Figure 6
From: pHUSH: a single vector system for conditional gene expression
Robust and titratable depletion of oncogenic B-Raf in melanoma tumor lines elicits the expected phenotypes in vitro and in vivo. (A) Knockdown of BRAF at Day 2 upon Dox addition as determined by western blotting in A375 cells. BRAF knockdown results in a significant reduction in phospho-ERK levels. (B) Proliferation assay reveals a growth stasis phenotype upon BRAF knockown in comparison to a shEGFP control in A375 cells. (C) Dose dependent decrease in VEGF secretion correlates with BRAF knockdown 24 and 48 h post Dox addition. (D) In vivo BRAF knockdown detected by non-invasive bioluminescence imaging. A previously characterized LOX-IMV1 clone containing pHUSH-Braf-shRNA [26] was re-engineered to express a Luc-BRAF transcipt fusion. After initiating a subcutaneous tumor model in Scid-beige mice, the expression level of a Braf targeting shRNA was indirectly monitored by bioluminescence imaging at 0, 0.01 mg/mL, 0.02 mg/mL, and 0.5 mg/mL Dox. (E) Day 25 tumors were harvested and equal total protein was assayed for luciferase enzyme activity in vitro.