Fig. 4: CTSB upregulation in response to MFF silencing enhances catabolism in melanosomal lumen.

A Gene ontology analysis (ShinyGO online tool S⁵⁵) of genes changed upon Mff and Drp1 silencing in melan-a; three independent experiments. Mff , but not Drp1 , silencing induces lysosomal and catabolism pathways in melanocytes. B Representative Western Blotting image of CTSB expression in melan-a treated with Mff, Drp1, and control siRNA. C Quantification of CTSB expression levels in silenced melan-a, normalized to β-actin. Mean ± SEM; from four independent biological experiments. Kruskal–Wallis test followed by Dunn’s multiple comparisons test, *p = 0.0252. D Representative images of control and Mff siRNA-treated melanocytes stained with HMB45 and anti-CTSB antibody. Scale bar, 10 μm, and for magnified regions, 1 μm. E Quantification of HMB45 colocalization with CTSB in melan-a melanocytes treated with control and Mff  siRNA. Mean ± SEM; n = 30 cells from three independent experiments. Two-tailed Mann-Whitney test, *p = 0.0189. F Representative images of the Magic Red assay for the measurement of CTSB degradative activity in control and Mff-silenced melan-a. Scale bar 200 µm. G Quantification of CTSB activity (Magic Red intensity, a.u. = arbitrary units) in Mff-silenced melan-a. Mean ± SEM; four independent experiments. Two-tailed Mann-Whitney test, *p = 0.0286. H Representative images of melan-a stained with Acridine Orange (AO) and treated with the indicated siRNA. AO-positive spots were detected by automated image analysis (Harmony® software). Scale bar 100 µm. I Quantification of AO intensity in melan-a treated with the indicated siRNA. Mean ± SEM, four independent experiments. Kruskal–Wallis test followed by Dunn’s multiple comparisons test, *p = 0.0407. J Representative Western Blotting image of control and double silencing of Mff and Ctsb in melan-a. K Automated detection and analysis (Harmony^® software) of HMB45-positive melanosomes was applied for quantification of their perimeter in melan-a cells treated with Mff and Ctsb siRNA. Mean ± SEM, four independent experiments. Two-tailed unpaired Student’s t test, *p = 0.0274. L Quantification of melanosome number in Mff and Ctsb-silenced melan-a cells. Mean ± SEM, four independent experiments. Two-tailed unpaired Student’s t test. *p = 0.0274.