Fig 2: M. tb virulent and avirulent strains increase the gene expression of the anti-apoptotic BCL-2 and decrease the pro-apoptotic BAX. Human MDMs were obtained after seven days in culture. 2 × 106 MDMs were infected at MOI 1 and MOI 10 with an avirulent (H37Ra) and at MOI 1 and MOI 5 with a virulent (H37Rv) strain of M. tb; 2 × 106 MDMs were not infected as a control (Uninf). At 24 h postinfection, cells were recovered and prepared for qPCR. Relative expression of BCL-2 (a), MCL-1 (b), BAX (c), and DIABLO (d) genes was assessed using qPCR. Bar graphs show the mean ± SD from four independent experiments (n = 4 donors). Statistical analysis was performed using Kruskal–Wallis analysis, followed by Dunn’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3: Sabizabulin induces apoptosis in BT474 and SKBR3 cells in a concentration-dependent manner. (A) A panel of apoptotic markers was profiled by western blotting after 24 h of treatment with colchicine or paclitaxel (100 nM) or sabizabulin (10, 20, 50, or 100 nM) in BT474 and SKBR3 cells. Signal from cleaved-PARP (anti-cleaved-PARP, CST 5625, 1:1000), phosphorylated BCL2 (pBCL2; anti-pBCL2, 2827 CST 1:1000) and cleaved caspase-3 (CST 9661, 1:500) was first normalized to β-actin (CST 3700, 1:20,000) by ImageJ densitometry, with the 100 nM sabizabulin-treated lane set to 1.0. (B) Cleaved-PARP and cleaved-caspase-3 levels in both HER2+ cell lines were determined by western blotting after treatment with 100 nM of sabizabulin for 12, 24, 48, and 72 h after normalization to β-actin. Data are representative of three independent biological replicate assays.

Fig 4: Depletion of Yip1A downregulates the expression of the anti-apoptotic proteins Bcl-2, cIAP2, and Mcl-1, and the phosphorylation of Bcl-2. (a) HeLa cells were transfected with control scramble siRNA or Yip1A siRNA. RT-PCR shows the relative mRNA levels of the indicated anti-apoptotic factors in control and Yip1A-knockdown cells at 48 after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments; **P<0.01. (b) RT-PCR shows the relative mRNA levels of the indicated proapoptotic factors in control and Yip1A-knockdown HeLa cells at 48 h after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments. (c) Western blotting shows the relative levels of anti-apoptotic Bcl-2, cIAP2, and Mcl-1 protein in control and Yip1A-knockdown cells at 48 h after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments; *P<0.05, **P<0.01. (d) Western blotting shows the relative levels of phosphorylated Bcl-2 (pBcl-2) protein in control and Yip1A-knockdown cells at 48 h after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments; **P<0.01. (e) CaSki cells were transfected with control scramble siRNA or Yip1A siRNA. Western blotting shows the relative levels of anti-apoptotic Bcl-2, pBcl-2, cIAP2, and Mcl-1 protein in control and Yip1A-knockdown cells at 72 h after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments; **P<0.01

Fig 5: M. tb virulence induces phosphorylation of BCL-2. Human MDMs were obtained after seven days in culture. 2 × 106 MDMs were infected at MOI 1 and MOI 10 with an avirulent (H37Ra) and MOI 1 and MOI 5 with a virulent (H37Rv) strain of M. tb; 2 × 106 MDMs were not infected as a control (Uninf). At 24 h postinfection, cells were recovered and prepared for Western Blot. Representative Western blot of MCL-1, BCL-2, p-BCL-2, and β-ACTIN (a). Band densities of MCL-1 (b), BCL-2 (c), and p-BCL-2 (d) were normalized against β-ACTIN and quantified by densitometry analysis with the ImageJ software. Results are shown in relative units (RU) of concentration. The bar graphs show the mean ± SD from two independent experiments (MCL-1 and BCL-2, n = 2 donors and two technical replicates; p-BCL-2, n = 3 donors and two technical replicates). Statistical analysis was performed using Kruskal–Wallis analysis, followed by Dunn’s post hoc test. * p < 0.05.