Fig 2: Premature NIX expression induces mitochondrial fragmentation and accelerates epidermal differentiation(A) WB of NIX and differentiation markers (Dsg1 and K10) in lysates from epidermal cultures grown for 3, 6, or 9 days and (bottom) quantification of relative NIX expression (mean ± SD; n = 3 expts., **p = 0.0071).(B) H&E staining of cultures ectopically expressing GFP versus GFP-NIX and (bottom) quantification of tissue thickness (mean ± SD, n = 201 fields, 6 expts., ***p < 0.0001) and dyskeratotic cells (mean ± SD, n = 150 fields, 6 expts., ***p < 0.0001).(C) TEM images of lower layers in control cultures, showing tubular mitochondria (green arrowheads), and in GFP-NIX-expressing cultures, showing mitochondrial constriction (blue arrowhead), partially engulfed mitochondria (magenta arrowheads), and mitophagic events (yellow arrowheads).(D) H&E staining of cultures treated with DMSO versus EUK134 and (bottom) IF of NIX and TOM20.(E) Quantification of tissue thickness in DMSO- and EUK134-treated cultures (mean ± SD, n = 22 fields, ***p < 0.0001).(F) WB of lysates from cultures treated with DMSO versus EUK134 and (bottom) quantification of relative NIX protein levels (mean ± SD, n = 5 expts., **p = 0.0006).(G) SDC images of undifferentiated NHEKs transduced with tubulin-mCh and GFP-Mito versus GFP-NIX and (right) quantification of mitochondrial length, perimeter, and AR of GFP-Mito- versus GFP-NIX-positive mitochondria (mean ± SD, n = 78 cells, 3 expts., ***p < 0.0001).(H) Diagram showing GFP-labeled NIX constructs.(I) SDC images of NHEKs transduced with GFP-labeled NIX constructs and incubated with TMRE.(J) Quantification of mitochondrial length in NHEKs expressing NIX constructs (mean ± SD; n = 163 cells, 4 expts., ***p < 0.0001).Dashed lines mark the bottom of the epidermis. White scale bars, 10 µm; H&E black scale bars, 50 µm; TEM black scale bars, 400 nm.

Fig 3: 4B3 antibody-induced Dsg3 depletion occurs from the non-desmosomal and desmosomal pools and cannot be rescued by efgartigimod. hTert cells were plated on 6-well plates, grown in KGM2 medium with 0.05 mM CaCl2 until confluent, and then switched to KGM2 with 2 mM CaCl2 for 24 h. Thereafter, the cells were either left untreated, or efgartigimod (25 µg/mL) was applied for 30 min prior to a 24 h treatment with 4B3 (50 µg/mL) antibodies. As controls, mock incubation (untreated) and an isotype-matched human IgG1 (50 µg/mL) were included. Sequential detergent extraction was performed, resulting in Triton-soluble (non-desmosomal) and Triton-insoluble (desmosomal) pools of proteins. (a) Western blot analysis of the fractions was performed to detect Dsg3 and Dsg1. Analysis of GAPDH level was included to ensure equal loading and the purity of the detergent insoluble fraction. Equal percentage of each fraction was loaded. Western blot signals for Dsg3 in the non-desmosomal (b) and desmosomal (c) pools were quantified using ImageJ software, normalized against GAPDH levels, and expressed as relative amounts compared to the untreated controls. The error bars represent the SD of four independent experiments. Statistical analysis was done using two-way ANOVA with Bonferroni’s post-test. Statistically significant differences are indicated by * = p = 0.05, ** = p < 0.01.

Fig 4: Loss of NIX results in impaired keratinocyte mitophagy and defective epidermal differentiation(A and B) H&E staining of organotypic cultures of control versus NIX-depleted THEKs (NIX KO-1 and KO-2), which exhibited swelling, vacuolization, and dyskeratotic cells (yellow arrowheads) in the upper layers.(C) Quantification of dyskeratotic cells in control versus KO cultures (mean ± SD, n = 53 fields, 3 expts., ***p < 0.0001).(D) WB of NIX in lysates from control and KO cultures.(E) IF of K10 and Dsg1, demonstrating altered keratin expression and cell morphology in NIX KO cultures; asterisks mark example suprabasal cells lacking cytoplasmic K10 staining.(F) Quantification of K10-negative cells in control versus KO cultures (mean ± SD, n = 20 fields, ***p < 0.0001).(G) Quantification of mean suprabasal cell roundness in control versus KO cultures (line, mean; box, 25th–75th percentile; whiskers, 5th–95th percentile; n = 376 cells from 20 fields, **p = 0.0003).(H) TEM images of control cultures, showing mitophagic events (green arrowheads) and clearance of membranous organelles in the uppermost layers; NIX-depleted epidermis showed retention of mitochondria (yellow arrowheads).(I) Quantification of relative electron density of upper layers in control versus KO cultures (mean ± SD, n = 63 fields, 2 KO lines, **p = 0.0015).Dashed lines mark the bottom of the epidermis. White scale bars, 10 µm; H&E black scale bars, 50 µm; TEM black scale bars, 100 nm.

Fig 5: Inhibition of RXR increased the expression levels of the desmosomal and TJ proteins. After densitometric analysis of western blotting, protein levels were normalized to the corresponding actin levels and showed relative to the amount present in the control cultures (C) below the blots. Protein levels of desmoglein (DSG)-1, DSG2, DSG3, plakoglobin (PG), claudin (CLDN)-1, CLDN4, CLDN6 and CLDN7 in HX 531-treated cultures (HX) were 4.5, 1.8, 4.1, 2.0, 1.5, 1.6, 1.2 and 1.2 times higher than those in control cultures, respectively. Protein levels of zonula occludens-1 (ZO-1) and E-cadherin were not changed by HX 531 treatment.