Fig 2: ML-323 induces hepatocarcinoma cancer (HCC) cell apoptosis via the ATF4-Noxa axis.A Annexin V-FITC /PI double-staining showing apoptotic Noxa-deficient HCC cells treated with ML-323. HCCLM3 and SMMC-7721 cells were transfected with control siRNA or Noxa siRNA and then treated with ML-323 for 48 h. B Western blot analysis showing the efficiency of siNoxa and its effects on cleaved-PARP levels. HCCLM3 and SMMC-7721 cells were treated as shown in (A). C Western blot analysis of Noxa-related transcription factors. HCCLM3 and SMMC-7721 cells were treated with DMSO or ML-323, and cell lysates were analyzed by western blotting using specific antibodies. D Annexin V-FITC /PI double-staining of ATF4-deficient HCC cells treated with ML-323. After transfection with control or ATF4 siRNA, HCCLM3 and SMMC-7721 cells were treated with ML-323 for 48 h. E Western blot analysis of the knockdown efficiency of siATF4 and its effect on the expression of Noxa and cleaved-PARP. F Western blot analysis of ubiquitinated proteins and ER stress-related proteins. HCCLM3 and SMMC-7721 cells were treated with ML-323. All the data represent at least three independent experiments (n = 3; error bars, SD). All data represented at least three independent experiments (n = 3; Error bars, SD).

Fig 3: Effect of exogenous H2O2 on LRRC8A mRNA accumulation, LRRC8A protein expression, swelling induced taurine release, and intracellular signaling associated with apoptosis and cell growth. Taurine release, mRNA accumulation and protein expression in A549WT were measured by tracer technique, qRT-PCR and SDS-PAGE/western blotting, respectively. (A) Fractional rate constant (min-1) for [3H]taurine release was determined in A549WT cells under isotonic conditions (321 mOsm) and following reduction in the osmolarity to 200 mOsm (shift in tonicity indicated by the arrow) in the absence (control, open circles) or presence of 100 µmol L-1 H2O2 (acute exposure, closed squares). Alternatively, cells were preexposed to isotonic growth medium containing 100 µmol L-1 H2O2 for 24 h prior to the release experiment (closed circles). Rate constants at each time point represent mean values from 7 (Control), 4 (preexposed) and 4 (acute exposure) sets of experiments. *indicates that maximal rate constant, obtained 6 min after hypotonic exposure, was significantly different from control cells (P < 0.05, paired Student's t-Test). (B) A547WT cells were preexposed to isotonic growth medium containing 100 µmol L-1 H2O2 for 6 or 24 h prior to taurine release experiments for determination of maximal rate constants (min-1), inactivation of the VRAC complex activity and LRRC8A protein expression. Values ± SEM for maximal rate constant, inactivation of taurine release pathway (4 sets with 6 and 24 h exposure) and protein expression (4/10 sets for 6/24 h exposure, respectively) are given relative to control cells not exposed to H2O2. Insert: Representative western blots for LRRC8A and GAPDH (Lane 1: Control cells; Lanes 2 and 3: Cells exposed to 25 µmol L-1 H2O2 for 6 and 24 h, respectively; Lanes 4 and 5: Cells exposed to 100 µmol L-1 H2O2 for 6 and 24 h, respectively). *indicates that values were significantly different from the control cells (P < 0.05, paired Student's t-Test). (C) LRRC8A and LRRC8D mRNA accumulation was determined in control A549WT cells and cells exposed for 24 h to 100 µmol L-1 H2O2. Values, calculated relative to untreated control cells, are given as means ± SEM of 4 sets of experiments. (D) A549WT cells were incubated in growth medium containing 100 µmol L-1 H2O2 for 24 h before proteins were extracted and tested for expression of phosphorylated p53 (P-p53), NOXA, p21, and GAPDH (house holding protein). Protein expression was normalized to GAPDH (9, 5, and 8 sets for P-p53, NOXA and p21, respectively), and presented as means ± SEM of values relative to non-treated control cells. Insert: Representative western blots for P-p53, p21, NOXA and GAPDH for (Lane 1: Control cells; Lanes 2 and 3: Cells exposed to 25 µmol L-1 H2O2 for 6 and 24 h, respectively; Lanes 4 and 5: Cells exposed to 100 µmol L-1 H2O2 for 6 and 24 h, respectively). *indicates that the protein expression of apoptotic markers was significantly different from control cells (P < 0.05, paired Student's t-Test). (E) A549WT cells were incubated in growth medium containing 100 µmol L-1 H2O2 for 24 h. Proteins were extracted at the time points indicated and tested for expression of total and phosphorylated Akt / FoxO3A. Protein expression ratios between phosphorylated and total proteins were calculated in 4 sets of experiments and data presented as mean values ± SEM of the ratios relative to control cells. Insert: Representative western blots for FosO3A, Akt, P-FosO3A, and P-Akt (Lane 1: Control cells; Lanes 2 to 6: Cells exposed to 100 µmol L-1 H2O2 for ½, 1, 3, 6, and 24 h, respectively). *indicates that the protein expression was significantly different from control cells (P < 0.05, paired Student's t-Test).

Fig 4: Combination therapy activates the integrative stress response and disrupts the NOXA.MCL-1.BIM axis, making stressed cells more prone to selection for apoptosis.Western blots in CHLA06, BT37, and CHLA05 cell models of AT/RT 24 h after DMSO control treatment and increasing fixed dose ratios of combination TAK-228 and Obatoclax treatment. Numbers above phospho-EI2Fa (Ser51) represent quantification of expression normalized to EI2Fa. Numbers above other blots represent quantification of protein expression normalized to ACTIN.

Fig 5: Delanzomib enhances Dox-induced apoptosis via p53 stabilization, p21, p27, PUMA, and NOXA upregulation, and p38/JNK activation in breast cancer cells. Four breast cancer cell lines (MCF-7, MDA-MB-361, MDA-MB-231, and HCC-1954) which represent the four major molecular subtypes of breast cancer, were cultured with Dox (0.05 µM) alone or in combination with delanzomib (0.1 µM) for 6, 8, or 12 hours. Untreated cells were used as controls. Whole-cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with indicated antibodies. a-tublin was used as a loading control.