Fig 2: Cellular HSP70 expression level in PEDV-infected cells. A–B Vero cells were infected with PEDV at an MOI of 1 and harvested at different times as indicated. Total RNA was isolated to analyse HSP70 and viral M gene mRNA levels by quantitative RT–PCR. The mRNA levels of HSP70 and viral M were normalized to the mRNA levels of GAPDH. C–D Cell extracts were analysed by Western blot using anti-HSP70, anti-PEDV, and anti-β-actin antibodies. β-actin served as an internal control. The protein levels of HSP70 and PEDV were quantified by determining band intensities and normalized to β-actin. E IPEC-J2 cells were infected with PEDV at an MOI of 0.1 or 1. Cells were harvested at 24 hpi. The cell extracts were analysed by Western blot using anti-HSP70, anti-PEDV, and anti-β-actin antibodies. β-actin served as an internal control. F The levels of HSP70 and PEDV protein in IPEC-J2 cells were quantified by determining band intensities, and subsequent normalization to the levels of data is the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: Quercetin and heat shock treatment. A–D Vero cells were heated (HS) or not (Ctrl) or treated with quercetin (100 μM; QCT) or not (DMSO). Twelve hours later, cells were inoculated with PEDV at an MOI of 1. Cells were harvested at 12 h and 24 h for Western blot analysis. The levels of PEDV N protein were quantified by determining band intensities, which were then normalized to the levels of β-actin. Data are the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) A Cell lysates collected at 12 hpi were subjected to Western blot assay with anti-HSP70, anti-PEDV, and anti-β-actin antibodies. B Cell lysates collected at 24 hpi were subjected to Western blot assay with anti-HSP70, anti-PEDV, and anti-β-actin antibodies. C–D Quantification of 12 h and 24 h results.

Fig 4: Colocalization of HSP70 and PEDV. Vero cells were infected with PEDV at an MOI of 1. Cells were fixed at 24 hpi, and double immunofluorescence analysis was performed with anti-PEDV (green) and anti-HSP70 (red) antibodies. Mock treatments served as negative controls. The nuclei were stained with Hoechst 333258 (blue). A Anti- HSP70 staining of mock-infected cells. B Anti-PEDV staining of mock-infected cells. C Merged image. D Anti- HSP70 staining of PEDV-infected cells. E Anti-PEDV staining of PEDV-infected cells. F Merged image.

Fig 5: Colocalization of PEDV M and HSP70. Cells were transfected with PEDV structure proteins. After 48 h, IFA was performed with anti-Myc (green) and anti-HSP70 (red) antibodies. Nuclei were stained with Hoechst 33,258 (blue). Colocalization between PEDV M and HSP70 was confirmed by confocal microscopy. A pCAGGS PEDV N-myc, B pCAGGS PEDV S-myc, C pCAGGS PEDV E-myc, D pCAGGS PEDV M-myc, and E non-transfected control. All images are merged.