Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: PI3K/AKT downstream Rac1-PAK1 signaling axis is not activated and involved in NDV entry into HD11 cells. (A) GTPase activity of Rac1 was analyzed by using the Rac1 activation assay kit, followed by Western blotting using an anti-Rac1 antibody. The level of GTP-Rac1 was normalized to the total Rac1. (B) The levels of p-PAK1 (T423) and PAK1 were analyzed by Western blotting using corresponding antibodies. GAPDH was used as a control. (C–G) Treatment with the Rac1 and PAK1 inhibitor, NSC23766 and IPA-3, showed no effect on NDV-induced LIMK1/CFN phosphorylation and entry of NDV. (C) HD11 cells were pretreated with NSC23766, IPA-3, or DMSO. Then the cells were inoculated or mock inoculated with NDV. Western blotting was used to determine the levels of p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN at 30 mpi with NDV. GAPDH was used as a control. (D–G) Treatment with the NSC23766 and IPA-3 had no effect on the adsorption and internalization of NDV. HD11 cells were pretreated with NSC23766 (D and E), IPA-3 (F and G), after which NDV adsorption and internalization assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Article Snippet: IPA-3 , PAK1 activation inhibitor , MCE , HY-15663.

Techniques: Activity Assay, Activation Assay, Western Blot, Control, Phospho-proteomics, Adsorption, Flow Cytometry

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