Fig 2: 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.

Fig 3: PLX4720 suppresses UV-induced apoptosis.The cSCC and HaCaT cell lines were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 µM PLX4720 and isolated for FACS analysis and protein extracts 24 hr later. (A) SRB1, (B) SRB12, (C) COLO16, and (D) HaCaT cells show at least 70% suppression of apoptosis in the presence of PLX4720 as measured by FACS for Annexin V+, TMRE-low cells (n = 6 for each cell line, ‘*’ denotes statistical significance at p<0.05). (E) A representative FACS plot for COLO16 is shown. Annexin V+, TMRE-low cells are contained in the upper left quadrant (boxed), which was significantly populated in UV-irradiated cells, but not in the absence of UV, or in the presence of PLX4720. (F) Western blots probed for the MAP kinases demonstrated strong phospho-JNK and phospho-p38 induction following irradiation and significant suppression by PLX4720. Phospho-ERK was slightly induced following irradiation, and at 24 hr, paradoxical hyperactivation in the presence of PLX4720 was observed, particularly in SRB1 and HaCaT cells. (G) Western blots showed that BIM was not upregulated in these BRAF-wild-type cells, consistent with intact ERK signaling. MCL1 was downregulated by irradiation and not modulated by PLX4720, whereas NOXA expression was strongly induced in irradiated cells and suppressed by PLX4720. (H) Western blots of BRAFV600E melanoma cell lines, A375 and WM35, demonstrated suppression of UV-mediated induction of phospho-JNK and phospho-p38 by PLX4720 at 24 hr. As expected, phospho-ERK is shut down in PLX4720-treated cells.DOI: http://dx.doi.org/10.7554/eLife.00969.003

Fig 4: Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in vivo.(A–D) cSCC samples from vemurafenib-treated patients and non-treated patients were analyzed by immunohistochemistry for phospho-JNK and cleaved caspase-3 expression. cSCC arising in vemurafenib-treated patients show decreased expression of phospho-JNK (B) and cleaved caspase-3 (D) as compared to sporadic cSCC in patient never treated with vemurafenib (A and C). Scale bar is 100 µm. (E) Comparisons of stained cells normalized to mm2 of tumor area revealed significant suppression of both phospho-JNK and cleaved caspase 3 expression in vemurafenib-treated cSCC (‘*’, p<0.05). (F and G) Hematoxylin-stained cryosections of skin harvested at 24 hr post-irradiation showed extensive apoptosis (arrowheads) with vacuolated blebbed cells and clumped pyknotic nuclei in control-treated mice (F) and significantly fewer apoptotic cells in PLX4720-treated mice (G). Scale bar is 50 µm. (H–I) Vehicle-treated (‘o’) and PLX4720-treated (‘+’) mice were unirradiated or irradiated once, and epidermis was harvested at 1 hr, 6 hr, and 24 hr post-irradiation. (H) Significant UV-induced upregulation of both phospho-JNK and phospho-p38 were observed within 1 hr, with significant suppression of phospho-JNK in PLX4720-treated mice by 6 hr and minimal suppression of phospho-p38. Phospho-ERK levels remained constant. The upstream regulators of JNK, MKK4 and MKK7, were both significantly activated within 1 hr of irradiation, and potently suppressed in PLX4720-treated mice. Cleaved caspase-3 levels increased within 6 hr and were suppressed in PLX4720-treated mice. (I) Noxa was induced most significantly at 6 hr and was potently suppressed by PLX4720 at all time points (‘***’, p<0.001).DOI: http://dx.doi.org/10.7554/eLife.00969.018