Pro-Proliferative Function of Mitochondrial Sirtuin Deacetylase SIRT3 in Human Melanoma  Jasmine George, Minakshi Nihal, Chandra K. Singh, Weixiong Zhong, Xiaoqi Liu, Nihal Ahmad  Journal of Investigative Dermatology  Volume 136, Issue 4, Pages 809-818 (April 2016) DOI: 10.1016/j.jid.2015.12.026 Copyright © 2015 The Authors Terms and Conditions

Figure 1 SIRT3 is overexpressed in human melanoma cell lines and clinical tissues. (a) qRT-PCR and (b) western blot analysis for SIRT3 expression in normal human melanocytes (NHEM), nontransformed but immortalized human melanocyte line (Mel-ST), and six human melanoma cell lines (SK-MEL-2, WM35, SK-MEL-28, G361, Hs294T, and A375). β-Actin and Gapdh were used as controls, respectively. (c) Representative images for SIRT3 immunohistochemical analysis in two melanoma tissue microarray containing a total of 4 normal skin, 24 nevi, and 96 malignant melanoma (stage II, III and IV) tissues. Positive SIRT3 staining is indicated by the intensity of the red color from the Vector Red chromagen used in staining. Individual tissue cores were blindly scored for SIRT3 staining intensity as negative (0), trace, weak (+1), moderate (+2), or strong (+3) staining in 50% of the cells examined. Representative cores for normal, nevus, and melanoma samples are shown. The magnification of large panels is ×10 and inset windows are ×40. The qRT-PCR and western blot data represent experiments performed in triplicates and the results are presented as means ± SEM. Statistical significance are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Gapdh, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real time-PCR; SEM, standard error of the mean; SIRT3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions

Figure 2 SIRT3 knockdown inhibits proliferation, colony formation, and migration of human melanoma cells. (a) Western blot analysis for confirming SIRT3 knockdown in SK-MEL-2 cells. Four independent shRNAs construct (1: TRCN0000038889, 2: TRCN0000038890, 3: TRCN0000038892, and 4: TRCN0000038893) were used for SIRT3 knockdown (shSIRT3) as described in the Materials and Methods section. Control cells were transduced with pLKO.1 empty vector (shNS). β-Actin was used as a loading control. (b) Western blot analysis of stable SIRT3 knockdown (shSIRT3 #1–#3) and control vector (shNS #1–#3) clones selected with puromycin (2 μg/ml) was performed to confirm the knockdown. β-Actin was used as a loading control. (c) Western blot analysis of stable SIRT3 knockdown (shSIRT3) in SK-MEL-2, G361, and SK-MEL-28 melanoma cells. β-Actin was used as a loading control. Proliferative potential was assessed at 24, 48, and 72 hours in stable cells using MTT assay. (d) Clonogenic cell survival of stable cells was assessed by colony formation assay. Representative images after 14 days (SK-MEL-2 cells) and 11 days (G361 and SK-MEL-28 cells) in culture are shown. (e) Cell migration was determined using an Ibidi Culture Insert. Representative images shown at 0, 24, and 48 hours were analyzed under a microscope. All experiments were performed in triplicates and the results are presented as means ± SEM. Statistical significance are indicated as *P < 0.05, ***P < 0.001, ****P < 0.0001. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SEM, standard error of the mean; shNS, nonspecific shRNA; shRNAs, short hairpin RNAs; SIRT 3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions

Figure 3 SIRT3 knockdown induces senescence-like phenotypes in human melanoma cells. (a) After stable SIRT3 knockdown in melanoma cells, the morphological changes were analyzed under a microscope (Nikon Digital Sight DS-Fi1 camera) and images were captured using NIS Elements AR 3.1 software. Representative images of control (shNS) and SIRT3 knockdown (shSIRT3) cells are shown. Scale bars = 100 μm. (b) SA-β-Gal staining in cells, showing accumulation of blue color in senescent cells. Representative images were analyzed under a microscope. Scale bar = 100 μm. (c) SAHF formation in cells was analyzed by DAPI staining of heterochromatin foci. Representative images are analyzed under a microscope. Scale bar = 10 μm. (d) qRT-PCR and (e) western blot analysis for SIRT3 and senescence associated proteins, p16INK4a and p21Waf1 levels in stable cells. β-Actin and Gapdh were used as controls. All experiments were performed in triplicates and the results are presented as means ± SEM. Statistical significance are indicated as *P < 0.05, **P < 0.01. DAPI, 4',6-diamidino-2-phenylindole; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real time-PCR; SA-β-Gal, senescence-associated beta-galactosidase; SAHF, senescence-associated heterochromatin foci; SEM, standard error of the mean; shNS, nonspecific shRNA; SIRT3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions

Figure 4 SIRT3 knockdown induces G1-phase arrest in human melanoma cells. (a) After SIRT3 knockdown in melanoma cells, they were subcultured, collected, and analyzed for cell cycle distribution using FACS analysis. (b) qRT-PCR and (c) western blot analysis for SIRT3, Cyclin D1, Cyclin E1, Cdk2, Cdk4, and Cdk6 expression levels in stable cells. β-Actin and Gapdh were used as controls, respectively. All experiments were performed in triplicates and the results are presented as means ± SEM. Statistical significance are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Cdk, cyclin-dependent kinase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real time-PCR; SEM, standard error of the mean; SIRT3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions

Figure 5 Exogenous SIRT3 overexpression increases growth and proliferation of Hs294T melanoma cells and normal immortalized Mel-ST melanocytes. (a) Western blot analysis of stable SIRT3 overexpression in Hs294T and Mel-ST cells transfected with control empty vector pcDNA 3.1(+), SIRT3-Flag (#1–#4) DNA, and mock control (no DNA) as described in the Materials and Methods section. β-Actin was used as a loading control. Stable cells were selected with G-418 (2 mg/ml) for 14 days for Hs294T and 12 days for Mel-ST. (b) Proliferative potential was assessed at 24, 48, and 72 hours in stable cells using MTT assay. (c) Clonogenic cell survival of stable cells was assessed by colony formation assay. Representative images after 10 days in culture are shown. The number of colonies were counted and plotted for quantitation. All experiments were performed in triplicates and the results are presented as means ± SEM. Statistical significance are indicated as ****P < 0.0001. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SEM, standard error of the mean; SIRT3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions

Figure 6 SIRT3 knockdown causes a decrease in tumor growth in the Nu/Nu xenograft mouse model. The melanoma cells (shNS and shSIRT3-SK-MEL-2 cells) were implanted and allowed to grow in Nu/Nu mice and tumorigenesis was assessed as described in the Materials and Methods section. (a) Average tumor volume (on a weekly basis), (b) tumor weight (at termination of experiment), and (c) Kaplan-Meier curve for target tumor size (on a weekly basis) are shown. (d) Pictures of resected tumors (at termination of the experiment) were captured using a digital camera. (e) Western blot analysis of SIRT3 expression in tumor tissues lysates was conducted; β-actin was used as loading control. Statistical significance are indicated as *P < 0.05, **P < 0.001, ***P < 0.0001. shNS, nonspecific shRNA; SIRT3, sirtuin 3. Journal of Investigative Dermatology 2016 136, 809-818DOI: (10.1016/j.jid.2015.12.026) Copyright © 2015 The Authors Terms and Conditions