Il 1β 200 01b

The specific reagents used in this study included antibodies against DNM1L (A2586), GAPDH (AC002), AKT (A3145), IKK (A2087), NF‐kBp65 (A2547), IL‐8 (A2541) from ABclonal (Wuhan, China); antibodies against p‐AKT (4060), p‐IKK (2697T), LC3B (3868T), COX‐2 (12282T) from Cell Signaling Technology; antibodies against NFKBIA (10268‐1‐AP) from Proteintech; the DNM1L inhibitor Mdivi‐1, a mitochondrial division inhibitor 1 (M0199) from Sigma‐Aldrich; IL‐1β (200‐01B) from Peprotech; MitoTracker Green, BCA protein assay kit and Annexin V‐FITC (C1048, P0010S, C1063) from Beyotime; DNM1L‐specific small interfering RNA (siRNA) and control scramble siRNA from Guangzhou RiboBio; the Genomic DNA Removal Kit, SYBR Green PCR Kit from ABM; the CCK8 kit from MEC; ROS Fluorescent Probe‐DHE from Vigorous; primers from Sangon Biotech; and SDS‐PAGE gels from Epizyme.

Recombinant human TNF‐α (315‐01A), IL‐6 (212–16), IFN‐γ (315–05), and IL‐1β (200‐01B) were purchased from Peprotech, Cranbury, NJ, and the JAK inhibitors, ruxolitinib (7064) and remdesivir (7226), from R&D Systems, Minneapolis, MN. Cells were stimulated with the cytokines IL‐6 (50 ng/ml), TNF‐α (20 ng/ml), and IFN‐γ (20 ng/ml) alone or a combination of TNF‐α + IFN‐γ (20 ng/ml of each) or all three (“cocktail” of IL‐6, TNF‐α, and IFN‐γ, 20 ng/ml of each) for 3, 5, or 7 days, unless indicated otherwise. For experiments with inhibitors, cells were cultured with or without JAK inhibitor (1 μM) or RDV (2 μM) for 30 min prior to cytokine stimulation. For conditioned medium (CM) experiments, cells were stimulated with or without cytokines for 3 days. Culture supernatants were then collected, centrifuged, filtered, and mixed with culture media at a 1:2 ratio. Early passage HUVECs were then cultured with control CM or cytokine‐induced senescent cell CM for 24 h.