Fig. 4:

Knockdown of IGFBP-3 increases expression of IGF-1R in whole cell lysates. hTCEpi cells were treated with siRNA oligonucleotides targeting IGFBP-3 in basal media. (A, B) An IGFBP-3 ELISA was used to confirm knockdown of IGFBP-3 in whole cell lysates (A, ***P<0.001, One-way ANOVA, post hoc multiple comparison test) and secretion of IGFBP-3 in conditioned media (B, ***P<0.001, One-way ANOVA, post hoc multiple comparison test). (C) Immunoblotting for IGF-1R in whole cell lysates demonstrated an increase in receptor expression after siRNA knockdown of IGFBP-3 in basal media compared to the non-targeting control. Rescue with 100 ng/ml exogenous rhIGFBP-3 further increased IGF-1R expression. (D) Immunoblotting for IGFBP-3 was used to confirm knockdown of IGFBP-3. β-actin was used as a loading control. hTCEpi cells were cultured in basal or growth media with or without 500 ng/ml of rhIGFBP-3 for 24 hours. Addition of rhIGFBP-3 in growth media did not alter IGF-1R expression compared to cells cultured in growth media alone. β-actin was used as a loading control. KGM: keratinocyte growth media; KBM: keratinocyte basal media; BP3: IGFBP-3; WCL: whole cell lysates; CTRL: control. Data representative of 3 independent experiments performed in triplicate. ELISA data presented as mean ± standard deviation.