Journal: Cell Death & Disease

Article Title: IL-17 promotes osteoclast-induced bone loss by regulating glutamine-dependent energy metabolism

doi: 10.1038/s41419-024-06475-2

Figure Lengend Snippet: A – C The expression of Asct2 and Gls1 at the mRNA and protein level on day 1, 3, and 5 during OC differentiation. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significance. D , E Glu regulate RANKL-induced osteoclastogenesis in a concentration-dependent manner. BMDMs were treated with different concentrations of Glu (0 mM,0.5 mM, 1 mM, 2 mM and 4 mM) or 5 μM V9302 for 5 days. TRAP-positive multinucleated (>3 nuclei) cells were counted as osteoclasts. * p < 0.05, ** p < 0.01, *** p < 0.001. F , G BMDMs were seeded on 0.2% collagen-gel-coated 6-well plates and stimulated with 30 ng/ml M- CSF and 75 ng/ml RANKL for 6 days. Then, the cells were digested and seeded onto the Osteo Assay stripwell plates. Mature osteoclasts were treated with various concentrations of Glu or 5 μM V9302 for 5 days. F-actin staining was then performed. * p < 0.05, ** p < 0.01, *** p < 0.001. H , I Pit formation assay in the Glu (0 mM,0.5 mM, 1 mM, 2 mM and 4 mM) or 5 μM V9302 treated group. Mature osteoclasts were cultured on Osteo Assay stripwell plates and treated with the medium containing RANKL and various concentrations of Glu for 2 days. The cells were then washed from the surface by using the 10% bleaching solution for 5 min. Resorption pits were captured with light microscopy and analyzed with Image J software. * p < 0.05, ** p < 0.01, *** p < 0.001. J – M BMDMs were cultured with the medium containing M- CSF, RANKL and various concentrations of Glu or were treated by 5 μM V9302 for 5 days. Relative mRNA expression levels of Nfatc1, Mmp9, Ctsk, and Acp5 versus β-Actin were quantified by qPCR. *compared to Glu (0 mM)/V9302 (0 μM) group; #compare to Glu (2 mM)/V9302(0 μM) group; * p < 0.05, ** p < 0.01, *** p < 0.001. # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: V9302 was purchased from MedChemExpress (HY-W015229, MedChemExpress, USA).

Techniques: Expressing, Concentration Assay, Staining, Tube Formation Assay, Cell Culture, Light Microscopy, Software

Journal: Cell Death & Disease

Article Title: IL-17 promotes osteoclast-induced bone loss by regulating glutamine-dependent energy metabolism

doi: 10.1038/s41419-024-06475-2

Figure Lengend Snippet: BMDMs were cultured with the medium that containing M-CSF (30 ng/ml) and RANKL (75 ng/mL) for 5 days with or without Glu deprivation, as well as treated with indicated stimulation, including IL-17(0.1 ng/ml), V9302 (5 μM) and α-KG (0.5 mM). A , B BMDMs were seeded in Seahorse XF analyzer culture plates and treated as described. Extracellular acidification rate (ECAR) was analyzed by XF Cell Mito Stress Assay. C , D BMDMs were seeded in Seahorse XFp analyzer culture plates and treated as described. Oxygen consumption rate (OCR) were analyzed by XF Cell Mito Stress Assay. E BMDMs were seeded in 6 well plates and treated as described. The levels of lactate were analyzed through Lactate Assay kit. F , G BMDMs were cultured with the Glu deprived medium that containing M-CSF (30 ng/ml) and RANKL (75 ng/mL) for 5 days, as well as treated with or without IL-17 (0.1 ng/ml) and α-KG (0.5 mM). TRAP-positive multinucleated (>3 nuclei) cells were counted as osteoclasts. H , I QPCR and Western blot assay examining the the expression of gene of osteoclast marker and IL-17 signaling pathway. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significance.

Article Snippet: V9302 was purchased from MedChemExpress (HY-W015229, MedChemExpress, USA).

Techniques: Cell Culture, Lactate Assay, Western Blot, Expressing, Marker