Figure 4.

RASAL2 inhibits BCa stemness and EMT via ERK/SOX2 signaling pathway. (a) Western blotting analysis of p-ERK1/2 and total ERK1/2 in 5637 cells transfected with RASAL2 shRNAs and scramble shRNA, or T24 cells infected with RASAL2 lentivirus and scramble control. (b) Tumorsphere assay of 5637 cells transfected with RASAL2 shRNAs after treatment with MEK1/2 inhibitor U0126 (10 μM). The tumorsphere number was counted and plotted, and percentage of tumorspheres with diameters <50 μm, 50–100 μm or >100 μm was calculated and plotted. The scale bar represents 100 μm, *P<0.05 versus DMSO. (c) Colony formation assay of 5637/shRASAL2 cells treated with MEK1/2 inhibitor U0126 (10 μM) or DMSO. The colonies number was counted and plotted. *P<0.05, **P<0.01 versus DMSO. (d) Representative pictures and quantification analysis of migration and invasion abilities of 5637/shRASAL2 cells treated with MEK1/2 inhibitor U0126 (10 μM) or DMSO. The scale bar is 100 μm, *P<0.05, **P<0.01 versus DMSO. (e) Western blotting analysis of CD44, SOX2, E-cadherin, vimentin, P-ERK and ERK in 5637 cells transfected with RASAL2 shRNAs after treatment with MEK1/2 inhibitor U0126 (10 μM). (f) Representative pictures and quantification analysis of migration and invasion assays in 5637/shRASAL2-2 cells transfected with SOX2 siRNAs and scramble siRNA. The scale bar is 100 μm, **P<0.01, ***P<0.001 versus control. (g) Colony formation assay of 5637/shRASAL2-2 cells transfected with SOX2 siRNAs and scramble siRNA. The colonies number was counted and plotted. *P<0.05 versus control. (h) Western blotting analysis of CD44, SOX2, E-cadherin and vimentin in 5637/shRASAL2-2 cells transfected with SOX2 siRNAs and scramble siRNA. GAPDH was used as internal loading control