Fig. 4: Sublethal doses of mitoTRAM-34 and rev-mitoTRAM reduce migration and anoikis resistance of B16F10 cells by affecting the cytoskeletal architecture.

a Representative images (left) and quantification (right) of wound scratch assays in B16F10 cells. Cells were treated with 0.5 µM mitoTRAM-34 (mitoT.), 5 µM rev-mitoTRAM (rev-m.), or 10 µM TRAM-34. The area of the gap has been highlighted in cyan for better visualization. The gap area was measured 0, 15, 20, and 24 h after performing the scratch using ImageJ and expressed as % of the initial gap area (mean + SEM, Two-Way Anova with Dunn’s posttest. N = 3–4. **p-value < 0.01, ***p-value < 0.001 compared to control). Scale bar is 250 µm. b Representative images showing the colony growth of B16F10 cells treated as in (a) in soft agar assays. The quantification on the right shows the total area of colonies measured with ImageJ and normalized to the control. However, small precipitates in TRAM-34-treated samples (likely due to the known low solubility of TRAM-34 [28]) were observed in soft agar assays therefore quantification of these samples are not reported in the graph. Mean + SEM of N = 3 is shown. Data were analyzed with One-Way Anova with Dunnett’s posttest (*p < 0.05, **p < 0.01 compared to the control). Scale bar is 5 mm. c The amount of ATP compared to the control in B16F10 cells treated for 24 h as indicated. 1 µg/ml oligomycin was used as negative control. Cells were cultured either in glucose or in galactose (that maximizes oxidative phosphorylation) to distinguish between total cellular and mitochondrial ATP production. Mean + SEM of N = 4 are shown (one-sample T test. *p < 0.05, **p < 0.01). Below, a representative Western Blot showing phosphorylated AMPK. β-actin was used as loading control. The quantification is shown in Fig. S4b. d Upper panel: Expression of HIF-1α and its target gene CA-IX in B16F10 cells after treatment for 24 h as indicated, analyzed by qRT-PCR. The normalized expression level relative to the control is reported (mean + SEM, N = 3). One-Way Anova with Dunnett’s posttest. ns: not significant). Below, representative Western Blot showing protein levels of HIF-1α and LOXL-2. β-actin was used as loading control. Quantification is shown in Fig. S4c. e Expression of genes related to epithelial-to-mesenchymal transition in B16F10 cells treated for 24 h as indicated. Data were analyzed as in (d). f Representative Western Blot of B16F10 cells treated for 24 h with 0.5 µM mitoTRAM-34 (mitoT.) or 5 µM rev-mitoTRAM (rev-m.) showing oxidative phosphorylation complexes I-V (CI-CV), mitochondrial outer membrane protein TOM-20 and β-actin (loading control). Quantification is shown in (g). g Quantification of the Western Blot shown in (f). TOM-20 was used for normalization and protein expression in treated samples is expressed as % compared to control (mean + SEM, N = 4. One-sample T test. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001). h Expression of BNIP-3 in B16F10 cells after treatment for 24 h as indicated, analyzed by qRT-PCR. The normalized expression level relative to the control is reported (mean + SEM, N = 3. One-Way Anova with Dunnett’s posttest. ***p < 0.001). i Representative Western Blots showing protein levels of BNIP-3 and CDC-42 treated as in (h). Quantification is shown in (j). j Quantification of the Western Blots shown in (i). Vinculin was used for normalization and protein expression in treated samples is expressed as % compared to control (mean + SEM, N = 3–4. One-sample T test. *p < 0.05, ***p < 0.001). k Confocal images of phalloidin staining in B16F10 cells treated for 24 h with 0.5 µM mitoTRAM-34 (mitoT.) or 5 µM rev-mitoTRAM (rev-m.). Multiple filopodial extensions are visible in control cells (indicated by white arrows) that are mostly lacking in treated samples. Scale bar is 15 µm. l Representative Western Blot showing protein levels of phosphorylated and total NF-κB in B16F10 cells treated as indicated for 24 h. Quantification is shown on the right panel. Vinculin was used for normalization and protein expression in treated samples is expressed as % compared to control (mean + SEM, N = 3. One-sample T test. *p < 0.05). m Quantification of wound scratch assays in B16F10 cells. Cells were treated with 0.5 µM mitoTRAM-34 (mitoT.) alone or together with NF-κB inhibitor TPCA-1 (0.5 µM), Rho/Rac/Cdc42 Activator I (2.5 µg/ml) or BNIP-3 overexpression (OE). The gap area was measured 0, 15, 20, and 24 h after performing the scratch using ImageJ and expressed as % of the initial gap area (mean + SEM, Two-Way Anova with Dunn’s posttest. N = 3–4. *p-value < 0.05, ***p-value < 0.001 compared to control). Representative images are shown in Fig. S4h. n As in (k), but cells were treated either with 0.5 µM mitoTRAM-34 (mitoT.) alone or together with 2.5 µg/ml Rho/Rac/Cdc42 Activator I (Cdc Act. I), which largely restores filopodal extensions (white arrows). Scale bar is 15 µm.