Skip to main content
. 2016 Apr 18;7(24):36168–36184. doi: 10.18632/oncotarget.8786

Figure 3. Rac1 basal activity promotes SOCE-dependent migration through the activation of the downstream effectors PI3K/Akt.

Figure 3

A. PI3K and Akt are involved in SOCE. Fluorescence measurement and relative fluorescence of Ca2+entry after intracellular calcium store depletion by Tg in cells treated for 1h with LY294002, a potent inhibitor of PI3K (upper panel,) or with MK2206, an inhibitor of Akt (lower panel). Data represent means ± SEM. *p<0.05 and ***p<0.001, sample significantly different from control (N=8, Mann–Whitney test). B. Left panel, Akt is involved in HCT-116 cell migration. Histograms showing cell migration with or without treatment by MK2206. Data are means ± SEM. ***p<0.001, sample significantly different from control (N=4, Mann–Whitney test). Right panel, phosphorylated Akt (P-Akt) levels (standardized based on total Akt) was determined by densitometry scanning to generate the values shown in the bar graph. Results are expressed as mean ± SEM. **p<0.01, sample significantly different from control (N=3, Kruskal-Wallis test). Inset, validation of P-Akt extinction by immunoblots after treatment with MK2206. C. Silencing of Orai1 and SK3 channels decrease Akt activation whereas siTRPC1 did not affect P-Akt. P-Akt levels (standardized based on total Akt) was determined by densitometry scanning to generate the values shown in the bar graph. Results are expressed as mean ± SEM. *p<0.05, sample significantly different from control (N=3, Kruskal-Wallis test). Immunoblots representing P-Akt and total Akt in cells under control condition and after transfection with siRNA for 24h. D. Inhibition of Rac1 by a dominant-negative form of Rac1(pRacN17) decreases SOCE (upper panel) and migration (lower panel) of HCT-116 cells. Fluorescence measurement and relative fluorescence of Ca2+entry after intracellular calcium store depletion by Tg in cells transfected with pRacN17and compared to a control condition (pGFP). Lower panel, histograms showing migration of HCT-116 cells transfected with P-RacN17-GFP plasmid or P-GFP plasmid after 24h. Data are means ± SEM. **p<0.01, sample significantly different from control (N=3, Mann–Whitney test). E. Calpain activity in HCT-116 cells. Inhibition of Akt by MK2206 decreases calpain activity. Results are expressed as mean ± SEM. *p<0.05 or ***p<0.001, sample significantly different from control (N=4, Kruskal-Wallis test).