Figure 3.
Impaired RSV-induced apoptotic cell death and reduced autophagy in patient primary fibroblasts. (A and B) Whole cell lysates from dermal fibroblasts from three controls (C, grey) and the patient (P, red) were analyzed for phosphorylation of ASK1/2 (pASK1/2), total ASK1, and vinculin (VCL) as loading control by immunoblotting (A). Graph depicting the densitometric quantification of pASK1/2 relative to total ASK1 (B). Bars show mean ± SD values of four experiments. (C–E) Primary fibroblasts from two controls (C, grey) and the patient (P, red) were stained with Annexin-V (apoptosis) and SYTOX Green (viability dye) to analyze induction of noninflammatory apoptotic cell death by flow cytometry. Cells were left untreated (UT) or treated with staurosporine (1 mM, 4 h) (C), mock transfected (transf ctrl) or transfected with PolyIC (tPolyIC, 500 ng/ml, 24 h) (D), or infected with mock virus (mock) or RSV (RSV-A strain Long, MOI 3, 24 h) (E). Bars show mean ± SD values of two experiments performed in duplicate and triplicate. (F–H) Primary fibroblast from three controls (C1–3) and the patient (P) were left untreated (UT), stimulated with rapamycin (Rapa, 500 nM, 24 h), HSV-2 (MS strain, MOI 1, 24 h), starved by culture in EBSS (4 h) or stimulated with H2O2 (300 nM, 30 min), in the absence or presence of chloroquine (+CQ, 20 μM, 4 h) to evaluate autophagy flux. Whole cell lysates were analyzed by immunoblot for LC3 lipidation and conversion of LC3-I into the smaller LC3-II with VCL as loading control. (H) Graph showing the ratio LC3-II/LC3-I as indicator for autophagy induction in primary fibroblasts from three controls (C, grey bars) and the patient (P, red). The ratio of LCR3-II/LC3-I was quantified using densitometry analysis of immunoblots in F and G. Bars show mean ± SD values. Statistics were calculated using the unpaired T test (B) and ordinary two-way ANOVA with Šídák’s multiple comparisons (C–E). ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: SourceData F3.

Impaired RSV-induced apoptotic cell death and reduced autophagy in patient primary fibroblasts. (A and B) Whole cell lysates from dermal fibroblasts from three controls (C, grey) and the patient (P, red) were analyzed for phosphorylation of ASK1/2 (pASK1/2), total ASK1, and vinculin (VCL) as loading control by immunoblotting (A). Graph depicting the densitometric quantification of pASK1/2 relative to total ASK1 (B). Bars show mean ± SD values of four experiments. (C–E) Primary fibroblasts from two controls (C, grey) and the patient (P, red) were stained with Annexin-V (apoptosis) and SYTOX Green (viability dye) to analyze induction of noninflammatory apoptotic cell death by flow cytometry. Cells were left untreated (UT) or treated with staurosporine (1 mM, 4 h) (C), mock transfected (transf ctrl) or transfected with PolyIC (tPolyIC, 500 ng/ml, 24 h) (D), or infected with mock virus (mock) or RSV (RSV-A strain Long, MOI 3, 24 h) (E). Bars show mean ± SD values of two experiments performed in duplicate and triplicate. (F–H) Primary fibroblast from three controls (C1–3) and the patient (P) were left untreated (UT), stimulated with rapamycin (Rapa, 500 nM, 24 h), HSV-2 (MS strain, MOI 1, 24 h), starved by culture in EBSS (4 h) or stimulated with H2O2 (300 nM, 30 min), in the absence or presence of chloroquine (+CQ, 20 μM, 4 h) to evaluate autophagy flux. Whole cell lysates were analyzed by immunoblot for LC3 lipidation and conversion of LC3-I into the smaller LC3-II with VCL as loading control. (H) Graph showing the ratio LC3-II/LC3-I as indicator for autophagy induction in primary fibroblasts from three controls (C, grey bars) and the patient (P, red). The ratio of LCR3-II/LC3-I was quantified using densitometry analysis of immunoblots in F and G. Bars show mean ± SD values. Statistics were calculated using the unpaired T test (B) and ordinary two-way ANOVA with Šídák’s multiple comparisons (C–E). ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: SourceData F3.

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