Fig. 3: Autophagy inhibition increases TMZ-induced apoptosis in C2C12 and RH30 cells.

a, b C2C12 and RH30 cells were treated with Baf-A1 (2, 4, 6, 8, and 10 nM) and cell viability was assessed after 72 h using MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.0001). c C2C12 and RH30 cells were treated with Baf-A1 (4 and 6 nM, 48, 72 h) and autophagy hallmark proteins were detected in both C2C12 and RH30 cell lines using immunoblotting. Baf-A1 induces accumulation of lipidated LC3β and decreases the degradation of p62 in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. d, e C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (4, 6 nM, 72 h) to evaluate autophagy inhibition in the presence of Baf-A1 treatment. TMZ + Baf-A1 treatment induces LC3β lipidation and reduces the degradation of p62 in C2C12 and RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f–h Representative figures of the flow cytometry histogram for RH30 are shown. Cells were treated with TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ. Percentage of sub-G1 abundance induced by TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ after 72 h has been showed. The Sub-G1 population showed an abundance of apoptotic cell death in each treatment. g, h C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and Baf-A1 (4 nM, 72 h) and then cell lysates were collected to examine the effect of TMZ, Baf-A1, and TMZ/Baf-A treatment on expression of Bcl2 family proteins (Bcl-2, Bcl-XL, Mcl-1, and Bax). k–r, k, l TMZ does not significantly change Mcl-1 expression in both C2C12 and RH30 cells. It is notable that Baf-A1 (6 nM)/TMZ co-treatment significantly (P < 0.01) decrease Mcl-1 expression in C2C12 cells while does not have any effect in RH30 cells. m, n TMZ does not significantly change Bcl-2 expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not significantly change Bcl-XL expression in both C2C12 and RH30 cells. o, p TMZ does not significantly change Bcl-XL expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not significantly change Bcl-2 expression in both C2C12 and RH30 cells. q, r TMZ does not significantly change Bax expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment significantly increase Bax-expression in C2C12 cells (P < 0.01) while does not have a significant effect on its expression in RH30 cells. s–u We have evaluated changes in mitochondrial membrane potential in the presence of TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination in C2C12 and RH30 cells. s Representative images show the mitochondrial membrane potential measured by TMRM. Red color denotes TMRM staining. t, u Measurement of the mean of TMRM fluorescence intensity shows that TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination does not change mitochondrial membrane potential in C2C12 t and RH30 u cells. The data are representative of the mean fluorescence in at least 100 cells in each cell type. The data were analyzed by Student’s t-test or ANOVA, followed by post hoc analysis. If p < 0.05, results were considered statistically significant