Fig 2: 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 3: 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 4: Apoptosis induction by MEKi+HDACi is dependent on BIM and BMF de-repression. (a) TPM cancer cells were treated with selective HDAC inhibitors. LN229 and SNU475 cells were treated with mocetinostat (MOC, a HDAC 1/2/3 inhibitor, 1 μM), entinostat (ENT, a HDAC 1/3 inhibitor, 2 μM), LMK235 (LMK, a HDAC 4/5 inhibitor, 1 μM) and nexturastat (NEX, a HDAC 6 inhibitor, 2 μM) for 24 hours with or without MEK inhibition (TR). Figure depicts immunoblots for markers of apoptosis (CC3, BIM and BMF). pERK and H3K27Ac were used to validate the effects of TR and HDAC inhibitors respectively. (b), (c) To test if the apoptosis induced by MEK1/2 inhibition + HDAC inhibition is dependent on BIM and BMF, cells were treated with 50 pmol BIM or BMF siRNA with or without 25 nM TR + 2.5 μM VOR followed by analysis of cleaved caspase 3 (CC3). Non-targeting siRNA pool was used as control. Also depicted in these panels is the quantification of CC3 (after normalization with actin) from three independent experiments. GAPDH or β-Actin were used as loading controls for immunoblots.

Fig 5: SNAI2 suppresses apoptosis and regulates BIM and BMF proximal promoters.(a) SNAI2 RNA expression levels in TERT promoter mutants (TPM) cancer cell lines (n = 55) or wild type (WT) cancer cell lines (n = 207). Data are from Cancer Cell Line Encyclopedia (CCLE, Broad Institute). N = 55 TPM cell lines and 207 wild type cell lines. (b) Dox-inducible SNAI2 overexpressing cell lines were induced with 200 ng/ml doxycycline for 96 hours with or without 50 pmol siRNA targeting SNAI2 (siSNAI2) to test the induction of apoptosis (cleaved caspase 3 (CC3) and cleaved parp (CP)) by immunoblots upon SNAI2 repression. (c) A101D and U87 cells were co-treated with the pan caspase inhibitors Z-VAD-FMK, 50 μM for the 96 hours, with or without siRNA against SNAI2 and immunoblotted for apoptosis, DNA damage markers and SNAI2. (d) RT-PCR data showing rescue of BIM mRNA upon siRNA-based knockdown of SNAI2 in LN229, A101D and SNU475 cells. (e,f) To test if SNAI2 localizes to BIM and BMF promoters and affects their expression, chromatin immunoprecipitation (ChIP) was performed. LN229 cells were treated with 50 pmol SNAI2 siRNA (siSNAI2) or control non-targeting (NT) siRNA and cultured for four days. Sheared chromatin was precipitated with antibodies against SNAI2, H3K27me3, or H3K27Ac. Non-specific IgG was used as a negative control. Samples were analyzed by qRT-PCR using primers designed to amplify known/predicted SNAI2 binding sites in BIM and BMF promoter. ChIP samples were normalized to input chromatin. (g) U87 cells were treated with 50 pmol siRNA against SNAI2 and rescued with a SNAI2-overexpressing plasmid that was resistant to the siRNA. Immunoblots depict rescue of SNAI2 and changes in histone-3 acetylation at K9 (H3K9ac). β-Actin was used as loading control for immunoblots.