Fig. 4: Calpain is a prerequisite for the release of cleaved IL-1α by human TH17 cells.
From: Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation
a, Immunoblot analysis of cell culture supernatants derived from TH17 cell clones that were restimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 5 d. b, Fold-change in relative fluorescence units (r.f.u.) after 1 h of incubation of TH17 cells with the calpain substrate Ac-LLY-AFC. TH17 cells were stimulated for 3 d with anti-CD3 and anti-CD28 monoclonal antibodies. c,e,g,h, ELISA of cell culture supernatants after stimulation of TH17 cells (c and e–g) with anti-CD3 and anti-CD28 monoclonal antibodies for 5 d and of monocytes (f) with LPS (24 h) and nigericin (30 min). Thapsigargin (g), 1 μM, was added on days 2 and 3 and EGTA (h) on day 0. d, Immunoblot analysis of human TH17 cell lysates. Human TH17 cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies and IL-1β in the presence or absence of calpain inhibitor II (10 mM) and analyzed on day 5. The data represent two experiments with two donors. e, TH17 cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 5 d after genetic depletion of CAPN1 or CAPN2 with CRISPR–Cas9 technology. Each circle indicates an independent blood donor. The data represent three independent experiments (b, c and e–h). P values were calculated using one-way ANOVA with Dunnett’s multiple-comparison test (c) or Fisher’s least significance difference test (e) or two-tailed, paired Student’s t-test (b and f–h).
