Figure 4 3-MA inhibited autophagy and

enhanced apoptosis induced by paclitaxel treatment in FLCN-deficient cells. A. Cells were pretreated with 5 mM 3-MA for 3 hours and subsequently treated with 100 nM paclitaxel or a control vehicle for 24 hours with or without bafilomycin A1 treatment. LC3 proteins were dramatically decreased after autophagy inhibitor 3-MA. B. Cells were treated with 3-MA and different concentrations of paclitaxel, MTT assay showed that cell viability was more significantly reduced in FLCN-deficient cells compared to 3-MA untreated cells (*: p < 0.05. UOK257 + Paclitaxel vs UOK257 + Paclitaxel + 3-MA; ACHN 5968 + Paclitaxel vs ACHN 5968 + Paclitaxel + 3-MA; n = 15). C. TUNEL assay showed that more Elafibranor apoptotic cells were detected among FLCN-deficient cells treated with 3-MA and paclitaxel selleck kinase inhibitor (*: p < 0.05. UOK257: Paclitaxel

vs UOK257+ 3-MA; ACHN 5968: Paclitaxel vs Paclitaxel + 3-MA; n = 15). Beclin 1 knockdown inhibited autophagy and sensitized FLCN-deficient cells to paclitaxel To further confirm the role of autophagy on cell death, we knocked down another autophagy marker, Beclin 1, in all four cell lines by the siRNA method. UOK257, UOK257-2, ACHN-sc, and ACHN-5968 cells were transfected with Beclin 1 siRNA or a negative control siRNA, respectively. We then AL3818 examined the effects of Beclin 1 knockdown on paclitaxel-mediated apoptosis and cell viability in these cells. Compared to the treatment with negative control siRNA, Beclin 1 siRNA remarkably abrogated the paclitaxel-induced LC3-II expression in FLCN-deficient UOK257 and ACHN-5968 cells regardless of bafilomycin A1treatment (Figure 5A). The knockdown of Beclin 1 led to a significant increase of apoptosis and inhibition of cell viability in FLCN-deficient cells, which was consistent with the results obtained through 3-MA treatment (Figure 5B, Figure 5C). These data indicated that autophagy provided

protection and survival advantage to FLCN-deficient cells against cell apoptosis and cell death induced by paclitaxel. Inhibition of autophagy could increase the paclitaxel-induced cytotoxicity to these cells PIK3C2G and might improve the efficacy of paclitaxel against these cancer cells. Figure 5 Beclin 1 knockdown inhibited autophagy and sensitized FLCN-deficient cells to paclitaxel. A. Cells were transfected with Beclin 1 siRNA or a random siRNA control for 24 hours and subsequently treated with 100 nM paclitaxel for 24 hours with or without bafilomycin A1 treatment, LC3 protein levels were detected using Western blot. Less LC3 proteins were detected in Beclin 1 siRNA treated cells. B. FLCN-deficient cells transfected with Beclin 1 siRNA or a random siRNA control were treated with different concentrations of paclitaxel. MTT assay showed that cell viability was obviously decreased after Beclin 1 siRNA treatment (*: p < 0.05.