Figure 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

Figure 4—figure supplement 1. Double staining of sporadic cSCC confirms phospho-JNK and cleaved caspase-3 expression within keratinocytes of tumors.

Sections were processed for standard immunohistochemistry and stained with primary antibodies against phospho-JNK, cleaved caspase-3 (Cell Signaling; peroxidase–DAB) as before, together with antibodies against cytokeratins 5/6 (clone D5/16 B4—Thermo; peroxidase–AEC). Results of the double staining show that in all cases, phospho-JNK staining and cleaved caspase 3 staining was observed exclusively in keratinocytes within tumors. Keratinocytes (CK5/6 +) are significantly larger and have more cytoplasm than macrophages or lymphocytes. Scale bar is 50 μM.

DOI: http://dx.doi.org/10.7554/eLife.00969.019