FIGURE 8.

IGFBP-3 increases mitophagy through increased interaction of sBNIP3L/NIX and LC3-II. 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. (A) Immunoprecipitation of sBNIP3L/NIX and immunoblotting for LC3-II confirmed a decrease in the interaction between LC3-II and sBNIP3L/NIX in cells co-treated with rhIGFBP-3 (p = .006 and p = .018 compared to knockdown and control, respectively). (B) Immunoprecipitation for LC3-II and immunoblotting sBNIP3L/NIX showed an increase in the interaction between LC3-II and sBNIP3L/NIX in knockdown cells (p = .024 compared to control). This was decreased by co-treatment with rhIGFBP-3 (p = .031 compared to knockdown). (C) Immunofluorescent labeling and quantification for sBNIP3L/NIX (green) and LC3-II (red) in hTCEpi cells. Nuclei were labeled with DAPI (blue). Findings paralleled the immunoprecipitation data shown in panels A and B. Scale bar: 10 μm. All data are expressed as mean ± standard deviation from three repeated experiments. One-way ANOVA with Student–Newman–Keuls post hoc multiple comparison test. KBM, keratinocyte basal media