atp stimulation  (InvivoGen)

Journal: bioRxiv

Article Title: Inhibition of PIM kinase in tumor associated macrophages suppresses inflammasome activation and sensitizes prostate cancer to immunotherapy

doi: 10.1101/2024.10.21.618756

Figure Lengend Snippet: A) Wild type (WT) and PIM triple knockout (TKO) BMDMs were stimulated with IFNγ (500 ng/ml) and LPS) (20ng/ml) for 24 hours or B) with IL-4 (20 ng/ml) for 24 hours, and lysates were collected for immunoblotting C) WT BMDMs were treated with PIM447 (3μM) and stimulated with IFNγ/LPS (M1) or IL-4 (M2). Lysates were analyzed by western blotting. D) WT BMDMs were pre-treated with PIM447 for 16 hours, followed by stimulation with LPS for 4 hours and ATP for 30 min to induce inflammasome activation. Lysates were analyzed by western blotting. E) Supernatant was collected from each cell condition and secreted IL-1β was quantified by ELISA. F) WT BMDM were pre-treated with PIM447, followed by LPS/ATP and immunoblotting was used to assess NF-κB proteins. G) mRNA analysis of TNFα.*p<0.05 Error bars = SEM.

Article Snippet: For inflammasome activation, BMDMs were treated with LPS (500ng/ml) (Invivogen) for 4 hours, followed by stimulation with ATP (2mM) (Invivogen) for 30 minutes.

Techniques: Triple Knockout, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay