Fig 2: MX2 sensitizes melanoma cells to targeted therapy. (A) GFP‐ and MX2‐overexpressing WM983b melanoma cells were treated with 2‐µM vemurafenib and WM1366—with 100‐nM trametinib for 72 h. Cell viability was evaluated using CellTiter‐Glo luminescent viability assay. Luminescent signal of treated cells was normalized to respective untreated controls. Each circle on histogram depicts a mean value of an independent experiment and each bar represents the mean + SD (n = 3). Statistical significance was evaluated using Welch's t test after log transformation of the data. (B) Cells were treated as in (A) and protein expression analysis by immunoblotting was performed to evaluate MAPK pathway activity and apoptosis. (C) GFP/MX2‐overexpressing melanoma cells were transfected with XAF1 targeting siRNA or scrambled control (siCTR) 24 h prior to 2‐µM vemurafenib treatment of WM983b sublines and 100‐nM trametinib treatment of WM1366 sublines. After 72 h, CellTiter‐Glo luminescent viability assay performed to assess cell viability and (D) immunoblotting analysis for protein expression. Each circle on histogram depicts mean value of an independent experiment and each bar represents the mean + SD (n = 3). Statistical significance was assessed by one‐way ANOVA with Tukey's multiple comparison test after log transformation of the data

Fig 3: ACF suppresses anti-apoptotic proteins MCL-1 and XIAP. (A) MDA-MB-231 and HS578T cells were treated with 0 or 5 µM ACF for 6 h and then western blot analysis for MCL-1, XIAP, BCL-2 and BCL-XL expression was performed. (B) MDA-MB-231 and HS578T cells were treated with 5 µM ACF and expression of cleaved PARP was assessed after 0, 1, 3, 6, 10 and 24 h using western blot analysis. (C) MDA-MB-231 and HS578T cells were treated using 0 or 5 µM ACF for 24 h and expression of XAF1 and XIAP was assessed using western blot analysis. (D) MDA-MB-231 and HS578T cells were treated using 5 µM ACF for 0, 1, 3, 6, 10 and 24 h and the expression of XAF1 and XIAP was assessed using western blot analysis. In all experiments, GAPDH or β-actin served as the loading control. ACF, acriflavine; MCL-1, myeloid cell leukemia sequence 1; BCL-2, B-cell lymphoma 2.

Fig 4: XAF1 expression is associated with a better melanoma survival. (A) Kaplan–Meier melanoma‐specific survival analysis of 703 primary melanomas and (B) Kaplan–Meier overall survival analysis of 360 TCGA metastatic melanomas stratified by median XAF1 RNA expression. Low is defined as bellow median. Analyses performed applying univariate Cox proportional hazard model. (C) Association between XAF1 and MX2 RNA expression tested by Pearson's correlation in primary melanomas from the LMC and (D) in metastatic melanomas from the TCGA. (E) The violin plots show the expression levels (log2(TPM +1)) of each XAF1 isoform in the TCGA skin cutaneous melanoma (SKCM) samples. Graph created using online tool Gepia2. 38 (F) Survival heatmap based on expression levels of different XAF1 isoforms in several cancer types. Heatmap displays hazard ratios in logarithmic scale (log10); red blocks denote higher risks, and blue blocks denote lower risks. Framed blocks represent significant results in prognostic analyses. Heatmap created with Gepia2 38 online tool

Fig 5: Characterization of XAF1 expression. (A) WM1366 and MM382 cells were transfected with control siRNA (siCTR) or MX2 specific siRNAs (siMX2_1 and siMX2_2). Forty‐eight hours post transfection, proteins and RNA were extracted and analyzed by immunoblotting and RT‐qPCR, respectively. Dot plot depicts % decrease of XAF1 mRNA expression compared to siCTR. Each dot represents mean value of an independent experiment (n = 3). β‐tubulin was used for protein loading control. (B) Immunoblot analysis of XAF1 and MX2 protein expression in normal human melanocytes (NHM), primary and metastatic melanoma lines (β‐tubulin used as a loading control; MX2 immunoblot has been previously published in Juraleviciute et al. 5 ) and (C) XAF1 mRNA expression analysis in the same panel of cell lines. Bar represents mean value + SD (n = 3), and circles depict mean value of each independent experiment. XAF1 mRNA expression is normalized to primary melanoma WM1366 cell line. (D) XAF1 mRNA expression in melanoma tumor samples derived from lymph node metastases. Tumors expressing lower XAF1 mRNA levels compared to primary WM1366 are below the line. (E) Expression of ISGs and STAT1 in WM1366 and MM382 melanoma cells after 72 h of XAF1 downregulation with siRNA followed by 90‐min treatment with 1000‐IU/mL IFNα (β‐actin used as a loading control)