Fig 2: (A) Increased expression of anti-apoptotic Mcl-1 after IRI in SOCS3-CKO mice.Total cell lysates were prepared from the left ventricles of wild-type (WT) or SOCS3-CKO mice pre-ischemia or 6 h after reperfusion, and western blots were probed with antibodies raised against Mcl-1, Bcl-xL, Bcl-2, Bad, and Bax. The graphs represent quantitative differences in expression among Mcl-1, Bcl-xL, Bcl-2, Bad, and Bax (n = 5 per group). *P < 0.05 vs. WT IRI. AU, arbitrary units; IRI, ischemia reperfusion injury. (B) Real-time PCR analysis of Mcl1 and Bcl2 mRNA expression in the hearts pre-ischemia and 3 h after reperfusion (n = 5 for each group). *P < 0.05 vs. WT after reperfusion.

Fig 3: Mcl-1 deficiency enhanced DEN-induced liver tumor development. At the age of 2 weeks, male Mcl-1fl/fl and Mcl-1Δhep mice were intraperitonially injected with PBS or DEN (20 mg/kg BW), and their livers were harvested after 4 or 6 months. (A,B) Representative images of the livers of PBS- or DEN-treated male Mcl-1 fl/fl and Mcl-1Δhep mice at the ages of 4 months (A) and 6 months (B). (C,D) Incidence rate of macroscopic (C) or microscopic (D) tumors at the age of 4 and 6 months (n ≥ 8 per group, chi-squared test). (E) Representative H&E staining images of liver sections from the DEN-treated male 6-month-old Mcl-1fl/fl and 6-month-old Mcl-1Δhep mice. The scale bar is 100 µm. (F) Ratio of liver weight to body weight (mean ± SEM, Mann–Whitney test). (G) Maximum tumor size (diameters by caliper) at the age of 6 months (mean ± SEM, Mann–Whitney test). (H) Mutation rate of B-Raf V637E in liver tumors (chi-squared test, *p < 0.05).

Fig 4: Hyperosmotic stress drives BCL-2 addiction of cancer cells. a–c HCT116 cells were challenged with KillerTRAIL (32 ng/ml) for 9 h in the presence and absence of NaCl (75 mM). After washing and lysis, western blot analyses were performed with antibodies specific for the indicated proteins. Detection of tubulin served as a loading control. The dashed line in c indicates that different cell lysates were used for BCL-W detection. The asterisk (*) in the MCL-1 blot indicates a defect in the CCD sensor of the western blot imaging system. All samples were run on the same gel, no gels were sliced. Data shown for a–c are representative of at least two experiments performed. d–g HCT116 and HCT116 BID knockout cells were challenged with the indicated concentrations of d ABT-737 (targeting BCL-2, BCL-xL, and BCL-W), e A1210477 (targeting MCL-1), f ABT-199 (targeting BCL-2), and g WEHI-539 (targeting BCL-xL) in the presence and absence of the indicated concentrations of NaCl. h HCT116 cells were challenged with the indicated concentrations of KillerTRAIL in the presence and absence of the BCL-xL inhibitor WEHI-539. For d–h, data points and mean ± SEM from three independent experiments are shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, *p < 0.0001

Fig 5: Diagram depicting the proposed mechanism of action of sorafenib in mesothelioma TICs. Sorafenib treatment causes the inhibition of MEK and ERK1/2 phosphorylation and the downregulation of Mcl-1 expression, leading to cell cycle arrest and activation of the apoptotic program. Sorafenib antitumor effects are mainly ascribed to a direct inhibition of FGFR1 activity rather than downstream effectors, such as Raf/Ras/MEK/ERK pathway. Thus TIC cultures autocrinally activating FGFR1 via the release of high amount of bFGF are likely to be more sensitive to the drug. bFGF basic fibroblast growth factor, FGFR FGF receptor, TIC tumor-initiating cell