Fig 2: LENOX promotes RAP2 interaction with DRP1 regulating its phosphorylation on serine 637 and mitochondrial fusion. A, MitoTracker CMXROS Red staining of 501Mel cells transfected with the indicated GapmeR or siRAP2C. MitoTracker signal is shown in grayscale and Hoescht in blue. Total mitochondrial count, area, perimeter, and mean form factor were calculated and compared by one-way ANOVA (Dunnett test). B, MitoTracker CMXROS Red staining of 501Mel cells with ectopic LENOX isoform expression. Mitochondrial parameters were calculated and compared as above. C–E, Phospho-DRP1 S637 and total DRP1 levels in 501Mel and A375 cell extracts, with H3 and ACTB as loading controls. F, Left, RAP2 IP from 501Mel cells expressing shRNAs revealed with antibodies to the indicated proteins. Right, LENOX and SAMMSON levels in the indicated IP fractions. G, IP of DRP1 from 501Mel cells (left) and LENOX and SAMMSON levels in the IP (right). H, PLA-mediated detection RAP2–DRP1 interaction in 501Mel cells. Appropriate negative controls using secondary antibodies only are shown. *, P < 0.033; **, P < 0.0021; ****, P < 0.0001.

Fig 3: LENOX and RAP2 modulate oxidative phosphorylation. A, Co-imaging of LENOX, p32, and RAP2 in 501Mel cells transfected with the indicated GapmeRs. B, RAP2-p32 colocalization in a human melanoma FFPE section. C, Levels of 16S rRNA, SAMMSON, and LENOX in mitochondrial and cytoplasmic fractions (top) and levels of RAP2, P32, and HSP60 proteins (bottom) D. Mitostress test of 501Mel cells 48 hours after transfection with indicated GapmeRs or siRAP2C. Experimental groups were compared by one-way ANOVA (Dunnett test). E, Mito Stress Test on cells with ectopic LENOX isoform expression. Experimental groups were compared as in D. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002.

Fig 4: LENOX and RAP2C promote metabolic switch upon BRAF inhibition. A and B, Mito Stress Test of 501Mel cells grown for 3 days with DMSO or vemurafenib (1 µmol/L) and transfected as indicated. Experimental groups were compared by one-way ANOVA (Tukey test). C, MitoTracker CMXROS Red staining of cells treated with DMSO or vemurafenib, transfected as indicated and analyzed by confocal microscopy as in Fig. 4A. D, Phospho-DRP1 S637 and total-DRP1 levels in cells treated as above. H3 was used as loading control. E and F, Proliferation and apoptosis of cells treated as indicated. Experimental groups were compared by one-way ANOVA. G and H, Crystal violet staining of GapmeR-transfected cells. Percentages of occupied areas were compared by one-way ANOVA. I, Growth of MEL006res PDX in mice treated with DT with or without nontargeting ASO (DT+GAP-CTR) or LENOX-targeting ASO (DT+GAP-LENOX). Tumor size was measured daily for 20 days. Statistics were calculated using two-way ANOVA (Šidák multiple comparisons test). J, Kaplan–Meier plot showing overall survival (OS) of mice described in I. DT, n = 5; DT+GAP-CTR, n = 3; DT+GAP-LENOX ASO, n = 4. Statistical analysis were calculated by log-rank (Mantel–Cox) test. *, P < 0.033; **, P < 0.0021; ***, P < 0.0002; ****, P < 0.0001.