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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Pigment Cell Melanoma Res. 2020 Jul 9;33(6):850–868. doi: 10.1111/pcmr.12905

Figure 4. Ablation of HMOX1 abrogates melanosphere formation.

Figure 4.

SK-Mel-5 and SK-Mel-28 cells were transfected with CRISPR/Cas9 constructs to ablate HMOX1 expression. Transfected cells were sorted by GFP expression and individual clones were expanded. (A) Complete loss of HMOX1 was validated by immunoblot analysis following overnight CoPP (10 μM) treatment to upregulate HMOX1. Quantitation of three blots is shown in the lower panels. (B) SK-Mel-5, SK-Mel-28 and their HMOX1-null counterparts, SK-Mel-5HMOX1- (5KO1), SK-Mel-5HMOX1- (5KO2) and SK-Mel-28HMOX1- (28KO1) were plated and used for melanosphere assay. (C) For MTT assay, 10,000 cells were plated per well of a 96-well plate and untreated or treated with 10 μM CoPP, 10 μM SnPP or 5 μM PLX-4032. Comparisons between wild-type and HMOX1-null lines are noted with asterisks. Comparisons amongst the various treatments are noted by # (#P < 0.001). Error bars represent the standard error of the mean (SEM) of three biological replicates for immunoblot quantitation, four for melanosphere assay and six for MTT assay. Statistical significance was determined using the standard Student’s t-test. *P < 0.05, **P < 0.01. Symbols were omitted where no significance was found.