Figure 9

Figure 9

(A,B) Representative confocal images depicting PIP3 (red) and PI3K (green) in MDA-MB-231 cells cultured under control conditions and after 96 h of ZFEs treatment. In control cells, PI3K was localized in the cytosol, positioned just behind the cell membrane, and exhibited co-localization with PIP3, suggesting the strong enzymatic activity of PI3K (A). In contrast, ZFEs treatment resulted in the perinuclear localization of PI3K, with the PIP3 signal nearly disappearing (B). Scale bars: 20 μm (C,D) Representative confocal images of PIP2 (red) and Cofilin (green) in MDA-MB-231 cells under control conditions and following 96 h of ZFEs treatment. Control cells displayed a dense distribution of Cofilin beneath the cortical ring, aligning with the F-actin pattern (C). After ZFEs treatment, Cofilin became dispersed throughout the cytosol of MDA-MB-231 cells. Additionally, an increased accumulation of PIP2 at the cell membrane, co-localizing with Cofilin, occurred (D). Scale bars: 10 μm. (E–H) Fluorescence intensity analysis from nucleus and cytoplasm of PI3K, PIP2, PIP3, and Cofilin in MDA-MB-231 cells. Fluorescence intensity was evaluated with ImageJ Software. A significant reduction in PI3K cytoplasm signal intensity was observed following treatment with ZFEs. In treated cells, PIP2 cytoplasm signal intensity increased, while the availability of Cofilin markedly decreased, both in the cytoplasm and in the nucleus. Moreover, PIP3 signal markedly decreased in the cytoplasm of treated cells. Data are presented as mean ± SD. Statistical analysis was performed with an unpaired two-tailed Student’s t-test. * p < 0.05; ** p < 0.01. (I,J) Western blot assay of PI3K (I) and PTEN (J) protein expression in MDA-MB-231 cells treated with 0.3 µg/mL ZFEs for 48 and 96 h. The analysis corroborated the data obtained from real-time PCR, revealing the decreased protein expression of PI3K in MDA-MB-231 cells after 96 h of ZFEs treatment. Analysis of PTEN expression showed no statistically significant difference following ZFEs treatment. Each sample was normalized against the relative expression of GAPDH. Data are presented as mean ± SD. * p < 0.05; *** p < 0.001.