Figure 1.
Expression of anti-apoptotic proteins and effects of Maritoclax on cell survival in melanoma cells.
A, Melanoma cells were harvested, lysed, and fractionated. The Western blot analysis was performed using mentioned antibodies and same blots were re-probed with either other antibodies or with anti-GAPDH to access loading. B, Melanoma cells were treated with increasing concentrations of Maritoclax (0.1–30 µM) for 24 and 48h. Cell viability was then analyzed by the MTT assay. IC50 values were calculated by non-linear regression analysis. C-D, Melanoma cells were treated with 5.0 µM of Maritoclax for 24h and apoptosis was determined by Live/Dead assay and Annexin-V- FITC binding through flow cytometry as described under Materials and Methods.
Figure 2.
Down regulation of Mcl-1 induces apoptosis in melanoma cells.
Melanoma cells were incubated with either indicated amount of Maritoclax for 24h to access dose response or with 5 µM Maritoclax for mentioned time points. After incubation, cells were harvested; total lysates were prepared, and fractionated. The Western blot analysis was performed using mentioned antibodies and same blots were re-probed with either other antibodies or with anti-GAPDH to access loading. The results shown are representative of two independent experiments. A-B, Maritoclax had no effect on Bcl-2 and Bcl-xL. C-D, Degradation of Mcl-1 is associated with apoptosis.
Figure 3.
Maritoclax degrades Mcl-1 by proteasomal activation and releases Bim and Bmf from the cytoskeleton.
A-B, Melanoma cells were treated with 5.0 µM of Maritoclax alone or a combination of 5.0 µM Maritoclax and 5 µM MG132 for 12 h and levels of Mcl-1, Bmf, Bim, and Noxa were assessed by immunoblot analysis. C, Melanoma cells were treated with Maritoclax (5.0 µM, 12h) alone, a combination of Maritoclax (5.0 µM) and MG132 (5.0 µM) for 12 h, and cytochalasin D (10 µM, 3h) alone. Subcellular fractions were prepared. Levels of Bmf, Bim, Cox IV, and GAPDH were assessed by immunoblot analysis. D, Melanoma cells were treated with either indicated amount of cytochalasin D or MG132 for 12h. Whole cell extracts were prepared. Levels of Bmf, Mcl-1, Caspase-3, and PARP cleavage were assessed by immunoblot analysis. Loading was confirmed by GAPDH.
Figure 4.
Maritoclax sensitizes melanoma cells to ABT-737.
A, UACC903 cells were treated with increasing amount of ABT-737 (0.1–30 µM) alone (open circle) or together with 2.0 µM Maritoclax (closed circle) and then incubated for 24 h. Cell viabilities were determined by MTT assay. B, UACC903 cells were treated with either 5.0 µM of ABT-737 alone or together with 2.5 µM Maritoclax and then incubated for 24h. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed through flow cytometer. C, Effect of Maritoclax on apoptosis. UACC903 cells were treated with either 5.0 µM of ABT-737 alone or in combination with 2.5 µM Maritoclax and incubated for 24h. Cells were stained with Annexin-V-FITC and then analyzed through flow cytometer. D-E, Melanoma cells UACC903 were treated with 5.0 µM of ABT-737 and 2.5 µM of Maritoclax together for mentioned time points. After incubation, cells were harvested; total lysates were prepared, and fractionated. The Western blot analysis was performed using mentioned antibodies and same blots were re-probed with either other antibodies or with anti-GAPDH to access loading.
Figure 5.
Maritoclax inhibits 3D spheroids and colony forming ability of melanoma cells.
A, UACC903 cells were grown as spheroids in Algimatrix for seven days and treated with either ABT-737 (5.0 µM) or Maritoclax (2.0 µM) alone or in combination for 3 days and on 10th day experiment was terminated. B-C, After termination of experiments spheroids were counted, size of spheroids was measured by using Image J software as described under Materials and Methods. Graph show corresponding quantification of spheroid growth using image analysis. D, Spheroids were isolated from matrix using dissolving buffer. Note the decrease of viable cells (calcein-AM) and increase of dead cells (ethidium bromide) in the treated spheroids only. E, UACC 903 cells were exposed to either 5.0 µM of ABT-737 or 2.5 µM Maritoclax alone or combination for 24h. Cells were re-plated after treatment for clonogenic assay. The effect of treatment was assessed on the basis of percent cell survival in comparison with the controls.