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. 2012 Oct 12;287(50):41667–41683. doi: 10.1074/jbc.M112.390781

FIGURE 2.

FIGURE 2.

STAT3 is required for up-regulation of GIV expression during wound healing and in invasive cancer cells. A, overexpression of myc-STAT3 increases both GIV mRNA and protein. Whole cell lysates of HeLa cells transiently overexpressing myc-STAT3 or vector control were analyzed for GIV protein (top) and mRNA (bottom) expression by immunoblotting (IB) and qPCR, respectively. Up-regulation of GIV protein (top) is specific because Gαi3 or tubulin remains unchanged. -Fold changes in GIV mRNA (bottom) are expressed as mean ± S.D. (n = 3). B, depletion of STAT3 reduces both GIV protein and mRNA levels in HeLa cells. HeLa cells were treated with Scr or STAT3 siRNA and analyzed for GIV protein (top) and mRNA (bottom) expression by immunoblotting and qPCR, respectively. Results are expressed as mean ± S.D. (n = 3). C, activation of STAT3 is required for up-regulation of GIV mRNA during wound healing. Whole cell lysates of HeLa cells stably expressing myc-tagged constructs, either the constitutively active STAT3-C mutant, the constitutively inactive, dominant negative STAT3-KR mutant, or vector control, were analyzed for myc-STAT3 and tubulin by immunoblotting (top) and for GIV mRNA by qPCR (bottom). -Fold changes in GIV mRNA are expressed as mean ± S.D. (n = 3). The p value was calculated by comparing GIV mRNA levels in HeLa cells expressing STAT3-C and STAT3-KR at 6 h postwounding. D and E, depletion of STAT3 reduces both GIV protein and mRNA in highly invasive MDA-MB 231 (D) and DLD-1 (E) cells. MDA-MB 231 and DLD-1 cells were treated with Scr or STAT3 siRNA, and whole cell lysates were analyzed for GIV protein (left) and mRNA (right) by immunoblotting and qPCR, respectively. Both GIV protein and mRNA levels were reduced in MDA-MB 231 and DLD-1 cells depleted of STAT3. Results are expressed as mean ± S.D. (n = 3). F and G, expression of siRNA-resistant mouse STAT3 in HeLa cells depleted of endogenous STAT3 increases both GIV mRNA (F) and protein (G) levels. HeLa cells were treated with control (Scr) or anti-human (h) STAT3 siRNA, transfected with vector or myc-STAT3 (mouse) as indicated, and subsequently analyzed for GIV mRNA by qPCR (F) and for GIV, STAT3, and tubulin by immunoblotting. Error bars represent S.D. *, p < 0.05; **, p < 0.01.