Fig. 3

S100A8/A9-RAGE signaling activates FAK in TNBC cells. A RAGE mRNA levels in MDA-MB231 and BT-549 cells after infection with pCDNA3 and pcDNA3.RAGE DNA vectors. B RAGE immunoblot in MDA-MB231 and BT-549 cells after infection with pCDNA3 and pcDNA3.RAGE DNA vectors. C pY397FAK, FAK, pS473AKT and AKT immunoblots in MDA-MB231 and BT-549 cells overexpressing RAGE and treated with 100 ng/ml rhS100A8/A9 for the indicated times. D pY397FAK, FAK, pS473AKT and AKT immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM RAGE antagonist FPS-ZM1. E pY397FAK, FAK, pS473AKT and AKT immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM FAK inhibitor VS-4718. F pY397FAK, FAK, pS473AKT and AKT immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM AKT inhibitor Ipatasertib. G pY397FAK and FAK immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM ROCK inhibitor Y-27632. H pY397FAK and FAK immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM Myosin II inhibitor Blebbistatin. I pY397FAK and FAK immunoblots in MDA-MB231 cells overexpressing RAGE and treated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in the presence of siRNA control and siRNA targeting Rho-A, respectively. J Y397FAK (green) and Nuclei (blue) confocal immunofluorescence staining performed in MDA-MB231 cells overexpressing RAGE and stimulated for 30 minutes with 100 ng/ml rhS100A8/A9 alone or in combination with 1 μM RAGE antagonist FPS-ZM1, 1 μM FAK inhibitor VS-4718 and 1 μM ROCK inhibitor Y-27632, respectively. K pY397 relative quantification in FA/cell. Error bars represent mean ± SD. * indicates p-value < 0.05. ꞵ-actin served as loading control for immunoblots. Results shown are representative of three independent experiments performed in triplicate