Duck enteritis virus infection suppresses viability and induces apoptosis and endoplasmic reticulum stress in duck embryo fibroblast cells via the regulation of Ca²⁺

Figures

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  Fig. 1.. Chinese standard challenge strain duck enteritis virus (DEV-CSC) infection induced apoptosis and inhibited viability in a time dependent manner. Duck embryo fibroblast (DEF) cells were infected with 10 multiplicity of infection (MOI) DEV-CSC for 2 hr, and cultured in normal medium for 0 hr, 24 hr, 48 hr and 72 hr. (A) The cell viability was assessed by cell counting kit 8 (CCK8) assay. (B–C) Cell apoptosis was estimated using flow cytometry and an annexin V fluorescein isothiocyanate (annexin V-FITC) apoptosis detection kit. Values are expressed as the mean ± SD (n=3). *: P<0.05, **: P<0.01, ***: P<0.001 vs. the 0 hr group.

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  Fig. 2.. Chinese standard challenge strain duck enteritis virus (DEV-CSC) infection induced apoptosis and Ca2+ accumulation inhibiting duck embryo fibroblast (DEF) cell viability. (A) Cell counting kit 8 (CCK-8) assay was used to determine the viability of DEF cells infected with 10 multiplicity of infection (MOI) DEV-CSC for 0, 2 and 4 hr, respectively, and then cultured in normal medium for 48 hr. (B–C) After treatment with 10 MOI DEV-CSC for 0, 2 and 4 hr, flow cytometry was used to evaluate apoptosis in DEF cells after culture in normal medium for 48 hr. (D) The cell viability was confirmed at 48 hr after infection with DEV-CSC at doses of 0–10 MOI. (E–F) Cell apoptosis was examined using flow cytometry and (G) Ca2+ mediated cellular fluorescence was analyzed using Fluo-4. Values are expressed as the mean ± SD (n=3). *: P<0.05, **: P<0.01, ***: P<0.001 vs. the control.

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  Fig. 3.. Different doses of Chinese standard challenge strain duck enteritis virus (DEV-CSC) induced unfolded protein response (UPR) in duck embryo fibroblast (DEF) cells. DEF cells were infected with 0–10 multiplicity of infection (MOI) DEV-CSC for 2 hr, and then cultured in normal medium for 48 hr. (A) Western blot was then used to evaluate the protein expression levels of C/EBP homologous protein (CHOP), glucose regulatory protein 78 (GRP78) and activating transcription factor 6 (ATF6) after 0–10 MOI DEV-CSC infection. The relative expression levels of CHOP (B), GRP78 (C) and ATF6 (D) were quantified based on their gray values. *: P<0.05, ***: P<0.001 vs. the control group.

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  Fig. 4.. Chinese standard challenge strain duck enteritis virus (DEV-CSC) induces apoptosis in duck embryo fibroblast (DEF) cells via changes in Ca2+. DEF cells were infected with 10 multiplicity of infection (MOI) DEV-CSC for 2 hr, and then cultured in normal medium supplemented with ethylene diamine tetraacetic acid (EDTA) for 48 hr. (A) The Ca2+ fluorescence of EDTA chelated DEF cells was evaluated using Fluo-4 while (B) cell viability was evaluated using the CCK-8 assay in DEF cells treated with 10 MOI or 10 MOI+EDTA; (C–D) Apoptosis in DEF cells treated with 10 MOI or 10 MOI+EDTA was also evaluated using flow cytometry. *: P<0.05, **: P<0.01, vs. the 10 MOI group.

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  Fig. 5.. Chinese standard challenge strain duck enteritis virus (DEV-CSC) induced endoplureasmic reticulum stress (ERS) in duck embryo fibroblast (DEF) cells via the regulation of Ca2+. DEF cells were infected with 10 multiplicity of infection (MOI) DEV-CSC for 2 hr, and then treated with ethylene diamine tetraacetic acid (EDTA) for 48 hr. (A) Western blot analysis of C/EBP homologous protein (CHOP), glucose regulatory protein 78 (GRP78) and activating transcription factor 6 (ATF6) expression in 10 MOI infected DEF cells with/without EDTA-treatment. (B) Relative expression of CHOP, GRP78 and ATF6 were quantified using their gray values. *: P<0.05, ***: P<0.001 vs. the 10 MOI group.

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  Fig. 6.. Chinese standard challenge strain duck enteritis virus (DEV-CSC) suppressed viability and accelerated apoptosis and endoplasmic reticulum stress (ERS) in duck embryo fibroblast (DEF) cells through the regulation of Ca2+. DEF cells were infected with 10 multiplicity of infection (MOI) DEV-CSC for 2 hr, and then were cultured with normal medium supplemented with 0 µM, 50 µM or 100 µM ethylene diamine tetraacetic acid (EDTA) for 48 hr. (A) Cell viability was then evaluated using cell counting kit 8 (CCK-8) assay and (B–C) apoptosis was quantified using flow cytometry. (D) After 2 hr of infection with 10 MOI DEV-CSC, DEF cells were treated with 0 µM, 50 µM or 100 µM EDTA for 48 hr. The expression of UPR related proteins were then determined by Western blot. *: P<0.05, **: P<0.01, ***: P<0.001 vs. the 0 µM group.