Journal: Oncogene

Article Title: Chk1 deficiency in the mouse small intestine results in p53-independent crypt death and subsequent intestinal compensation

doi: 10.1038/onc.2008.482

Figure Lengend Snippet: (A-D) IHC analysis of intestinal tissue sections from wild-type ( AhCre + Chk1 +/+ ) and experimental ( AhCre + Chk1 F/F ) mice with antibodies detecting phosphorylated-H2A.X (A), phosphorylated-ATM (B), p53 (C) and p21 (D). Each row of panels represents analysis using a single antibody on a wild-type mouse (left), and an AhCre + Chk1 F/F mouse at day 1 (middle) and day 2 PI (right). Bar , 25μm.

Article Snippet: The following antibodies were used for immunhistochemistry: anti-ATM (1:300; Rockland), anti-Caspase 3 (1:750; R&D systems), mouse anti-p53 (1:50; Labvision), mouse anti-H2A.X (Ser139) (1:200, Upstate), mouse anti-BrdU (1:100; Becton Dickinson), rabbit anti-p21 (1:500, Santa Cruz).

Techniques:

Journal: Oncogene

Article Title: Chk1 deficiency in the mouse small intestine results in p53-independent crypt death and subsequent intestinal compensation

doi: 10.1038/onc.2008.482

Figure Lengend Snippet: (A-D) IHC analysis of intestinal tissue sections from wild-type ( AhCre + Chk1 +/+ ) and experimental ( AhCre + Chk1 F/F ) mice with antibodies detecting phosphorylated-H2A.X (A), phosphorylated-ATM (B), p53 (C) and p21 (D). Each row of panels represents analysis using a single antibody on a wild-type mouse (left), and an AhCre + Chk1 F/F mouse at day 1 (middle) and day 2 PI (right). Bar , 25μm.

Article Snippet: The following antibodies were used for immunhistochemistry: anti-ATM (1:300; Rockland), anti-Caspase 3 (1:750; R&D systems), mouse anti-p53 (1:50; Labvision), mouse anti-H2A.X (Ser139) (1:200, Upstate), mouse anti-BrdU (1:100; Becton Dickinson), rabbit anti-p21 (1:500, Santa Cruz).

Techniques:

Journal: Nature

Article Title: RHOJ controls EMT-associated resistance to chemotherapy

doi: 10.1038/s41586-023-05838-7

Figure Lengend Snippet: a , Western blot analysis of phosphorylation of ATM/ATR substrates in EPCAM + , EPCAM − and Rhoj -KO EPCAM − cells untreated and treated with cisplatin/5FU for 12 h and 24 h. n = 3. b , c , Western blot analysis ( b ; n = 3) and FACS analysis quantification ( c ; n = 5) of the percentage of cells expressing γ-H2AX in EPCAM + , EPCAM − and EPCAM − Rhoj -KO cells, 12 h and 24 h after cisplatin/5FU administration. β-Actin was the loading control. n values represent the number of independent primary cultured cell lines. d , e , Representative cell cycle FACS profile ( d ) and quantification ( e ) of the percentage of cells in G0/G1, S and G2/M phase 12 h and 24 h after cisplatin/5FU treatment of EPCAM + , EPCAM − and EPCAM − Rhoj -KO BrdU-7AAD-labelled cells. n values indicate the number of independent experiments. f , Representative stretched DNA fibres labelled with CldU and IdU from EPCAM + , EPCAM − and EPCAM − Rhoj -KO cells untreated and treated with cisplatin/5FU for 12 h. n = 6. Scale bar, 10 μm. g , Fork rate (FR) values in EPCAM + , EPCAM − and EPCAM − Rhoj -KO cells untreated and treated with cisplatin/5FU for 12 h. Data are median. Statistical analysis was performed using ANOVA, with condition, experience and their interaction; the P value was adjusted using the two-way post hoc Sidak test. n values represent the number of forks pooled from six independent experiments. h , The percentage of origin of firing in EPCAM + , EPCAM − and EPCAM − Rhoj -KO cells untreated and treated with cisplatin/5FU for 12 h. From left to right, total number of DNA fibres scored: n = 3,020, 3,123, 3,065, 3,091, 3,078 and 3,069, in six independent experiments. For c , e and h , statistical analysis was performed using Kruskal–Wallis tests followed by two-sided Mann–Whitney U -tests; P values were adjusted for multiple comparisons using Bonferroni correction. For the box plots, the centre line shows the median, the box limits represent the 25th and 75th percentiles, and the whiskers show the minimum and maximum values.

Article Snippet: For immunostaining, the following primary antibodies were used: goat GFP (Abcam, ab6673, 1:500), chicken K14 (Thermo Fisher Scientific, MA5-11599, 1:2,000), rabbit vimentin (Abcam, ab92547, 1:500), rabbit active caspase-3 (R&D, AF835, 1:600), rabbit 53bp1 (Novus, NB100-304, 1:200), mouse phospho-histone H2A.X (Ser139) (Millipore, 05-636, 1:500), mouse RAD51 (Santa-Cruz Biotechnology, sc-398587, 1:50), rat RPA32/RPA2 (Cell Signaling, 2208, 1:500), Rhodamine Phalloidin (Thermo Fisher Scientific, Arg415, 1:400), rabbit HA (Abcam, ab9110, 1:1,000), rat phospho-histone H3 (S28) (Abcam, ab10543, 1:2,000), rabbit Ki-67 (Abcam, ab15580, 1:400).

Techniques: Western Blot, Expressing, Cell Culture, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

doi: 10.1093/nar/gku217

Figure Lengend Snippet: Spironolactone treatment leads to persistent DNA damage. ( a ) U2OS cells were treated with Spiro (40 μM) or DMSO for 24 h followed by treatment of Phleomycin for 1 h and harvested after 0, 2, 6 or 24 h after this treatment. Cells were lyzed in RIPA buffer and analyzed by western blot against the proteins RPA, H2AX and their phosphorylated versions (S4/8 for RPA and S139 for H2AX). Tubulin was used as a loading control. ( b ) Left: U2OS cells were treated with Spiro or DMSO for 1 h, Phleomycin-treated for 1 h and left for recover for 6 h in the presence of Spiro or DMSO. Next, the cells were fixed and co-stained for 53BP1, γH2AX and DAPI. On the right, quantification of foci formation by γH2AX (top) and by 53BP1 (bottom) from two different immunofluorescence experiments. Cells were divided into subgroups according to the amount of foci (0–5, 6–20 or >20) per cell. Statistical significance was calculated using the one tailed heteroscedastic t-test (* p < 0.05).

Article Snippet: Antibodies used are rabbit anti-53BP1 (Bethyl Laboratories), mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck Millipore), rabbit anti-RAD51 PC130 (Calbiochem), rabbit anti-BRCA1 sc642 (Santa Cruz), mouse anti-CtIP clone 14–1 (Active Motif) and mouse anti-RPA32 NB600–565 (Novus).

Techniques: Western Blot, Staining, Immunofluorescence, One-tailed Test