Hydroxychloroquine Modulates Metabolic Activity and Proliferation and Induces Autophagic Cell Death of Human Dermal Fibroblasts  Bettina Ramser, Agatha Kokot, Dieter Metze, Nina Weiß, Thomas A. Luger, Markus Böhm  Journal of Investigative Dermatology  Volume 129, Issue 10, Pages 2419-2426 (October 2009) DOI: 10.1038/jid.2009.80 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Hydroxychloroquine (HCQ) suppresses metabolic activity, proliferation, and extracellular signal-regulated kinase ERK1/2 activation, but not the S6 ribosomal protein or Akt phosphorylation in human dermal fibroblasts (HDFs). (a) Metabolic activity as measured by XTT test. Cells were incubated with HCQ for 48hours; *P<0.0001, **P<0.00001. (b) Cell proliferation as measured by 3[H]-thymidine incorporation. HDFs were incubated with HCQ for 48hours; *P<0.05, **P<0.01, ***P<0.001 versus control. (c) HCQ time-dependently decreases ERK1/2 phosphorylation at Thr202/Tyr204. Cells were treated with HCQ (30μM) or with PD98059 (10μM) as indicated. ERK1/2 phosphorylation was determined by western immunoblotting using a phosphospecific antibody (Ab) followed by stripping and reprobing with an anti-ERK1/2 Ab. (d) Effect of combined HCQ and PD98059 treatment on metabolic activity of HDFs. Cells were treated with HCQ alone or in combination with PD98059 (5μM) for 48hours, followed by XTT test; *P<0.05, **P<0.001, ***P<0.00001 versus control. (e and f). HCQ does not alter Akt (Thr308 and Ser473) and S6 ribosomal protein phosphorylation. Cells were treated with basic fibroblast growth factor (bFGF) (10ng/ml), HCQ (30μM), and everolimus (EV, 1nM) or with platelet-derived growth factor (PDGF) (20ng/ml), HCQ (30μM), and wortmannin (100nM) for 10minutes. Phosphorylation of S6 ribosomal protein and Akt was determined by western immunoblotting using phosphospecific Abs. Equal amounts of proteins were ensured by stripping and reprobing with an anti-α-tubulin Ab. Journal of Investigative Dermatology 2009 129, 2419-2426DOI: (10.1038/jid.2009.80) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Hydroxychloroquine (HCQ) induces phosphatidylserine translocation but no DNA fragmentation in HDFs. (a and b) Annexin-V staining was determined by FACS analysis. Cells were treated with HCQ as indicated for 24hours. Staurosporine (STS) (0.5μM) was used as positive control; *P<0.01, **P<0.002, ***P<0.0001. (c) Effect of HCQ on mono- and oligonucleosome formation in HDFs after 24hours as determined by cell-death detection ELISA; *P<0.001, **P<0.0001. (d) Effect of HCQ on DNA fragmentation. Cells were treated with HCQ or STS (0.5μM) for 12hours, followed by DNA fragmentation assay; *P<0.0001. Journal of Investigative Dermatology 2009 129, 2419-2426DOI: (10.1038/jid.2009.80) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hydroxychloroquine (HCQ) induces autophagic cell death in human dermal fibroblasts (HDFs). (a and b) Vacuolization of HDFs and HaCaT cells exposed to HCQ. Cells were treated with the drug as indicated for 24hours, followed by phase-contrast microscopy. Note that the MEK inhibitor PD98059 (10μM) did not induce vacuoles; scale bars: 10μm. (c) Vacuolization of HDFs exposed to HCQ as shown by Giemsa staining. For induction of classical apoptosis STS (0.1μM) was used; scale bar: 10μm. (d)Autophagic vacuoles in HCQ-treated HDFs as shown by electron microscopy. Cells were exposed to STS (0.1μM) or HCQ (30μM) for 8hours; scale bars: 1μm. (e) Immunofluorescence analysis of LC3B in HCQ-treated HDFs. Cells were exposed to 30μM HCQ for 16hours, stained with the cytoplasmic F-actin marker fluorescein phalloidin (green), an antibody (Ab) against LC3B (red), or 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue); scale bar: 10μm. (f) HCQ induces LC3B-II expression in HDFs. Cells were treated with 10 or 30μM HCQ for 16hours, followed by western immunoblotting using an Ab against LC3B and re-probing with an anti-α-tubulin Ab. Journal of Investigative Dermatology 2009 129, 2419-2426DOI: (10.1038/jid.2009.80) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Autophagic cell death by hydroxychloroquine (HCQ) in human dermal fibroblasts (HDFs) is associated with upregulation of Beclin-1. (a) Cells were exposed to 30μM HCQ for 6hours, followed by real-time PCR. Gene expression was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene; *P<0.0001. (b) Increased Beclin-1 protein levels in HDFs after HCQ treatment. Cells were treated with 30μM HCQ for 12 and 16hours followed by western immunoblotting using an antibody (Ab) against Beclin-1 and Bcl-2. Equal loading was ensured by membrane stripping and reprobing with an anti-panERK Ab. (c) Subcellular distribution of Beclin-1 in HDFs exposed to HCQ as shown by immunofluorescence. Cells were exposed to 30μM HCQ for 24hours and incubated with an anti-Beclin-1 Ab. HDFs were subsequently incubated with a Texas red–conjugated secondary Ab and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI); scale bar: 10μm. Journal of Investigative Dermatology 2009 129, 2419-2426DOI: (10.1038/jid.2009.80) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions