Extended Data Fig. 5: Overexpression of SAC2, but not SAC1 prevents endosomal PI4P accumulation, endosomal recruitment of NLRP3 and inflammasome activation.
From: Distinct changes in endosomal composition promote NLRP3 inflammasome activation
a, Confocal images of HeLa cells expressing mCherry, mCherry-tagged hSAC1 (mCh-SAC1) or mCherry-tagged hSAC2 (mCh-SAC2) treated with vehicle (Ctrl), 15 µM nigericin or 45 µg ml−1 CL097 for 60 min. Cells were co-stained with antibodies against PI4P and EEA1. Scale bar: 10 µm. b, Quantification of PI4P levels on endosomes in HeLa cells (upper panel, Means ± SD, N = 3, n = 90 cells for each group) and NLRP3 puncta-containing HeLa cells (lower panel, means of percentage ± SD, N = 4, n = 100 cells for each group) in experiments shown in panel a. c, Immunoblotting of supernatants and lysates from THP-1 cells stably expressing mCherry, mCherry-tagged hSAC1 (mCh-SAC1) or mCherry-tagged hSAC2 (mCh-SAC2). Cells were treated with vehicle (Ctrl), 10 µM nigericin for 45 min, 50 µM CL097 for 45 min or 100 µM R837 for 2 hours. Antibodies recognizing both p45 and p20 fragments of caspase-1 (CASP1), p31 and p17 fragments of IL-1β, NLRP3 and mCherry were used. Tubulin was chosen as a loading control. d, Cellular uptake of Sytox Green and ELISA analysis of IL-1β secretion of THP-1 cells stably expressing mCherry, mCherry-tagged hSAC1 (mCh-SAC1) or mCherry-tagged hSAC2 (mCh-SAC2). Cells were treated as described for panel c. Means ± SD, N = 3. Data were analyzed with an unpaired two-sided t-test (b, d). Data shown in a, c are representative of three independent experiments.
