Skip to main content
. 2019 Nov 25;11(12):1858. doi: 10.3390/cancers11121858

Figure 2.

Figure 2

Figure 2

FoxO3a restores the sensitivity of tamoxifen resistant BCCs to tamoxifen. TamR/TetOn-V (A) and TamR/TetOn-AAA (C) cells were serum-starved for 24 h and then switched to 5% PRF-CT plus 1 µM 4-OHT and treated or not up to 72 h with Dox (3 µg/mL). MCF-7 cells were transfected with a Scramble siRNA (siScramble) (E) as control, or a siRNA for FoxO3a (siFoxO3a) (F). After 6 h, cells were serum-starved for 16 h and then shifted to 5% PRF-CT +/−4-OHT (1 µM) up to 72 h. 4-OHT treatment was renewed every day. Cells were then harvested by trypsinization and counted using trypan blue dye exclusion assay. Data represent the mean ± SD of three independent experiments. * p < 0.05 vs. relative untreated cells. The error bars indicate SD. Duplicate experiments were subjected to WB analysis to assess FoxO3a expression in Dox treated TamR/TetOn-V and TamR/TetOn-AAA cells (B,D), and in MCF-7 FoxO3a silenced cells (G). (H) TamR/TetOn-V and TamR/TetOn-AAA cells were treated as above described for 72 h and (I) siFoxO3a and siScramble MCF-7 cells, treated as above-described for 72 h were subjected to cell cycle analysis (see Materials and Methods). The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle are reported. The results are expressed as the mean from three independent experiments (Figure S5). A duplicate set of cells was subjected to WB to assess FoxO3a induction in Dox treated TamR/TetOn-AAA cells (J) and in MCF-7 FoxO3a silenced cells (K). TamR/TetOn-V and TamR/TetOn-AAA cells treated as before for 72 h and processed for TEM analysis (L) and TUNEL assay (M). siFoxO3a and siScramble MCF-7 cells were treated as before and subjected to TEM (O), and TUNEL analyses (Q). In TEM micrographs, scale bars: 10 µm. Original magnification: ×1200. In TUNEL assay, DAPI was used to counterstain the nuclei. Apoptotic cells were photographed at 10x magnification and then counted using Image J software. The graph on the right represents the corresponding apoptotic index (% apoptotic cells/total cell number in the field). Duplicate experiments were subjected to WB analysis to assess the expression of indicated proteins in Dox treated TamR/TetOn-V and TamR/TetOn-AAA cells (N) and in MCF-7 FoxO3a silenced cells (P). In WBsβ, Actin and GAPDH were used as loading controls.