Fig. 3
The ATP-P2RX6-Ca2+ signaling mediated the MAPK family ERK1/2 activation to promote RCC migration/invasion. a Intracellular Ca2+ levels were determined in SN12-PM6 to evaluate the P2RX6 effect. Arrows indicate the points at which ATP (10 μM) was added. b The net change in Ca2+ levels was normalized to (Fmax-F0)/F0. c Migration assay showed Ca2+ inhibitor verapamil (2uM) could block ATP-P2RX6 induced cell migration in 786-O and OS-RC-2 cells. d Transwell assay showed Ca2+ inhibitor verapamil (2uM) could block ATP-P2RX6 induced cell migration in 786-O and OS-RC-2 cells. e Quantitative analysis for Fig. 3d. f Western Immunoblotting for MAPK family ERK1/2 and p-ERK1/2 expression using sh-P2RX6 1# and 2# treat with/without DHT, GAPDH as an internal control. g WB for MAPK family ERK1/2 and p-ERK1/2 expression using verapamil treat with/without over-express P2RX6, GAPDH as an internal control. h Immunofluorescence showed that ATP could stimulate the expression of p-ERK1/2 in both the cellular cytoplasm and nucleus. i Migration assay showed ERK1/2 inhibitor SCH772984 (0.1uM) could block over-expression P2RX6 induced cell migration in 786-O and OS-RC-2 cells. j Transwell assay showed ERK1/2 inhibitor SCH772984 (0.1uM) could block over-expression P2RX6 induced cell invasion in 786-O and OS-RC-2 cells. k Quantitative analysis for Fig. 3j. N.S. = not significant. * P < 0.05, ** P < 0.01, *** P < 0.001