Fig 2: Stromal HO‐1 modulates melanoma pigmentation. (a) Proliferation of B16‐F10‐Luc cells during co‐culture with Hmox1 +/+ and Hmox1 −/− MSCs, measured using IVIS Lumina imaging system (each bar represents mean ± SEM, n = 4, representing MSC isolations from 4 separate mice per genotype, *p < 0.05, **p < 0.01). (b) qRT‐PCR analysis of Tyr expression in melanoma cells after 6 days of co‐culture with MSCs of different genotypes; values normalized to Hmox1 +/+ MSC co‐cultures (each bar represents mean + SEM, n = 2, *p < 0.05). (c) Experimental scheme of the fibrin bead assay with B16‐F10 melanoma cells and Hmox1 +/+ or Hmox1 −/− MSCs. (d) Growth dynamic of melanoma spheres in the fibrin gel, quantified using ImageJ (each bar represents mean ± SEM, n = 4, *p < 0.05). (e) Representative macroscopic (top) and microscopic (bottom) images of melanoma spheres after 6 days of fibrin bead co‐culture. (f) qRT‐PCR analysis of Tyr expression in B16‐F10 melanoma cells after 6 days of fibrin bead co‐culture with Hmox1 +/+ or Hmox1 −/− MSCs; values normalized to Hmox1 +/+ MSCs co‐culture (each bar represents mean + SEM, n = 4).

Fig 3: HO‐1 deficiency alters melanosome content and trafficking in B16‐F10 melanoma cells. (a) Flow cytometry analysis (FSC/SSC) of cell size and granularity; high granularity indicates high melanosome content. Representative dot plots. (b) Melanin content measured as % of SSChigh cells after 4‐day melanogenesis induction with or without 10 μM vitamin B3 (each bar represents mean + SEM, n = 3, **p < 0.01). (c) Spectrophotometric quantification of extracellular melanin content in cell culture media from cells treated with 10 μM vitamin B3 (each bar represents mean + SEM, n = 3, *p < 0.05).

Fig 4: Correlation of HMOX1 expression with pigmentation‐associated pathways in skin cutaneous melanoma (SKCM) and uveal melanoma (UVM). (a) Summary of significant correlations from the TCGA Firehose Legacy datasets (SKCM and UVM). (b) Representative scatterplots showing correlations between HMOX1 and selected genes in SKCM (top) and UVM (bottom): ATP6V0D2 (melanosome acidification), ATOX1 (copper handling), and SLC29A3 (lysosome–melanosome crosstalk), using mRNA expression (RNA Seq V2 RSEM) with both axes displayed on a log2 scale. Lower table contains Pearson correlation coefficients and p‐values for the selected correlations.

Fig 5: HO‐1 status does not affect iPSC differentiation toward melanocytes. (a) Timeline of the melanocyte differentiation protocol. (b) Phase‐contrast microscopic images of cells migrating from EBs at Days 6 and 15 of differentiation (representative images, 100×). (c) qRT‐PCR analysis of Mitf expression at Day 0 and at Day 11 of differentiation compared with Melan‐A melanocytes; Eef2 was used as a housekeeping gene (each bar represents mean + SEM, n = 3–4, **p < 0.01 vs. Hmox1 +/+;***p < 0.001 Melan A vs. Hmox1 −/− at Day 0; ## p < 0.01 vs. Day 0; #### p < 0.0001 vs. Day 0). (d–g) qPCR analysis of melanocyte markers at Day 40 of differentiation, in Hmox1 +/+ and four independent Hmox1 −/− iPSC‐derived lines; Eef2 was used as a housekeeping gene (each bar represents mean + SEM).