Fig 2: RBBP5 suppresses the growth of melanoma in vivo. (a) Images of nude mice at the end of experiment; (b) tumor volume (mm3) was measured to draw tumor growth curves and (c) tumour weight (mg) was measured to evaluate the growth of xenograft tumors; (d) Western blotting was used to detect the expression of marker proteins in the Wnt/ß-catenin signalling pathway (ß-catenin and c-myc) and EMT (N-cadherin, E-cadherin, and MMP-7). (e) Western blotting was used to detect the expression of H3K4me3 and p16 in mouse xenograft tumour tissues; IHC was used to detect the expression of RBBP5, H3K4me3, and N-cadherin in mouse xenograft tumour tissues. (f) Representative images and (g–i) quantification results were shown. Scale bar: 20 µm. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: A model of the biological function and mechanism of RBBP5 in melanoma.

Fig 4: RBBP5 inhibits the Wnt/ß-catenin signalling pathway and EMT. (a–d) The effect of RBBP5 silencing on the Wnt/ß-catenin signalling pathway and EMT marker expression was evaluated by western blotting in A375 and A2058 cells. (e–h) The effect of RBBP5 overexpression on the Wnt/ß-catenin signalling pathway and EMT marker expression was evaluated by western blotting in A375 and A2058 cells. Data are represented as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and ***p < 0.001; ns, no significance.

Fig 5: RBBP5 was downregulated in melanoma. (a, b) Western blot to explore the expression of RBBP5 in three pairs of melanoma tissue compared with nevi and adjacent tissue. (c, d) Western blotting and (e) qRT-PCR were performed to verify the mRNA and protein expression of RBBP5 in two melanoma cell lines (A375 and A2058) compared with a human immortalized keratinocyte (HaCaT) cell line. (f) IHC of RBBP5 was performed in 106 cases of melanoma tumor tissues, 100 cases of adjacent tissues, and 23 cases of nevi tissues. Representative images with different tissues stained are shown. Scale bar: 200 µm and 50 µm. (g, h) The relative score of RBBP5 was quantified by the grade of staining intensity and the percentage of positively stained cells. A375 and A2058 cells were transfected with empty vector (shRBBP5-vector) or shRNAs targeting RBBP5 (shRBBP5), and the relative mRNA level of RBBP5 was measured by qRT-PCR. GAPDH was used as an internal control (j). The protein expression of RBBP5 was detected by western blotting. GAPDH was used as a loading control (k, l). A375 and A2058 cells were transfected with RBBP5 empty vector (RBBP5-vector) or RBBP5 overexpression lentivirus (RBBP5), and transfection efficiency was verified by q-PCR (i) and western blotting (m, n). GAPDH was used as a loading control. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, no significance.