Fig 2: TR1 knockdown melanocytes have decreased pigmentation. (a) TR1 activity in TR1ctrl and TR1low cell lines, n = 3. (b) Cell pellets from TR1ctrl (left), and TR1low cells (right). (c) Western blot analysis of protein expression for TYRP1, TR1, and TYR in TR1ctrl and TR1low cells. (d) Tyrosinase activity was measured for the TR1ctrl and TR1low cells, n = 3. (e) mRNA analysis by qPCR for TR1low cells relative to TR1ctrl with gene expression normalized to RPLP0 using the ??CT method. Bars represent the mean log2 of the change in gene expression inTR1low cell cells relative to TR1ctrl. p < 0.001 for TXNRD1; p = 0.053 for TYRP1; p = 0.22 for TYR; p = 0.01 for DCT; p = 0.45 for MITF. All cells evaluated were propagated in complete melanocyte medium then moved to minimal medium for 72 h before analysis (see Section 2). Full Western blot images can be found at Supplementary Materials.

Fig 3: Disruption the gene encoding TR1 (TXNRD1) using CRISPR/Cas9 results in loss of expression of the melanocyte-specific isoform of MITF (MITF-M) in melanoma cells. (a) Analysis of the PIG1 parent cell line and M14 melanoma cells with wild-type TR1 treated with H2O2 under reducing and non-reducing conditions gives results similar to those found for the TR1low and TR1ctrl cell lines, but M14TXNRD1+/- cells have no MITF-M (b) Map of exons used in MITF-A and the shorter melanocyte specific MITF-m. (c) RNAseq analysis shows that M14 cells with wild-type TR1 express exon 1M, while M14TXNRD1+/- cells do not (red text). Both cell lines contain transcripts mapping to exons 1A, 1B1b, and 2. Full Western blot images can be found at Supplementary Materials.

Fig 4: Acute effects of TR1, TRX1 and GSH inhibition on the expression of melanin synthesis proteins. (a) Growth of TR1low cells is sensitive to depletion of GSH by 72 h treatment with BSO, while TR1ctrl cells are unaffected (b) Western blot analysis comparing expression of TYRP1, TYR, and MITF after 48-h treatment with 10 µM BSO, 60 µM ATG or both BSO and ATG in the TR1ctrl and TR1low cells. (c) Western blot analysis comparing protein expression of MITF, TYRP1, and TR1 in TR1ctrl and TR1low cells after 24 and 48 h of 5 µM PX12 treatment. (d) GSH was measured in a portion of the cells treated with PX12 in (c). The amounts of reduced GSH are increased in PX12-treated TR1ctrl cells and TR1low cells at 48h compared to 24h (p < 0.003 for both cell lines). In all treatments GSH in TR1ctrl cells is significantly lower than in TR1low p < 0.001. Full Western blot images can be found at Supplementary Materials.

Fig 5: Non-transformed cells are killed by 4~5 µM AF, but spared by TrxR1 knockdown plus Bz or 2 µM AF plus Bz.A, B MCF10A cells were treated with the indicated concentrations of AF for 24 h. C, D MCF10A cells pretreated with the indicated doses of z-VAD, Nec, Fer, 3-MA, Baf, or CHX were further treated with AF for 24 h. E, F MCF10A cells transfected with siNC or siTrxR1 for 24 h were further treated with the indicated doses of Bz for 24 h. G, H MCF10A cells were treated with the indicated concentrations of AF and/or Bz for 24 h. A, C, E, G Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. * p < 0.001; ns (non-significant). B, D, F, H Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 µm.