Fig. 1: PLX8394 decreases laminin-332 synthesis and inhibits Smad2 phosphorylation both in 3D spheroids and in 2D monolayer cultures in RT3 cells.

See also Supplementary Fig. S1. A HaCaT and RT3 cells were treated with a panel of inhibitors for 24 h in 2D monolayer conditions, followed by 3D spheroid formation. The inhibitors (LY294002 (PI3K inhibitor); PD98059, trametinib (MEK inhibitors); dabrafenib, PLX4720, PLX8394 (BRAF inhibitors) were added daily for 3 days. Laminin-332, phosphorylated ERK1/2 (p-ERK1/2), and total ERK1/2 (tot-ERK1/2) levels were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332 and p-ERK1/2 levels corrected for β-actin or tot-ERK1/2 is shown below the blots. Values are relative to the levels (1.0) of DMSO control in HaCaT or RT3 cells. B RT3 cells were treated with dabrafenib (50 nM), PLX4720 (10 μM), or PLX8394 (10 μM) for 24 h in 2D monolayer conditions, followed by 3D spheroid formation with skin primary fibroblasts. The spheroids were grown for 24 h or 48 h before harvesting for western blotting. The levels of laminin-332, p-ERK1/2, tot-ERK1/2, p-Smad2, and tot-Smad2 were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332, p-ERK1/2, and p-Smad2 levels corrected for β-actin, tot-ERK1/2, or tot-Smad2 is shown below the blots. Values are relative to the levels (1.0) of DMSO control in 24 h or 48 h samples. C A panel of primary (UT-SCC-12A, UT-SCC-91A, UT-SCC-105, UT-SCC-118) and metastatic (UT-SCC-7, UT-SCC-59A, UT-SCC-115) cSCC cell lines were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown 3 days. The levels of laminin-332, p-Smad2, and p-ERK1/2 were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332, p-Smad2, and p-ERK1/2 levels corrected for β-actin, tot-Smad2, or tot-ERK1/2 is shown below the blots. Values are relative to the levels (1.0) of control samples (without PLX8394) of each cell line. RT3 cells were treated with increasing concentrations of PLX8394 for 4 h (D) or 18 h (E). The cells were then subjected to TGF-β (10 ng/ml, 30 min) and harvested for western blotting. p-Smad2 and tot-Smad2 levels were analyzed by western blotting. Densitometric quantitation of p-Smad2 levels corrected for tot-Smad2 is shown below the blots. Values are relative to the level (1.0) of DMSO plus TGF-β treated samples.