Fig. 3

HDAC6 controls CD47 expression in melanoma cells. A Comparison of basal expression levels of Cd47 in SM1 and B16 mouse melanoma cells by flow cytometry. BCd47 gene expression analysis of SM1 and B16 cells upon stimulation with IFNγ (100 ng/ml) in the absence or presence of 5 µM of NextA or Tubastatin A (TubA), evaluated by qRT-PCR. C Cell surface expression of Cd47 in SM1 cells upon stimulation with IFNγ in the absence or presence of NextA evaluated by flow cytometry. D Western Blotting analysis of HDAC6 after performing a partial knockdown (KD) in B16 cells compared to the non-target control (NT). Acetylated tubulin is used as a marker for HDAC6 inhibition, and alpha tubulin is a loading control. E qRT-PCR evaluating Hdac6 expression in B16 NT and HDAC6 KD cells. F-G qRT-PCR (F) and flow cytometry (G) analyses of Cd47 expression in NT and HDAC6 KD B16 cells. H Flow cytometry evaluating basal cell surface expression of CD47 in the human melanoma cell line WM164 NT and HDAC6 KD. I CD47 expression analysis of WM164 NT and HDAC6 KD cells upon IFNγ stimulation, evaluated by qRT-PCR at different timepoints. J qRT-PCR evaluating CD47 expression in human melanoma cells WM1361A upon IFNγ stimulation and in the presence of 2.5, 5, and 10 µM of NextA. K qRT-PCR evaluating CD47 expression in WM793 human melanoma cells upon IFNγ stimulation and in the presence of 5 µM of NextA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant