1. MicroRNA-211 Loss Promotes Metabolic Vulnerability and BRAF Inhibitor Sensitivity in Melanoma 
Anupama Sahoo, Sanjaya K. Sahoo, Piyush Joshi, Bongyong Lee, Ranjan J. Perera 
Journal of Investigative Dermatology 
Volume 139, Issue 1, Pages (January 2019)
DOI: /j.jid
Copyright © 2018 The Authors Terms and Conditions

2. Figure 1
miR-211 deletion inhibits melanoma growth, invasion, and colony formation. (a) Schematic showing the position of two guide RNAs that bind –205 bp (gRNA1) upstream and +93 bp downstream (gRNA2) of the pri-miRNA211 locus (110 bp) and primer locations for detection of genomic deletion of pri-miR-211. (b) PCR showing the deletion allele (900 bp) and wild-type allele (1,310 bp) indicated with red arrowheads. (c) Mature miR-211 expression in the miR-211 hemizygous knockout clone SK-P8-2. (d) Proliferation of SKMEL28 and SK-P8-2 cells at the indicated time points. (e) Cell invasion assayed in SKMEL28 and SK-P8-2 cells. Original magnification ×4. (f) Colony-forming assay in SK-P8-2 and SKMEL28 cells. MTT assay was used to determine cell number (right). Original magnification ×4. (g) Western blot analysis of the indicated proteins in SKMEL28 and SK-P8-2 cells. Scale bars: 1 cm = 200 µm Graphs shown are mean ± standard deviation and are representative of at least three independent experiments. Student t test: ∗∗∗P < 0.001, ∗∗∗∗P < bp, base pair; gRNA, guide RNA; miR, microRNA; p-, phosphorylated; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

3. Figure 2
Impaired mitochondrial respiration and complex formation in miR-211–deleted melanoma cells. (a) Oxygen consumption rate (OCR) was analyzed using the Seahorse XF analyzer (Seahorse Bioscience, North Billerica, MA) in SKMEL28 and SK-P8-2 cells. (b) Ratio of mitochondrial ND1 genomic DNA to nuclear β-globulin genomic DNA in SKMEL28 and SK-P8-2 cells. (c) Targeted metabolomic analysis of SKMEL28 and SK-P8-2 cells. (d) Mitochondrial complex expression in SKMEL28 and SK-P8-2 cells. (e) Ratio of NAD/NADH and NADP/NADPH levels in SKMEL28 and SK-P8-2 cells. (f) Intracellular ROS levels were measured in SKMEL28 and SK-P8-2 cells after treatment with 2′7′-dichlorofluoresicin diacetate by FACS analysis. Graphs shown are mean ± standard deviation and are representative of at least three independent experiments. Student t test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < gDNA, genomic DNA; miR, microRNA; NAD, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; ns, not significant; ROS, reactive oxygen species.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 3
MiR-211 loss influences vemurafenib sensitivity in melanoma cells. (a) SKMEL28, SK-P8-2, 501-Mel, and 501-Mel-P5-5 cells were treated with vemurafenib (Vem) (2 μmol/L) for 24 hours and analyzed for miR-211 expression. (b) miR-211 expression in vemurafenib-resistant SKMEL28-P1 cells. (c–f) SKMEL28 and SK-P8-2 cells treated with DMSO only or with vemurafenib (2 μmol/L) for 24 hours and analyzed for (c) OCR, (d) mitochondrial complex expression by Western blotting, and (e) quantitative PCR analysis and (f) cell viability after 48 hours. (g–i) SKMEL28-P1 cells were edited by the CRISPR-Cas9 system and analyzed by quantitative PCR for (g) miR-211 expression, (h) mitochondrial complex expression, and (i) OCR. Graphs shown are mean ± standard deviation and are representative of at least three independent experiments. Student t test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < M, mol/L; miR, microRNA; ns, not significant; OCR, oxygen consumption rate; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

5. Figure 4
miR-211 deficiency impairs melanoma metabolism and growth in vivo. (a–c) Tumor volume and size of SKMEL28 and SK-P8-2 xenografts in SCID mice. (d) miR-211 levels in tumor xenografts measured by quantitative real-time PCR. (e) Representative images of immunohistochemical analysis of Ki67 in SKMEL28 and SK-P8-2 xenografts. Percentage of Ki67-positive cells in a given area (bottom). Scale bars for original magnification ×1 = 3 mm; scale bars for original magnification ×20 = 100 μm. (f) Western blot analyses of mitochondrial complex proteins in SKMEL28 and SK-P8-2 xenograft tissues. Representative blots (top) and their quantitative analysis (bottom). (g) Metabolic analysis of SKMEL28 and SK-P8-2 xenograft tissues showing TCA cycle intermediates and glycolytic intermediates. Graphs shown are mean ± standard deviation and are representative of at least three independent experiments. Student t test was performed to detect differences between the samples as indicated. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P <  M, mol/L; miR, microRNA; ns, not significant; T, time; TCA, tricarboxylic acid.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions