Figure 6.

Silencing ALG-2 prevents ESCRT-III-mediated membrane repair and curtails epithelial viability

TR146 cells were treated with ALG-2 siRNA or scrambled siRNA (control).

(A and B) ALG-2 silencing verified by immunoblotting using vinculin as loading control. Data in B are means ± SEM of 6 separate determinations.

(C) Representative micrographs of TR146 cells treated with 30 μM candidalysin. Membranes were stained with FM4-64 and PM rupture was detected by endomembrane staining and using DAPI. Insets: mitochondrial membrane potential assessed with rhodamine-123. Outlines of TR146 cells indicated by dotted lines. Scale bar represents 5 μm; dashed line indicates the position where the XZ image on top was constructed.

(D) Quantification of PM blebbing from experiments like those in C. Data are means ± SEM of ≥3 individual experiments.

(E) Representative time courses of [Ca²⁺]_c determinations made using GCaMP6s following 30 μM candidalysin treatment in Ca²⁺-containing medium. TG (500 nM) was added where indicated.

(F) Estimation of the [Ca²⁺]_c changes induced by 10 μM candidalysin. Standard deviation of the [Ca²⁺]_c changes induced by 10 μM candidalysin in cells treated with control or ALG-2 siRNA. Data are means ± SEM of at ≥3 individual experiments.

(G) Representative micrographs of TR146 cells treated with 10 μM candidalysin and stained with propidium iodide to identify dead cells, then permeabilized with 0.2% Triton X-100 and counter-stained to visualize all nuclei using Sytox Green. Scale bar represents 200 μm.

(H) Quantification of cell death from experiments like those in (C). Data are means ± SEM of ≥3 individual experiments. See also Figure S3.