Fig. 4: TG2 expression is required for pigmentation in human melanoma cell lines, and in vivo zebrafish and mouse models.

a Immunoblot analyses and relative densitometry of melanogenesis-related targets (TG2, Tyrosinase, and DCT) in human melanoma cell lines Mel JuSo, IPC-298 and SK-MEL-3. GAPDH was used as loading control (number of independent biological replicates = 3). Statistical significance is specified with asterisks (^*p < 0.05, ^***p < 0.001, ^****p < 0.0001). Data are represented as mean ± SEM. b Relative mRNA levels quantified by qRT-PCR analysis of TG2, DCT and TYRP1 expression in human melanoma cell lines Mel JuSo, IPC-298 and SK-MEL-3. β-actin was used as house-keeping gene in qRT-PCR (number of independent biological replicates = 3). Statistical significance is specified with asterisks (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001). Data are represented as mean ± SEM. c Photos of zebrafish morphology at 48 hpf comparing melanophores formations in TG2 KD and Ctrl morphants and relative quantification. The zebrafish larvae were injected with 0.1 pmol of zTg2b antisense morpholino/embryo. Ctrl Morpholino (CtrlMO) was used as reference. Images were acquired with the same exposure, at 3.2X magnification. Statistical significance is specified with asterisks (^****p < 0.0001). Data are represented as mean ± SEM. d Histology of mice skin: representative light micrographs of paraffin sections from C57BL/6 WT (A) and KO (B) mice skin, stained with hematoxylin and eosin, where the surface cornified layer and the numerous hair follicle are clearly identifiable; no detectable abnormalities are present in KO (B). C, and D depict magnifications. Melanin granules are visible along the dendritic extensions of melanocytes (arrowheads) are found in WT skin (shown in C). In TG2 KO skin melanin granules are mostly found in the perinuclear region of melanocytes (arrowheads) (shown in D). Scale bar: A, B = 150 µm; C, D = 12.8 µm.