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Contrary to mock-treated macrophages, LeTx induced a gradual increase in cell volume starting approximately 13 min before cell rupture (Fig. 6a, b, Supplemental Fig. 7a and Supplemental Movie 13).

In order to obtain standard spherical particles and decrease defects on the surface, the AgNP-decorated SiNW array was annealed in N2 at 200°C for 90 min before cell fabrication.

We serum starved the cells for 24 h and added IGF1 either during the whole starvation period or during the last 10 min before cell lysis (Figure 5C).

To relate the intracellular tTG distribution to membrane compartments, the intracellular membranes were labeled for 10 min with 10 µg/ml styryl dye FM4-64FX 30 min before cell fixation and labeling with anti-tTG antibody.

Approximately 90 min before cell harvest, the lymphocyte culture was supplemented with 500 ng/ml colcemid.

Samples were incubated in the dark for 30 min before cell cycle analysis.

The kinetics of lysosome destabilization in FlaTox-treated C57BL/6J (B6) macrophages was closely aligned with that of LeTx-intoxicated cells, with LysoTracker signal decay occurring around 6 9 min before cells became Sytox Green-positive (Fig. 4c, d).

Under these conditions, an increased Fluo4 signal was noted 6 9 min before cells became PI positive (Fig. 7c, d, Supplemental Fig. 10b and Supplemental Movie 16), in line with FlaTox-induced cell volume increase being slightly delayed relative to LeTx-treated cells (Fig. 6).

Quantification of data from 21 cells from several independent experiments showed that Annexin-V staining was observed in B6Nlrp1b+ BMDMs treated with PA+LFn-FlaA (here called FlaTox) approximately 3 min before cells became PI positive (Fig. 2c, d, Supplemental Fig. 1b and Supplemental Movie 5).

Propidium iodide (PI) (1.4 μg/ml) was added 5 min before cells were analyzed using an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).

Preincubation with JNK inhibitor, SP600125 (40  μM), or/and ERK inhibitor, PD98059 (60  μM), was carried out for 30 min before cells were treated with 50  μM FK506.