Journal: Genes & Development

Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression

doi: 10.1101/gad.351985.124

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Rabbit polyclonal TFIID–TBP (N12) , Cell Signaling Technology , 44059 , AB_2799258.

Techniques: Software, Transduction, Virus, Subcloning, Bacteria, Recombinant, Bradford Assay, Protease Inhibitor, RNA Extraction, Reporter Assay, Bicinchoninic Acid Protein Assay, Quantitative RT-PCR, Negative Control

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

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 , ATM suppresses the induction of genomic amplifications by DSBs. The histogram shows the percentage of A si SI-ER-U2OS cells with genomic amplifications as determined by PI-FACS 72 h following treatment with 4-OHT. Where indicated, the cells were pre-treated with the ATM specific inhibitor KU3355 5 or 24 h prior to treatment with 4-OHT. The results represent the average of three independent experiments ± S.D. *** p < 0.001. b , ATM inhibition does not stimulate rereplication induction by MLN4924. The histogram shows the percentage of control (pX330) U2OS cells or U2OS cells with ATM deletion (1ATM) with rereplication as determined by PI-FACS 48 h following treatment with 0.1 μM MLN4924. The results represent the average of three independent experiments ± S.D. ns: non-significant. c , d , Deletion of RNF8, RNF168, or RIF1 suppresses 53BP1 recruitment to DSBs. c, Representative immunofluorescence images of 53BP1 showing formation of 53BP1 foci following exposure of control (pX330) U2OS cells or U2OS cells deleted of RNF8, RNF168, or RIF1 to 5 Gy and analysed 1 h post-exposure. d , Quantitation of the number of 53BP1 foci per cell in control (pX330) cells or U2OS deleted of RNF8, RNF168, or RIF1. The results represent the average of a minimum of 100 nuclei in each of three independent experiments for each condition ± S.D. ** p < 0.01, *** p < 0.001. g , MDC1 and RNF168 suppress IR-induced genomic amplifications. Histograms showing the percentage of control (pX330) 293T cells or 293T cells deleted of MDC1 or RNF168 with genomic amplifications as determined by PI-FACS 48 h following the exposure to 5 Gy. The results represent the average of three independent experiments ± S.D. ** 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: Inhibition, Control, Immunofluorescence, Quantitation Assay