Down-Regulation of SOX2 Underlies the Inhibitory Effects of the Triphenylmethane Gentian Violet on Melanoma Cell Self-Renewal and Survival Silvia Pietrobono,

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Down-Regulation of SOX2 Underlies the Inhibitory Effects of the Triphenylmethane Gentian Violet on Melanoma Cell Self-Renewal and Survival Silvia Pietrobono, Andrea Morandi, Sinforosa Gagliardi, Gianni Gerlini, Lorenzo Borgognoni, Paola Chiarugi, Jack L. Arbiser, Barbara Stecca Journal of Investigative Dermatology Volume 136, Issue 10, Pages 2059-2069 (October 2016) DOI: 10.1016/j.jid.2016.06.610 Copyright © 2016 The Authors Terms and Conditions

Figure 1 GV inhibits melanoma sphere self-renewal and survival. (a) Primary and secondary melanoma spheres. Melanoma cells were treated for 72 hours during primary sphere formation and left untreated to form secondary spheres. (b) Representative phase-contrast images of secondary SSM2c spheres as indicated in a. Scale bar = 100 μm. (c) Size of primary and secondary spheres treated with increasing doses of GV. (d) Quantification of BrdU incorporation and (e) of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic cells in SSM2c primary spheres after GV treatment. (f) Percentage of caspase 3-positive melanoma cells after GV treatment. (g) Western blot of SOX2 in adherent melanoma cells and spheres after GV treatment. HSP90 was used as a loading control. Data shown are mean ± standard error of the mean. n = 3. ∗P ≤ 0.05 versus control. 7-AAD, 7-amino actinomycin D; GV, gentian violet; N, number. Journal of Investigative Dermatology 2016 136, 2059-2069DOI: (10.1016/j.jid.2016.06.610) Copyright © 2016 The Authors Terms and Conditions

Figure 2 GV reduces the number and self-renewal of ALDHhigh melanoma stem cells. (a, b) Quantification and representative images of ALDHhigh cells after GV treatment for 24 hours. ALDH inhibitor diethylaminobenzaldehyde was used as negative control. (c) Western blot of BCL2 and SOX2 in ALDHhigh and ALDHlow subpopulations after GV treatment. (d) Number of secondary spheres in ALDHhigh and ALDHlow SSM2c cells treated with GV. (e) Cytometric analysis of apoptotic cells in ALDHhigh and ALDHlow fractions 48 hours after GV treatment. (f) Representative images of sorted ALDHhigh and ALDHlow subpopulations. Sorting gates were set at least one logarithm apart from the relative baseline fluorescence (drawn after the incubation with DEAB). (g) Western blot of cleaved caspase-3 in ALDHlow- and ALDHhigh-sorted cells, as shown in f. HSP90 was used as loading control. Data shown are mean ± standard error of the mean. n = 3. ∗P ≤ 0.05 versus control. ALDH, aldehyde dehydrogenase; Cleav, cleaved; DEAB, diethylaminobenzaldehyde; GV, gentian violet; N, number. Journal of Investigative Dermatology 2016 136, 2059-2069DOI: (10.1016/j.jid.2016.06.610) Copyright © 2016 The Authors Terms and Conditions

Figure 3 SOX2 mediates the inhibitory effect of GV on melanoma stem cell self-renewal. (a) Western blot of SOX2 in M33x and SSM2c spheres transduced with LV-c or LV-SOX2. HSP90 was used as loading control. (b) Primary and (c) secondary sphere assay shows that ectopic SOX2 counteracts the decrease in the number of spheres induced by GV treatment. (d) Size of primary SSM2c spheres transduced with LV-c and LV-SOX2. (e) Quantification of the percentage of early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic cells in SSM2c spheres transduced with LV-c and LV-SOX2. Data shown are mean ± standard error of the mean. n = 3. ∗P ≤ 0.05 versus control. GV, gentian violet. Journal of Investigative Dermatology 2016 136, 2059-2069DOI: (10.1016/j.jid.2016.06.610) Copyright © 2016 The Authors Terms and Conditions

Figure 4 GV impairs melanoma cell viability by promoting mitochondrial apoptosis and G2 cell cycle arrest. (a) Cell viability assay 48 hours after GV treatment. Controls (DMSO) were set to 100%. (b) Cell cycle analysis after GV treatment (48 hours). (c) Western blot of cyclin B1, BCL2, BAX, total and cleaved caspase-3, and p53 in melanoma cells treated with GV for 48 hours. HSP90 was used as loading control. Densitometric quantification of BAX/BCL2 ratio is shown. (d) Cytometric analysis of apoptotic cells after GV treatment for 48 hours. (e) Western blot of cytochrome C in cytosolic extracts 48 hours after GV treatment. Fibrillarin (nuclear) and GAPDH (cytosolic) were used as loading control. (f) Quantification of mitochondrial ROS in cells treated with GV, stained with MitoSOX (Thermo Fisher Scientific, Waltham, MA) and analyzed by fluorescence-activated cell sorting. Data shown are mean ± standard error of the mean. n = 3. ∗P ≤ 0.05 versus control. Cleav, cleaved; Cytc, cytochrome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, gentian violet; ROS, reactive oxygen species; WCE, whole cell extract. Journal of Investigative Dermatology 2016 136, 2059-2069DOI: (10.1016/j.jid.2016.06.610) Copyright © 2016 The Authors Terms and Conditions

Figure 5 GV inhibits STAT3/SOX2 axis. (a) Immunoprecipitation of endogenous EGFR in A375 cells. (b) Western blot of pSTAT3Y705 and STAT3 in adherent cells and spheres treated with GV, for 24 and 72 hours, respectively. HSP90 was used as loading control. (c) STAT3 cellular localization 24 hours after GV treatment. GAPDH (cytoplasm) and fibrillarin (nuclear) were used as loading control. (d) Chromatin immunoprecipitation on SOX2 proximal promoter. IgG was used as negative control and set to 1. Data represent mean ± standard error of the mean. n = 3. (e) Quantification of dual-luciferase reporter assay. Relative luciferase activities were firefly/Renilla ratios, with the level induced by control equated to 1. Data represent mean ± standard error of the mean. n = 4. (f) Western blot of SOX2 and BCL2 in SSM2c transfected with Myc-STAT3wt, Myc-STAT3Y705E, or Myc after 250 nmol/L of GV for 24 hours. (g, h) Primary spheres formation and representative images in cells transfected with Myc-STAT3wt, Myc-STAT3Y705E, or Myc. Scale bar = 100 μm. ∗P < 0.05 versus control. (i) Proposed mechanism of action of GV. ∗P < 0.05 versus control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, gentian violet; Rel, relative; STAT3, signal transducer and activator of transcription-3. Journal of Investigative Dermatology 2016 136, 2059-2069DOI: (10.1016/j.jid.2016.06.610) Copyright © 2016 The Authors Terms and Conditions