Fig. 8.

Knockdown of endogenous IGF-IR desensitizes GH-induced STAT5 activation in β-cells. A, The efficacy of knockdown. MIN6 cells were transiently transfected with pGIPZ-IGF-IR shRNAmir-1, 2, or 3, or pGIPZ-NS (nonsilencing control) plasmid DNA for 72 h. Cell extracts were analyzed by immunoblotting with anti-IGF-IR and anti-β-actin, respectively. Mock, Transfection control without shRNAmir. WB, Western blot. B, Infection of MIN6 cells. The lentiviruses containing IGF-IR shRNAmir-1 or NS shRNAmir were produced in HEK293T cells and used to transduce MIN6 cells. Images were taken 6 d after infection. Upper panels, TurboGFP; lower panels, phase contrast. C, Stable knockdown of IGF-IR (IGF-IR KD). The lentivirally infected cells were selected with puromycin for 4 wk. Cell extracts were immunoblotted with anti-IGF-IR and anti-β-actin, respectively. D and E, IGF-IR knockdown desensitizes GH-induced STAT5 activation. Serum-starved MIN6, IGF-IR KD, and NS control cells were stimulated with vehicle (−), bGH (500 ng/ml), or IGF-I (20 ng/ml) for 15 min. Cell extracts were immunoblotted with anti-pSTAT5 and anti-β-actin, respectively (D). Pooled data of anti-pSTAT5, as in D, from three independent experiments were densitometrically analyzed and statistical results are plotted (E). Data are mean ± sem (n = 3). *, P < 0.05 (IGF-IR KD vs. either MIN6 or NS control).