Autophagy is altered during the transition from anagen to catagen.

<p>(A and B) Confocal fluorescence analysis revealed a marked reduction in the number of LC3B-positive fluorescent dots during the transition from anagen to catagen. (A) Representative confocal images of anagen and early/middle catagen organ-cultured HFs probed with specific anti-LC3B antibody/Alexa555 (red). Nuclei were stained with Hoechst 33342 (blue). (B) Quantification of the number of LC3B-flurescent dots per nuclei in the hair matrix region of anagen and early/middle catagen HFs, shown as the average of LC3B-fluorescent dots/nuclei ± SEM. **<i>P</i> < 0.01, catagen versus anagen. (C and D) In line with a reduction in autophagy-dependent protein degradation during HF cycle progression into catagen, the protein levels of SQSTM1 significantly increases in catagen. (C) Representative confocal images of anagen and early/middle catagen organ-cultured HFs probed with specific anti-SQSTM1 antibody/Alexa488 (green). Nuclei were stained with Hoechst 33342 (blue). (D) Quantification of SQSTM1-fluorescent signal in the hair matrix region of anagen and catagen HFs. Fluorescent intensity of anagen HFs was set to 1. Shown as relative integrated density ± SEM. *<i>P</i> < 0.05, catagen versus anagen. (E) Quantitative assessment of autophagic structures in TEM sections from anagen and catagen HFs showing the relative percentage of AVs per field. Shown as average ± SEM. **<i>P</i> < 0.01, catagen versus anagen. All quantifications were performed only on epithelial HF cells, specifically the hair matrix cells and precortical matrix, while the connective tissue sheath and dermal papilla were excluded from analysis. The underlying numerical data and statistical analysis for panels B, D, and E 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>. AV, autophagic vacuole; HF, hair follicle; LC3B, Light Chain 3B; MK, matrix keratinocyte; SQSTM1, sequestosome 1; TEM, transmission electron microscopy.