Figure 7. Changes in the Cell Proliferation of Tzb-refractory Cells Upon siRNA-induced Knock Down of the Autophagosome Marker LC3.

Tzb-refractory TzbR POOLs were mock transfected, transfected with a non-specific control siRNA Pool (negative control) or transfected with siRNA-targeting LC3. 72 h after transfection, one set of mock-, non-specific negative control-, and RNAi LC3-transfected cells were used for immunoblotting analyses of LC3 expression. A second set of cells were harvested, re-cultured in 24-well plates at a density of 10,000 cells/well and treated with regular medium in the absence or presence of Tzb (1 µg/mL Tzb in SKBR3 parental cells and 10 µg/mL Tzb in TzbR POOLs). There were no significant changes in cell numbers when TzbR cells were treated with Tzb as single agent (omitted). siRNA-induced blockade of LC3 expression significantly reduces cell proliferation rates in TzbR POOLs. More importantly, supra-additive growth inhibitory interactions occur in LC3-depleted Tzb-treated TzbR POOLs. The data presented are mean of number cells ×10⁵/well (circles) and 95% confidence intervals (bars) of three independent experiments made in duplicate after 3, 6 and 9 days. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions (i.e. LC3 siRNA±Tzb) and control cells (i.e. siRNA [-]) are shown. All statistical tests were two-sided.