000, ab32074; Abcam), MDC1 (1:25,000, ab11171; Abcam), LIG1 (1:1000, 18051-1-AP; Proteintech), and LIG4 (1:1000, HPA001334; Sigma Aldrich).
Techniques: Control, Clone Assay, CRISPR, Western Blot, Quantitation Assay, Inhibition
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