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. Author manuscript; available in PMC: 2018 Jul 18.
Published in final edited form as: Oncogene. 2018 Jan 18;37(25):3384–3398. doi: 10.1038/s41388-017-0110-2

Figure 5. PL/APR-246 treatment promotes DNA damage.

Figure 5

(a) 8-oxo-dG in UMSCC10A cells treated with PL and/or APR-246. UMSCC10A cells growing on fibronectin-coated coverslips were treated with 10 μM PL and/or 25 μM APR-246 in serum-free and phenol-red-free medium for 16 h at 37°C, fixed in absolute methanol (- 20°C, 20 min) and permeabilized with 0.1% Triton X-100 at room temperature for 15 min. After blocking, the cells were stained with FITC-conjugated avidin for 1 h at 37°C. Fluorescence is captured with an Olympus IX51 fluorescence microscope. (b, c)Comet assay showing elevated DNA damage in cells treated with PL and APR-246. UMSCC10A cells were treated with 10 μM PL and/or 25μM APR-246 for 16 h. The cells were then trypsinized and washed with PBS. Two thousand cells were mixed with 100 μl low melting agarose for alkaline comet assay. Cells in the gel were stained and visualized with epifluorescence microscopy (b). (c) Percentage of DNAs in the tail (damaged DNA) was calculated. *P < 0.01, n = 3. (d, e) Expression of γ-H2AX in PL/APR-246-treated cells. UMSCC10A cells were treated with 10 μM PL and/or 25 μM APR-246 for 16 h. After the treatment, the cells were collected for the immunofluorescent analysis of γ-H2AX foci formation (d) or immunoblot analysis of γ-H2AX and H2AX expression (e).