1. Hypothalamic–Pituitary–Thyroid Axis Hormones Stimulate Mitochondrial Function and Biogenesis in Human Hair Follicles 
Silvia Vidali, Jana Knuever, Johannes Lerchner, Melanie Giesen, Tamás Bíró, Matthias Klinger, Barbara Kofler, Wolfgang Funk, Burkhard Poeggeler, Ralf Paus 
Journal of Investigative Dermatology 
Volume 134, Issue 1, Pages (January 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Mitochondrial-encoded subunit 1 of cytochrome c oxidase (MTCO1) expression. Hair follicles (HFs) were treated with (a) vehicle, (b) triiodothyronine (T3; 100 pM), (c) thyroxine (T4; (100 nM), (d) thyrotropin (TSH; 10 mU ml−1), or (e) thyrotropin-releasing hormone (TRH; 30 nM) for 24 hours and immunostained for MTCO1 (nuclear counterstain: hematoxylin) and evaluated by quantitative immunohistomorphometry. DP, dermal papilla; HM, hair matrix; ORS, outer root sheath. Bar=100 μm. MTCO1-IR compared with vehicle, measured in the ORS (f) and HM (h). Treatment groups were normalized to the vehicle arbitrarily set to 100. n=6–11 HFs (two subjects). (g) MTCO1 mRNA steady-state levels (qRT–PCR) in HFs treated with T3 (100 pM) or T4 (100 nM) for 24 hours. Data from 15 HFs (1 subject). Mean±SEM (one-way analysis of variance (ANOVA)), *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Mitochondrial transcription factor A (TFAM) expression. Hair follicles (HFs) treated with (a) vehicle, (b) triiodothyronine (T3; 100 pM), (c) thyroxine (T4; 100 nM), (d) thyrotropin (TSH; 10 mU ml−1), or (e) thyrotropin-releasing hormone (TRH; 30 nM) for 24 hours and immunostained for TFAM (nuclear counterstain: 4′,6-diamidino-2-phenylindole (DAPI)) and evaluated by quantitative immunohistomorphometry. DP, dermal papilla; HM, hair matrix; ORS, outer root sheath. Bar=100 μm. Increased IR compared with vehicle (arbitrarily set to 100), measured in the ORS (f) and HM (h). n=5–10 HFs (two subjects). (g) TFAM mRNA mRNA steady-state levels (qRT–PCR) in HFs treated with T3 (100 pM) or T4 (100 nM) for 24 hours. n=15 HFs (1 subject). Mean±SEM (one-way analysis of variance (ANOVA)), **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Mitochondrial biogenesis. Hair follicles (HFs) treated with (a, g) vehicle, (b, h) triiodothyronine (T3; 100 pM), (c, i) thyroxine (T4; 100 nM), (d, j) thyrotropin (TSH; 10 mU ml−1), or (e, k) thyrotropin-releasing hormone (TRH; 30 nM) for 24 hours. (a–f) Number of ultrastructurally detectable mitochondria (transmission electron microscopy, TEM), particularly in the perinuclear region of human ORS keratinocytes. n=10–12 keratinocytes (six subjects). N, nucleus; red arrows indicate exemplary mitochondria. Green bar=5 μm. (g–k) Porin-IR. DP, dermal papilla; HM, hair matrix; ORS, outer root sheath. Yellow bar=100 μm. (l, m) Porin-IR was assessed by quantitative immunohistomorphometry. Data are reported as percentages, normalized to the vehicle. n=5–10 HFs (2 subjects). Mean±SEM (one-way analysis of variance (ANOVA)), *P<0.05, ***P<0.001.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Thyrotropin (TSH) effects in the presence of thyrotropin receptor (TSHR) antagonist K1-70. Hair follicles (HFs) treated with (a, e, i) vehicle, (b, d, f, h, l, n) TSH (10 mU ml−1), and/or (c, d, g, h, m, n) K1-70 (0.1 ng ml−1) for 24 hours and immunostained for (a–d) mitochondrial-encoded subunit 1 of cytochrome c oxidase (MTCO1; nuclear counterstain: hematoxylin), (e–h) mitochondrial transcription factor A (TFAM), and (i–n) porin (nuclear counterstain: 4′,6-diamidino-2-phenylindole, (DAPI)), and evaluated by quantitative immunohistomorphometry. DP, dermal papilla; ORS, outer root sheath. Bar=100 μm. Increased immunoreactivity (IR) compared with vehicle (arbitrarily set to 100) for (o) MTCO1, (p) TFAM, and (q) porin. n=8–24 HFs (two subjects). Mean±SEM (one-way analysis of variance (ANOVA)), **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Mitochondrial activity. (a) Scheme of calorimetric chip. (b, c) Δ Heat development of the hair follicles (HFs) in μV. Stabilization of the metabolism, consist in a constant signal observed over several hours. n=8 HFs (two subjects/treatment). Arrows indicates start of treatment with (b) triiodothyronine (T3) (100 pM) and (c) thyrotropin (TSH; 10 mU ml−1) in green (■) and red lines (▼). Gray line (•) represents vehicle. (d, e) HFs treated with T3 (100 pM), thyroxine (T4; 100 nM), thyrotropin-releasing hormone (TRH; 30 nM), and TSH (10 mU ml−1) for 24 hours. (d) Complex I activity (n=6 subjects, 8 HFs/subjects, mean±SEM, P<0.001); (e) complex IV activity in human HF (n=6 subjects, 8 HFs/subject, mean±SEM, P<0.001). (f) Outer root sheath (ORS) keratinocytes treated as d and e for 5 and 24 hours. Percentage of adenosine triphosphate production normalized to vehicle, arbitrarily set to 100. n=8–16 wells, mean±SEM (one-way analysis of variance (ANOVA)). *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Oxidative stress in human uman outer root sheath (ORS) keratinocytes. ORS keratinocytes were seeded into 96-plates. After reaching 70% confluence, cells were treated (a–c) with triiodothyronine (T3) (100 pM), thyroxine (T4; 100 nM), thyrotropin-releasing hormone (TRH; 30 nM), or thyrotropin (TSH; 10 mU ml−1) for 24 hours. (a) Graph shows relative fluorescence units (RFU) detected by dichlorofluorescein measurement reported in percentage; vehicle (basal level) is set to 100. Mean±SEM (one-way analysis of variance (ANOVA)). *P<0.05, ***P<0.001, n=6–24 wells. (b, c) qRT–PCR. Graphs show mRNA steady-state levels for (b) catalase and (c) superoxide dismutase 2 (SOD2) in human ORS keratinocytes treated with T3 (100 pM) or T4 (100 nM) for 24 hours. Mean±SEM (one-way ANOVA), *P<0.05, **P<0.01, n=3 wells.
Journal of Investigative Dermatology  , 33-42DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions