alkaline comet assay kit  (Beyotime)

Journal: MedComm

Article Title: The SUMOylated RREB1 interacts with KDM1A to induce 5‐fluorouracil resistance via upregulating thymidylate synthase and activating DNA damage response pathway in colorectal cancer

doi: 10.1002/mco2.70105

Figure Lengend Snippet: Ras‐responsive element binding protein 1 (RREB1) activates Chk1‐mediated DNA damage response (DDR) to promote the 5‐fluorouracil (5‐FU) resistance in colorectal cancer (CRC). (A) Immunofluorescence (IF) assay to evaluate the effect of RREB1 and RREB1 3KR on the recruitment of 53BP1 in HCT116 cells and SW480 cells under treatment of 5‐FU for 24 h. Scale bars: 10 µm. (B–D) RREB1 overexpression or knockdown in HCT116 cells were treated with 50 µM 5‐FU for 24 h. Then, the expression of p‐Chk1 and γH2AX were also detected by western blot. (E) RREB1 accelerates the release of Chk1 from chromatin under 5‐FU treatment. The cytosol and chromatin were isolated using a kit from Beyotime. And the level of Chk1 in cytosol and in chromatin was measured by western blot. (F, G) We treated RREB1‐ or RREB1 3KR ‐overexpressed cells in HCT116 and SW480 with or without 10 µM MK‐8776, a Chk1 inhibitor, in combination with 50 µM 5‐FU for 24 h. Then the level of γH2AX was detected by western blot (F) or IF assay (G). Scale bars: 10 µm. (H) Comet assay was applied to evaluate the effect of Chk1 inhibitor MK‐8776 on 5‐FU‐induced DNA damage. Fifty µM 5‐FU in combination with or without 10 µM MK‐8776 were treated for 24 h. The tail DNA was measured at least in 10 cells using CASP software. Scale bars: 100 µm. (I) Colony formation assay under 1 µM 5‐FU treatment or under 1 µM 5‐FU plus 1 µM MK‐8776 for 14 days in HCT116 cells with overexpressing Flag‐RREB1 or Flag‐RREB1 3KR . * p < 0.05, ** p < 0.01, **** p < 0.0001, ns, not significant.

Article Snippet: DNA damage caused by 5‐FU in CRC cells was measured with an alkaline comet assay kit (4250‐050‐K, Trevigen).

Techniques: Binding Assay, Immunofluorescence, Over Expression, Knockdown, Expressing, Western Blot, Isolation, Single Cell Gel Electrophoresis, Software, Colony Assay