Human Hair Follicle Stem/Progenitor Cells Express Hypoxia Markers

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Presentation transcript:

Human Hair Follicle Stem/Progenitor Cells Express Hypoxia Markers Michelle Rathman-Josserand, Gaïanne Genty, Jennifer Lecardonnel, Sandrine Chabane, Annabelle Cousson, Jean François Michelet, Bruno A. Bernard Journal of Investigative Dermatology Volume 133, Issue 8, Pages 2094-2097 (August 2013) DOI: 10.1038/jid.2013.113 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunolabeling of human hair follicles from scalp tissue sections. (a) Rabbit anti-GLUT1 1/100 (Chemicon-ab1341, Temecula, CA) followed by Cy 3-conjugated donkey anti-rabbit 1/100 (Jackson Laboratories-ab711-165-152, Bar Harbor, ME). Antibodies were diluted in PBS/0.05% Tween-20/10% serum; 30-minute incubations at RT. (b) Rabbit anti-CA IX 1/500 (Abcam-ab15086, Cambridge, UK) and mouse anti-K19 1/100 (Thermo Scientific, Waltham, MA) followed by goat anti-rabbit Alexa Fluor 488 1/400 (Molecular Probes-A11008, Eugene, OR) and goat anti-mouse IgG2a FITC 1/100 (Caltag Laboratories-M32301, Invitrogen, Carlsbad, CA). DAPI nuclei counterstaining. (b’) Magnification of image a inset. (c) Mouse anti-CD34 1/10 (Becton Dickinson-MY10, Franklin Lakes, NJ) followed by goat anti-mouse IgG1 Alexa Fluor 488 1/600 (Invitrogen-A21121). Rabbit anti-CA IX 1/2000 followed by goat anti-rabbit IgG Alexa Fluor 546 1/400. Antibodies were diluted in Thermo Scientific Pierce Enhancer (Waltham, MA) (46644); sequential 1-hour incubations at RT. DAPI nuclei counterstaining. (c’) Magnification of image c inset. CA IX, carbonic anhydrase IX; DAPI, 4,6-diamidino-2-phenylindole; GLUT1, glucose transporter 1; K19, keratin 19; PBS, phosphate-buffered saline; RT, room temperature. Bar =50μm. Journal of Investigative Dermatology 2013 133, 2094-2097DOI: (10.1038/jid.2013.113) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Impact of hypoxia and ER3721 on clone-forming efficiency and on hypoxia-inducible transcription factor 1α (HIF1α) protein levels. (a) Clone-forming efficiency test (CFE). Outer root sheath (ORS)-derived keratinocytes were plated at 1,000cells per Petri dish and cultured for 10 days in CFE conditions in the presence of (A) 21% O2, atmospheric conditions (B) 3% O2, hypoxia or (C) 21% O2 with 300μM of ER3721. The experiment was performed in triplicate and the average number of clones per Petri dish is cited. (D–F) Microscopic analysis of individual clones, magnification × 20. (G) Number of clones quantified according to clone size (mm). (b) Western blotting of HIF1α on cell extracts from ORS-derived keratinocytes cultured with 3% O2 or 21% O2 in the presence or absence of 300μM of ER3721 for 24, 48, or 72hours. Immunodetection was performed using mouse anti-HIF1α antibody clone mgc3, MA1-516 (Thermo Scientific). Journal of Investigative Dermatology 2013 133, 2094-2097DOI: (10.1038/jid.2013.113) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions