Fig. 5 | Bone Research

Fig. 5

From: HuR-mediated nucleocytoplasmic translocation of HOTAIR relieves its inhibition of osteogenic differentiation and promotes bone formation

Fig. 5

HuR is essential for the nucleocytoplasmic translocation of HOTAIR. a RNA-fluorescent in situ hybridization (FISH) was conducted to detect HOTAIR localization using digoxigenin (DIG) labeling probes specific for HOTAIR sequences (red). Nuclei were stained with Hoechst (blue). Scale bar, 20 μm. b Detection of U6 and 18 S RNA using RNA probes labeled with Cy3 (red). Nuclei were stained with Hoechst (blue). Scale bar, 20 μm. c RNA FISH to analyze the HOTAIR localization in BMSCs cultured with osteogenic medium for 0 days and 3 days. Scale bar, 20 μm. d KEGG analysis of the HOTAIR binding proteins in BMSCs through biotin-labeled full-length HOTAIR or biotin-labeled control RNA-based RNA pulldown by mass spectrometry. e Western blot analysis of HOTAIR binding protein HuR in BMSCs by biotinylated RNA pulldown. f qRT‒PCR analysis of HOTAIR levels by RNA immunoprecipitation in BMSCs with a HuR antibody, and rabbit IgG was used as a control. g Western blot analysis of HuR protein levels in BMSCs cultured with osteogenic medium for 0 days and 3 days. h Structured illumination microscopy (SIM) images for the colocalization of HuR and HOTAIR in BMSCs cultured with osteogenic medium for 0 days and 3 days. FISH was conducted to detect HOTAIR using DIG labeling probes specific for HOTAIR sequences and TRITC secondary antibody (red). Immunofluorescence was performed to detect HuR using a HuR antibody and FITC secondary antibody (green). Scale bar, 10 μm. i The distribution of HOTAIR in BMSCs cultured with osteogenic medium for 0 days and 3 days. Scale bar, 10 μm. All data are the mean ± s.e.m. from three independent experiments. Two-tailed unpaired Student’s t test was used for statistical evaluations of two group comparisons. **P < 0.01

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