Fig. 7
O-GlcNAcylation target NUP153 is essential for MYC nuclear accumulation and osteoclastogenesis. a Levels of O-GlcNAcylation on NUP153 in early and late stages of osteoclastogenesis by immunoprecipitation (n = 6 per group). b MS2 spectrum of O-GlcNAcylated peptide in NUP153 from RANKL and TNFα costimulated RAW264.7 cells. c Western blot analysis for chromatin-bound MYC in the cells with Nup153 knockdown or OSMI-1 (n = 3 replicates). d Representative immunofluorescence images of confocal microscopy and quantification of the median MYC intensity within the nuclear volume in the cells treated with OSMI-1 and Nup153 siRNA (n ≥ 30 cells per group). Spearman correlation analysis on the intensities of MYC and NUP153 in cells treated with Nup153 siRNA (n = 20 cells). Unless specified, cells were treated with vehicle and non-targeting siRNA. e Representative images for TRAP staining on cells transfected with Nup153 siRNA. f Conceptual scheme of the functional role of O-GlcNAcylation modulation of NUP153 during osteoclastogenesis. The illustration was created by using images from Servier Medical Art (http://smart.servier.com/) and Scheng23 (https://commons.wikimedia.org/wiki/File:O-GlcNAc_cycling-1.png), licensed under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) and CC BY-SA 4.0 licenses (https://creativecommons.org/licenses/by-sa/4.0/), respectively. Bar graphs are shown as median ± IQR. Horizontal scale bars represent 5 µm (d) or 50 µm (e) in IF images. Statistical significance was determined by two-way (a) or one-way ANOVA (c, d). P-values and Spearman’s ρ are presented in the graphs. Linear regression and its 95% confidence interval (dashed line) are presented in the correlation plots (d). MFI, median fluorescence intensity
