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. 2021 Sep 13;118(38):e2009309118. doi: 10.1073/pnas.2009309118

Fig. 1.

Fig. 1.

PP2A acts as a human/mouse conserved ON switch to license NLRP3 responses downstream of multiple TLRs. (A) BMDMs were primed with 100 ng/mL ultrapure LPS, 1 µg/mL Pam3CSK4, and 100 µg/mL zymosan for 4 h or 2.5 µg/mL Poly(I:C) for 1 h, followed by stimulation with 5 µM nigericin for 1 h or 70 µM R837 for 3 h or 150 µg/mL MSU for 5 h. Cells were treated with the PP2A inhibitor okadaic acid (1 µM) for 15 to 30 min before nigericin, R837, or MSU stimulation. After stimulation, cell supernatants were collected and cells lysed. (B–D, F, and G) IL-1β cytokine secretion was measured in the BMDM supernatants using ELISA. Because IL-1β is not transcriptionally induced after 1 h of Poly(I:C) priming [optimal priming time for Poly(I:C)], inflammasome-dependent cell death was used as a readout instead, measured by LDH release assay in E. (H–J) Caspase-1 was detected in BMDM supernatants (SN) and cell lysates (XT) using Western blot. One representative Western blot is shown. IL-1β data are shown as mean + SEM from pooled results of three independent experiments, except in B where data are shown as mean + SEM from two independent experiments and E where LDH data are mean + SD of triplicates from one experiment and in F where IL-1β data are mean + SD of triplicates from one experiment representative of four. (K) IL-1β cytokine secretion after PP2A knockdown was measured in iBMDM supernatants using ELISA. Data are shown as a mean + SD of triplicates from one representative of three independent experiments. In conditions where not enough cells were available for triplicates, duplicates were seeded. (L) HMDMs were primed for 4 h with 100 ng/mL LPS, followed by stimulation with 5 µM nigericin for 1 h. Cells were treated with the PP2A inhibitor okadaic acid (1 µM) 15 min before adding nigericin. Supernatants were collected and IL-1β cytokine secretion was measured using ELISA. (M) Caspase-1 was detected in HMDM cell lysates (XT) using Western blot, with one representative Western blot shown. Data in L are shown as mean + SEM from pooled results of two independent experiments with four donors, with one representative Western blot shown in M. **P < 0.01, ***P < 0.001, ****P < 0.0001.