Figure 4. SQLE inhibition impaired the WIP1-ATM axis.

A and B, SQLE inhibition led to a decrease in IR-induced p-ATM (S1981) levels and phosphor-ATM-1981 foci (30 min post-2Gy IR) in MCF-7 and HCC-38 cells, as detected by WB and immunofluorescence staining. TF was given 24 h prior to irradiation. The percentage of cells with positive p-ATM (S1981) foci were presented. The cells with ≥10 foci were counted as positive. C, SQLE inhibition by TF decreased IR-induced p-ATM (S1981) levels at different time points after IR, as measured by immunofluorescence staining of MCF-7 cells. TF was given 24 h prior to irradiation. D, Representative immunofluorescence images of p-ATM (S1981) foci (magnification, 63× oil). E, ATM inhibition (10 μM) abrogated the TF-induced reduction in HR activity in MCF-7 and H1299 cells, as detected by the HR-DrGFP reporter assay. F-H, SQLE knockdown or pharmaceutical inhibition increased WIP1 protein expression in cells with or without wild-type. I and J, SQLE inhibition by SQLE knockdown or inhibitors did not affect WIP1 mRNA expression. K, Schematic representation of sucrose gradient preparation, polysome fractionation, and fraction analysis. L, Polysomes profiling. M, SQLE inhibition by TF increased polysome-associated WIP1 mRNA in MCF-7 cells. WIP1 mRNA was detected by qRT-PCR. The TF-treated MCF-7 cells showed significant increments in the WIP1 mRNA-associated polysomes. TF was given 24 h prior to irradiation (A-D). Data are presented as the mean ± SD in (B, C, E, I and J) and as the mean ± SEM in (M). All data in (I and J) are normalized with shCon or Con group. Statistical significance was determined by two-way (C) or one-way ANOVA (B, E, I and J) followed by Bonferroni’s post-hoc analysis for multiple comparisons. The Student’s t-test was used for the data in (M) for each group comparison. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.