Fig. 4: O-GlcNAcylation of MITF at S49 is required for its nuclear accumulation.

a MITF was purified from HEK293T cells and analyzed by LC-MS/MS analysis to identify the potential O-GlcNAcylation sites. The peptide of MITF from amino acids 34–56 with O-GlcNAcylation identified by LC-MS/MS was shown (note: S49 is a potential O-GlcNAc site). b HEK293T cells expressing indicated MITF or its O-GlcNAc mutants from plasmids were harvested 48 h after transfection, followed by co-IP and FLAG-IPs were then immunoblotted for indicated proteins. (n = 3 independent experiments). c Identification of the O-GlcNAcylation modification of MITF by in vitro O-GlcNAcylation assays. Recombinant GST-MITF, GST-MITF-S49A, and His-OGT were purified from E. coli. (n = 3 independent experiments). d MCF-7 PR cells were transfected with indicated plasmids for 48 h before being harvested for co-IP. (n = 3 independent experiments). FLAG-IPs were then immunoblotted for indicated proteins. e Immunofluorescence to examine the MITF localization with indicated treatment in MCF-7 PR cells (n = 3 independent experiments). The scale bar represents 10 μm. Right, the quantification of results is shown on the left. f MCF-7 PR cells were transfected with indicated plasmids for 48 h, then cytoplasmic and nuclear fractions were extracted and subjected to immunoblotting for indicated proteins. (n = 3 independent experiments). g MCF-7 PR cells were transfected with the indicated plasmids for 48 h, and then the nuclear fraction of cell lysates was mixed with biotin-tagged MITF DNA-binding motif, followed by streptavidin pull-down. Streptavidin pull-down was then immunoblotted for indicated proteins. (n = 3 independent experiments). h MCF-7 PR cells were transfected with indicated plasmids after depletion of endogenous MITF by shRNA, followed by qPCR to examine the expression of indicated genes (n = 3 independent experiments). i Cell viability was examined in MCF-7 PR cells with indicated treatments (n = 3 independent experiments). j, k Representative images (j) and growth curves (k) of MCF-7 PR xenograft tumors with indicated treatments for 3 weeks. n = 6 mice/group. **p ≤ 0.01. All error bars are expressed as mean ± SEM. Two-tailed Student’s t tests were employed for statistical evaluation. Source data are provided as a Source Data file.