Figure 7. Autophagy-Deficient Endothelial Cells Are More Susceptible to α-toxin in vitro.

(A) Percent cell death measured by the lactate dehydrogenase (LDH) assay in cultured endothelial cells harvested from WT and Atg16L1^HM mice inoculated with α-toxin or control suspension. Data represents 5 independent experiments, each using cells pooled from 2 WT or Atg16L1^HM mice. Data represent mean and SEM.

(B) Representative images of ethidium bromide incorporation by endothelial cells from WT and Atg16L1^HM mice inoculated with α-toxin as an assay for pore formation. Scale bar=10μm.

(C) Quantification of mean fluorescent intensity (MFI) per cell from (B). n=44 cells from 3 WT mice and 83 cells from 3 Atg16L1^HM mice. Data represent mean and SEM.

(D–F) Representative Western blots for Adam10 and actin in whole cell lysates prepared from endothelial cells (D) and bone marrow cells (E) harvested from WT and Atg16L1^HM mice. Quantification of Adam10 levels normalized to actin in endothelial cells shown in (F). n=5–6 mice/group. Data represent mean and SEM.

(G) Representative immuno-fluorescence staining of Adam10 in endothelial cells from WT and Atg16L1^HM mice. Scale bar=10μm.

(H) Quantification of mean fluorescent intensity (MFI) per cell from (E). n=63 cells from 3 WT mice and 137 cells from 3 Atg16L1^HM mice. Data represent mean and SEM.

(I) Adam10 mRNA expression in endothelial cells from WT and Atg16L1^HM mice normalized to GAPDH. n=5 mice/group.

(J–K) Representative Western blot (J) and quantification (K) for Adam10 and actin in whole cell lysates from WT endothelial cells treated with PBS or 20mM ammonium chloride (NH₄Cl) for 24 hours. n=7 replicates/group from 2 independent experiments (see supplemental methods for full experimental design).

(L–M) Representative Western blot (L) and quantification (M) for Adam10 and actin in whole cell lysates from WT endothelial cells treated with PBS 5μM Lys05 for 24 hours. n=6–9 replicates/group from 2 independent experiments.

(N–O) Representative Western blot (N) and quantification (O) for Adam10 and actin in whole cell lysates from WT endothelial cells treated with complete media (+serum) or media without serum (− serum) for 6 hours to induce autophagy. n=6–7 replicates/group from 2 independent experiments.

(P–Q) Representative Western blot (P) and quantification (Q) for Adam10 and actin in whole cell lysates from WT endothelial cells treated with PBS or 100μM epirubicin for 6 hours. n=4–5 replicates/group from 2 independent experiments.

(R) Survival curve of curve of WT mice treated i.p. with epirubicin (0.6μg/g body weight) or PBS vehicle control and inoculated intranasally with a 20μl suspension of filter-sterilized S. aureus culture supernatant containing α-toxin (+toxin). n=19–20 from 4 independent experiments.

*p<0.05, **p<0.01, ****p<0.0001