Fig. 4.

SPHK1 and MTA3 are positively correlated with PD-L1 expression in melanoma patient samples. a Representative images of tetrachromatic immunohistochemical staining of DAPI, SPHK1, PD-L1, and MTA3 expression in a metastatic melanoma tissue array. Scale bars, 200/50 μm in insets. b Quantification and correlation analysis of SPHK1, PD-L1, and MTA3 based on Log₂-transformed fluorescent intensities of multiplex IHC staining. See also Supplementary Table 6. MTA3 or c-Myc expression was analyzed by western blot including images (c) and quantification (d), or RT-PCR (e; n = 3) in melanoma cell lines treated with PF543 or DMSO. The plot (d) was generated from three independent experiments and showed as mean ± SEM. f Melanoma cells transfected with siSPHK1 or scrambled negative control siRNA were visualized by western blot. See also Supplementary Fig. 7. Data represent mean ± SEM. ns non-significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined by Dunnett’s multiple comparisons test (d, e)