Fig 2: The BQZC-activated AMPK–mTOR–ULK1/BMF pathway is pivotal in BQZC-induced autophagy-dependent apoptosis in lung cancer cells. (A) The A549 cells were treated with BQZC, both in the presence and absence of Rap, for 24 h. Subsequently, the expression levels of Bax, BMF, and LC3B-II/I were assessed utilizing Western blot analysis. (B) The A549 cells were treated with BQZC, both in the presence and absence of ComC, for 24 h. Subsequently, the expression levels of Bax, BMF, and LC3B-II/I and the protein expression levels of AMPK–mTOR–ULK1 phosphorylation were assessed utilizing Western blot analysis. β-actin served as a loading control. (C) The impact of Rap and ComC on BQZC-induced apoptosis is shown, and cell apoptosis rates were measured using flow cytometry. (D) The expression of BMF, p-AMPK, p-mTOR, and p-ULK1 in tumor tissues was detected using Western blotting. β-actin was used as a loading control. All data are presented as mean ± SD, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. BQZC treatment.

Fig 3: A schematic of BQZC-induced apoptosis and autophagy in lung cancer cells. BQZC induced apoptosis in A549 cells by activating the intrinsic pathway mediated through mTOR–BMF–Bax signaling cascades, simultaneously inducing autophagy via AMPK–ULK1 signaling. Arrows denote activation, whereas bars signify inhibition.

Fig 4: AKT1 inhibition augments RocA-induced apoptosis by inducing BMF expression. (A) A375 cells were treated with RocA (50, 100, and 200 nM) and MK-2206 (1.25, 2.5, and 5 μM) for 48 h, or transfected with eIF4E siRNA for 16 h, followed by treatment with RocA (50, 100, and 200 nM) for 48 h. Cell apoptosis was evaluated using the TUNEL assay. ns, not significant, *p < 0.05. (B–D). A375 cells were transfected with c-Myc siRNA, c-Jun siRNA, or BMF siRNA for 16 h, followed by co-treatment with RocA (50, 100, and 200 nM) and/or MK-2206 (1.25, 2.5, and 5 μM) for 48 h. Protein levels of c-Myc (B), c-Jun (C), and BMF (D) were analyzed by western blotting. (E) Protein band intensities were quantified using Image J software, and the relative expression ratios of c-MYC/β-actin, c-BMF/β-actin, and c-Fos/β-actin were calculated. Data are presented as the mean ± SEM of two independent experiments. ns, not significant, *p < 0.05, ***p < 0.001. (F) A375 cells were transfected with EIF4E siRNA for 16 h and then co-treated with RocA (50, 100, and 200 nM) for 48 h. eIF4E expression levels were analyzed by western blotting

Fig 5: RocA promotes the reactivation of AKT1, leading to AKT1-driven downregulation of c-Myc, c-Jun, and the pro-apoptotic BH3-only protein BMF. A375 cells were treated with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h. (A). Alternatively, cells were co-treated with 100 nM RocA and 2.5 μM MK-2206, (B) U0126, (C) or eIF4E siRNA (D) for the same time. The protein levels of AKT1 and its downstream targets were analyzed by western blotting