Fig. 3: OGT interacts with MITF and promotes its nuclear translocation.
From: O-GlcNAcylation of MITF regulates its activity and CDK4/6 inhibitor resistance in breast cancer
a Immunofluorescence to examine the MITF localization in cells as indicated (n = 3 independent experiments). The scale bar represents 10 μm. Right low, quantification of data shown on the left. b Mass spectrometry analysis to identify MITF-associated proteins in MCF-7 PR cells. c Co-immunoprecipitation (co-IP) to detect the interaction of indicated proteins in MCF-7 PR cells. (n = 3 independent experiments). d In vitro O-GlcNAcylation assay to examine O-GlcNAcylation of MITF. Recombinant GST-MITF and His-OGT were purified from E. coli. (n = 3 independent experiments). e HEK293T cells were transfected with the indicated plasmids for 48 h before being harvested for co-IP assay. FLAG-IPs were immunoblotted for indicated proteins. (n = 3 independent experiments). f HEK293T cells were transfected with the indicated siRNAs and plasmids for 48 h before being harvested. FLAG-IPs were immunoblotted for the indicated proteins. (n = 3 independent experiments). g MCF-7 PR cells were transfected with the indicated siRNAs for 48 h, followed by immunostaining to examine the localization of MITF (n = 3 independent experiments). The scale bar represents 10 μm. Right up, cells shown in the left image were harvested and immunoblotted for indicated proteins; Right low, quantification of data shown in the left. h MCF-7 PR cells were transfected with the indicated siRNAs and plasmids for 48 h before being harvested. FLAG-IPs were immunoblotted for the indicated proteins. (n = 3 independent experiments). i, j MCF-7 PR cells were treated as indicated for 48 h and then harvested for co-IP. FLAG-IPs were immunoblotted for indicated proteins. (n = 3 independent experiments). k MCF-7 PR cells were transfected with indicated plasmids, and the nuclear fractions were subjected to the biotin pull-down assay, followed by immunoblotting for indicated proteins. (n = 3 independent experiments). Upper, sequence of MITF-DNA-binding motif tagged with biotin. The red color represents the E-box of the MITF binding motif. l, m Cell viability of MCF-7 PR cells (l) and T-47D PR cells (m) treated with increasing concentration of palbociclib for 3 days after MITF depletion by siRNA (n = 3 independent experiments). **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.
