Intrafollicular autophagic flux is active in MKs.

<p>(A) Representative confocal images of organ-cultured anagen HFs treated for 4 h with CQ (10 μM) or vehicle (water). An overlay of LC3B/Alexa555 (red) and Hoechst 33342 (blue) is shown (scale bar 20 μm). The boxed regions show high magnification images of LC3B-fluorescent dots in MKs (scale bar 5 μm). (B) CQ treatment significantly increased the number of LC3B-flurescent dots per nuclei in the hair matrix region compared with vehicle. Quantification was only performed on epithelial HF cells, specifically the hair matrix cells and precortical matrix, while the connective tissue sheath and dermal papilla were excluded from analysis. Shown as average of LC3B-fluorescent dots/nuclei ± SEM. **<i>P</i> < 0.01, CQ versus vehicle. (C) Representative TEM images from organ-cultured anagen HFs showing a continuous autophagic flux in MKs. (I–III) AVi’s with typical, well-visible bilayers separated by a narrower electron-lucent cleft (arrowheads). Predominantly, these AVi’s contained morphologically intact cytosol and organelles. Note the well-defined mitochondria in III (asterisk). (IV) AVd’s characterized by a partially or completely degraded internal membrane and electron dense cytoplasmic material and/or organelles at various stages of degradation (asterisks). IVa and IVb are higher magnifications of the boxed regions in IV. (V) Putative autophagolysosome characterized by lamellar internal membranes (arrowhead). Scale bars are 0.5 μm in C-I, C-III, C-IV, C-Iva, and C-IVb; 1 μm in C-II and C-V. (D) Quantitative assessment of autophagic structures in TEM sections showing the relative percentage of AVi and AVd in anagen HFs. Shown as average ± SEM. **<i>P</i> < 0.01, AVd versus AVi. The underlying numerical data and statistical analysis for panels B and D are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002864#pbio.2002864.s005" target="_blank">S1 Data</a>. AVd, late/degradative autophagic vacuole; AVi, initial autophagic vacuole; CQ, chloroquine; cyt, cytoplasm; HF, hair follicle; LC3B, Light Chain 3B; MK, matrix keratinocyte; n, nucleus; TEM transmission electron microscopy.