Figure 7.

Silencing of L-GILZ decreases DEX anti-proliferative activity and the expression of p53, p21 and PUMA in MCF-7 cells. (a) Lysates from MCF-7 cells that were treated with DEX (10⁻⁷ M) for 24 h or left untreated were (b) immunoprecipitated with anti-p53 antibodies, and co-immunoprecipitated L-GILZ was detected by Western blot using anti-GILZ antibody. (c) Co-localization of endogenous L-GILZ and p53 proteins in MCF-7 cells that were treated with DEX (10⁻⁷M) for 24 h or left untreated was detected by in situ PLA. Images are representative of three experiments with consistent results. Graph shows RCP values normalized to untreated control (100%), representing the mean of three separate experiments. **DEX-treated versus control cells. (d) MCF-7 cells were transfected with L-GILZ (siL-GILZ) or scramble-L-GILZ (siCtrl) siRNAs 24 h before DEX treatment (10⁻⁷M) for 48 h. Cell cycle analysis according to PI staining was performed. A representative experiment is shown (DNA content is on x axis, and number of nuclei is on y axis). (e) L-GILZ, p53, p21 and PUMA mRNA levels were determined by real-time PCR in untransfected (Ctrl), siCtrl and siL-GILZ MCF-7 cells treated 24 h after silencing with DEX (10⁻⁷ M) for 3 h. Fold increase is relative to the respective untreated control. ‘***' indicates the direct comparison of L-GILZ versus scramble-L-GILZ siRNAs