Fig. 5

Dependence of PD-1 biologic activities on SHP2/BRAF/MEK cascade. a. Effects of siRNA targeting SHP2 (siSHP2–100 nM) or the relative control on SHP2 protein expression levels assessed by western blot in 8505c cells stably transfected with PD-1 or the empty vector (one representative clone is shown). b. DNA synthesis of stably transfected 8505c pCMV3 PD-1 cells (mean of 3 clones) compared to empty vector transfected cells treated with siSHP2 or siCTR assessed by BrdU incorporation. Data are presented as mean ± SD of 5 independent experiments. c. DNA synthesis of 8505c cells stably transfected with PD-1 (pCMV3 PD-1 compared to pCMV3) and treated for 18 h with SHP099 (350 nM) assessed by BrdU incorporation. The mean of 3 clones is shown for each condition. Data are presented as mean ± SD of 5 independent experiments. d. DNA synthesis of stably transfected 8505c pCMV3 PD-1 cells (3 clones) compared to empty vector transfected cells treated for 18 h with Vemurafenib (Vemu - 10 μM) or Selumetinib (Selu - 10 μM) assessed by BrdU incorporation. Data are presented as mean ± SD of 5 independent experiments. e. Percent of migrated 8505c cells over control toward sPD-L1 (1 μg/ml) or medium alone (10% FBS) following treatment with SHP099 (350 nM), Vemurafenib (Vemu - 10 μM) or Selumetinib (Selu - 10 μM). Data are presented as mean ± SD of 5 independent experiments. * P < 0.05 compared to the relative control. § P < 0.05 compared to NT or siCTR