FIGURE 2.

IGFBP-3 blocks autophagy in epithelial cells. Cells were transfected with siRNA oligonucleotides targeting IGFBP-3. Non-targeting oligonucleotides were used as a control. Cells were then cultured in KBM with or without 500 ng/ml rhIGFBP-3 for 24 h. Ten nM bafilomycin (Baf-1) was used to block lysosomal fusion. Lysates were immunoblotted for autophagy markers P62 and LC3-II. Low exposure (Low Exp) was used to visualize LC3-II in cells treated with Baf-1 due to robust protein accumulation. High exposure (High Exp) was used to visualize cells not treated with Baf-1 due to low accumulation of the LC3-II protein. (A) In hTCEpi cells, in the absence of Baf-1, no differences were observed in LC3-II. Treatment with rhIGFBP-3 did decrease LC3-II levels in Baf-1-treated cells (p = .004 and p = .002 compared to knockdown and control, respectively). (B) In HCECs, there was a similar decrease in LC3-II after treatment with rhIGFBP-3 in Baf-1-treated cells (p = .006 and p = .003 compared to knockdown and control, respectively). Data expressed as mean ± standard deviation from 3 repeated experiments. One-way ANOVA with Student–Newman–Keuls post hoc multiple comparison test. KBM, keratinocyte basal media