Journal: Bone

Article Title: HIF-1α mediates osteoclast-induced disuse osteoporosis via cytoophidia in the femur of mice.

doi: 10.1016/j.bone.2022.116648

Figure Lengend Snippet: Fig. 4. HIF-1α regulated the IMPDH and CTPS cytoophidia in osteoclasts. (A) The RNA-seq analysis of the osteoclasts induced by BMMs from the control and CKO mice indicated that the mRNA expression level of IMPDH1 and CTPS increased slightly. (B) Western blotting quantified the protein expressions of CTPS1 and IMPDH2, which showed that their expressions increased with the HIF-1α accumulation (n = 3 per group). (C) The IMPDH2 and CTPS1 immunofluorescence co-staining on the 2nd, 3rd, 4th and 6th day of induction with RANKL showed that these nucleic acid metabolic enzymes aggregated in the cytoplasm with special shapes (white arrowheads). The positive signal is in the shape of a dot, rod, short line, and ring. (D) In vivo, IMPDH2 and CTPS1 co-staining was performed. In osteoclasts, the dot and linear microstructure are visible. Moreover, the expression of these enzymes in the osteoclasts was significantly higher in the unloaded control group than the expression in the unloaded CKO group. (E) The IMPDH2 immunofluo rescence staining indicated that IMPDH2 cytoophidia (white arrowheads) increased in the CoCl2 group whereas the IMPDH2 cytoophidia decreased in the HIF-1α KO groups treated with or without CoCl2. (F) The CTPS1 immunofluorescence staining indicated that the CTPS1 cytoophidia increased in the CoCl2 group whereas the CTPS1 cytoophidia decreased in the HIF-1α KO groups treated with or without CoCl2. (G) The quantitative analysis of the IMPDH2 expression in the four groups. (H) The quantitative analysis of the CTPS1 expression in the four groups. Bars = 100 μm. The data were expressed as the median and interquartile range. Significance is presented in graphs and was determined by Student's t-test. Ab breviations: CTPS, cytidine-5-triphosphate synthase; DMSO, dimethyl sulfoxide; IMPDH, inosine 5-monophosphate dehydrogenase; MPA, mycophenolic acid.

Article Snippet: The primary antibodies used were mouse Hypoxyprobe antibody (HypoxyprobeTM-1Kit, USA), mouse anti-HIF-1α (BF8002, 1:200; Affinity, China), rabbit anti-HIF-1α (ab179483, 1:200; Abcam, USA), mouse anti-CTSK (sc-48353, 1:200; Santa Cruz, USA), rabbit antiCTP synthase (15914-1-AP, 1:200; Proteintech, China), rabbit antiIMPDH2 (12948-1-AP, 1:200; Proteintech, China) and rabbit anti-cMyc (ab32072; 1:200; Abcam, USA).

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Immunofluorescence, Staining, In Vivo