Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Article Title: p16INK4a downregulation alleviates temporomandibular joint osteoarthritis combined with type 2 diabetes by driving M2 polarization.
doi: 10.1016/j.biopha.2025.118172
Figure Lengend Snippet: Fig. 3. MS37452 and P16KD restrain inflammatory angiogenesis in inflammatory microenvironment of TMJ. (A) A diagram showing how to collect conditioned medium (CM) from M0. M0 cells were divided into three groups: Ctrl, P16KD, and MS37452. M0 were stimulated with or without TNFα ± GP during culture for 24 h. Next, medium was changed to fresh RPMI-1640 + 10 % FBS for 24 h and M0 CM was collected. At last, M0 CM was co-cultured with HUVECs. Then, HUVECs were used for subsequent transwell assay, scratch assay, tube formation assay, western blot and RT-qPCR. (B) Representative images and quantification of HUVECs transwell assay (Ctrl: M0 VS TNFα+GP: t = 66.86, df = 4. Ctrl-TNFα+GP VS P16KD-TNFα+GP: t = 77.36, df = 4. Ctrl-TNFα+GP VS MS37452-TNFα+GP: t = 65.01, df = 4). (C) Representative images and quantification of HUVECs scratch assay (Ctrl: M0 VS TNFα+GP: t = 23.12, df = 4. Ctrl-TNFα+GP VS P16KD-TNFα+GP: t = 23.14, df = 4. Ctrl-TNFα+GP VS MS37452-TNFα+GP: t = 22.50, df = 4). (D) Representative images and quantification of HUVECs tube formation assay (Ctrl: M0 VS TNFα+GP: t = 139.4, df = 4. Ctrl-TNFα+GP VS P16KD-TNFα+GP: t = 115.5, df = 4. Ctrl-TNFα+GP VS MS37452-TNFα+GP: t = 116.8, df = 4). (E) Western blot analysis of CD31 and VEGFA in HUVECs from different groups. (F) RT-qPCR analysis of CD31 and VEGFA in HUVECs from different groups (Relative expression of CD31 mRNA: Ctrl: M0 VS TNFα+GP: t = 66.96, df = 4. Ctrl-TNFα+GP VS P16KD-TNFα+GP: t = 80.36, df = 4. Ctrl-TNFα+GP VS MS37452-TNFα+GP: t = 86.18, df = 4. Relative expression of VEGFA mRNA: Ctrl: M0 VS TNFα+GP: t = 61.40, df = 4. Ctrl-TNFα+GP VS P16KD-TNFα+GP: t = 75.08, df = 4. Ctrl-TNFα+GP VS MS37452-TNFα+GP: t = 72.31, df = 4). Each experiment was performed three to five times. The data are presented as mean ± SD. P16KD, p16 knockdown; Ctrl, control; M0, human THP-1 derived macrophages; GP, high glucose and high palmitoleic acid. *P < 0.05 represent significant differences between the indi cated columns.
Article Snippet: After that, membranes were incubated at 4 ◦C overnight with primary antibodies against inducible nitric oxide synthase (INOS) (Proteintech Group, Rosemont, IL, USA), arginase 1 (Arg1) (Proteintech Group, Rosemont, IL, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Rosemont, IL, USA), transferrin receptor (TFRC) (Proteintech Group, Rosemont, IL, USA), vascular endothelial growth factor A (VEGFA) (Proteintech Group, Rosemont, IL, USA), alkaline phosphatase (ALP) (Proteintech Group, Rosemont, IL, USA), runt-related transcription factor 2 (RUNX2) (Proteintech Group, Rosemont, IL, USA), cluster differentiation 31 (CD31) (Proteintech Group, Rosemont, IL, USA), ferritin heavy chain (FTH1) (Proteintech Group, Rosemont, IL, USA).
Techniques: Cell Culture, Transwell Assay, Wound Healing Assay, Tube Formation Assay, Western Blot, Quantitative RT-PCR, Expressing, Knockdown, Control, Derivative Assay
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