Figure 3. The mitochondrion-localized autophagy receptor NIX is upregulated in the upper epidermal layers.

(A) Diagram of the epidermis, showing increasing NIX expression in the upper layers as keratinocytes differentiate.

(B) IF of NIX in normal human skin and organotypic cultures.

(C) IF of endogenous NIX and TOM20 in organotypic epidermis.

(D) WB of NIX in lysates from undifferentiated NHEKs versus epidermal cultures grown for 6 or 9 days (below, relative band intensity normalized to TOM20).

(E) Diagram depicting regulation of NIX, a transcriptional target of HIF-1a. Inhibition of prolyl hydroxylases (PHs) by DMOG or chelation of iron using DFP relieves repression of HIF-1a, which leads to increased NIX.

(F) WB and quantification of NIX in lysates from NHEKs treated with DMSO or DMOG (mean ± SD, n = 4 expts., *p = 0.039).

(G) IF of NIX and TOM20 in undifferentiated NHEKs treated with DFP.

(H) IF of NIX and TOM20 in NHEKs treated with DMSO, CoCl₂, or EUK134.

(I) Quantification of NIX IF (relative to TOM20) in NHEKs treated with DMSO, CoCl₂, or EUK134 (mean ± SD, n = 33 fields, p < 0.0001).

(J) Stimulated emission-depletion (STED) microscopy images of undifferentiated NHEKs transduced with GFP-NIX and immunostained for TOM20.

Dashed lines mark the bottom of the epidermis. White scale bars, 10 μm.