Nigericin

Nigericin was from Abcam (Cambridge, UK), valinomycin from Fluka (Buchs, Switzerland). All other chemicals were from Sigma-Aldrich (Buchs, Switzerland).

Primary monocytes were seeded into 96‐well plates at 5 × 10³ cells/well, in RPMI 1640, GlutaMAX medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Lonza). When indicated, primary monocytes were incubated for 3 h in the presence of LPS (10 ng/ml, InvivoGen). Unless otherwise indicated, primary monocytes were then treated for 90 min with nigericin (5 μM, InvivoGen), ATP (2.5 mM, Sigma) (Mariathasan et al, 2006), staurosporine (1.25 μM, Tocris), UCN‐01 (12.5 μM, Sigma), or Ro31‐8820 (100 μM, Tocris). When indicated, monocytes were treated with colchicine (1 μM or at the indicated concentration, Sigma), MCC950 (10 μM, Adipogen AG‐CR1‐3615), paclitaxel (Taxol, 5 μM, Sigma), nocodazole (5 μM, Sigma), z‐YVAD‐FMK, z‐IETD‐FMK, z‐DEVD‐FMK (at the indicated concentrations, Bachem, #4027532, #4034771, #4027402, respectively), VX‐765 (InvivoGen), 30 min before addition of UCN‐01, TcdB (Abcam, #ab124001, 125 ng/ml), and TcdA (1 μg/ml) or nigericin. TcdA was purified from Clostridioides difficile VPI10463 strain, as previously described (von Eichel‐Streiber et al, 1987; Popoff, 1987). Following the incubation, cells were centrifuged and supernatants were collected.