Fig. 1:

IGF-1R, CAV-1, and CLTC regulate IGFBP-3 secretion in conditioned media. An IGFBP-3 ELISA was used to analyze the concentration of secreted IGFBP-3. (A) hTCEpi cells treated with siRNA oligonucleotides targeting IGF-1R significantly decreased IGFBP-3 secretion into culture media compared to the non-targeting control (**P<0.001, One-way ANOVA, Holm-Sidak post hoc multiple comparison test). (B, C) hTCEpi cells were treated with siRNA oligonucleotides targeting CAV-1 and CLTC. Knockdown of each protein increased secretion of IGFBP-3 in basal media. Unlike the non-targeting control, secreted levels of IGFBP-3 were unchanged by the addition of 10 μg/ml of human recombinant insulin in basal media (***P<0.001, One-way ANOVA, Holm-Sidak post hoc multiple comparison test). (D) Immunoblotting for IGF-1R, Cav-1 and CLTC confirmed knockdown in whole cell lysates. IGFBP-3 immunoblotting of whole cell lysates paralleled secreted levels of IGFBP-3. β-actin was used as a loading control. KGM: keratinocyte growth media; KBM: keratinocyte basal media; IGF-1R: insulin-like growth factor type 1 receptor; IGFBP-3: insulin-like growth factor binding protein-3; Cav-1: caveolin-1; CLTC: clathrin; CTRL: control; ins: insulin. Data representative of 6 independent experiments performed in triplicate. ELISA data presented as mean ± standard deviation.