Fig 2: HSPA8 contributes to PRRSV infection along with CD163. (A) HSPA8 co-localized with internalized PRRSV in BHK-21 cells. The cells were inoculated with PRRSV (10 MOI) at 37°C for 0.5 h, and fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and stained with anti-HSPA8 pAbs (catalog no. 10654-1-AP; red) and anti-PRRSV GP5 MAb (green). The cell nuclei were stained with DAPI (blue). Images were acquired on the Zeiss confocal microscope. The co-localization was assessed by determination of Pearson’s correlation coefficient. Scale bars, 10 µm. (B-C) HSPA8 contributed to PRRSV infection. BHK-21 cells were transfected with HSPA8-Myc and/or CD163 for 36 h and then infected with PRRSV (1 MOI) for 24 h. The cells were subjected to IB using anti-Myc MAb, anti-CD163 pAbs, anti-actin MAb, and anti-PRRSV N MAb (B). PRRSV RNA abundance was determined by RT-qPCR (C). Data represent means ± SD from three independent experiments. *, P < 0.05.

Fig 3: HSPA8 is important for PRRSV attachment and internalization in CRL-2843-CD163 cells. (A, B) HSPA8 was expressed both on the cell surface and in the cytoplasm of CRL-2843-CD163 cells. CRL-2843-CD163 cells were fixed with 4% PFA, stained with anti-HSPA8 MAb (catalog no. 66442-1-AP; red) and anti-ATP1A1 pAbs (green), respectively. Cell nuclei were stained with DAPI (blue) by confocal microscopy. Scale bars, 10 µm (A). WCLs and membrane extracts of CRL-2843-CD163 were subjected to IB using anti-HSPA8 pAbs (catalog no. 10654-1-AP), anti-ATP1A1 pAbs and anti-GAPDH MAb (B). (C) HSPA8 pAbs inhibited PRRSV infection. CRL-2843-CD163 cells were incubated with HSPA8-GST pAbs and GST pAbs at 1:16 dilution in DMEM at 37°C for 1 h. Then the cells were washed with PBS and inoculated with PRRSV (0.3 MOI) at 4°C for 1 h. After three washes, the cells were again incubated with DMEM containing the corresponding antibodies for 24 h. PRRSV RNA abundance was determined by RT-qPCR. Data represent means ± SD from three independent experiments. **, P < 0.01. (D) Soluble HSPA8 protein inhibited PRRSV infection. PRRSV at 0.3 MOI was incubated with HSPA8-GST or GST protein at the final concentration of 2 µM at 37°C for 1 h, and then inoculated in CRL-2843-CD163 cells and harvested at 24 h. PRRSV RNA abundance was determined by RT-qPCR. Data represent means ± SD from three independent experiments. **, P < 0.01. (E) HSPA8 inhibitor suppressed PRRSV infection. CRL-2843-CD163 cells were treated with apoptozole (25 µM) and infected with PRRSV at 0.3 MOI for 1 h. After three washes, the cells were again cultured for 24 h. PRRSV RNA abundance were determined by RT-qPCR. Data represent means ± SD from three independent experiments. *, P < 0.05.

Fig 4: Validation of signature genes in the MAPK-RAP1A dataset. (A) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different TNM stages. (B) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different T stages. (C) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different N stages. (D) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different M stages.

Fig 5: Legumain promotes chaperone-mediated autophagy of GPX4.Primary lgmnWT and lgmnKO mRTECs were treated with 1 µM erastin for 24 hours or incubated under normoxic or hypoxic conditions for 6, 12, and 24 hours. A Western blot assay of GPX4. B Quantification of GPX4 mRNA levels by qPCR. Primary lgmnWT and lgmnKO mRTECs were pretreated with MG132, CQ, and Baf A1 for 2 hours before treatment with 1 µM erastin for 24 h. C Western blot using an antibody against GPX4. D mRTECs were treated with 1 µM erastin for 24 h or incubated under hypoxic conditions for 24 h. Lysosomes and lysosome-free cell lysates were collected and the protein level of GPX4 was measured by western blotting. Lamp1 and ß-actin were used as loading controls of lysosomal and non-lysosomal proteins, respectively. E, F Primary mRTECs from lgmnWT and lgmnKO mice were infected with control or legumain-overexpressing lentiviral constructs for 48 h and then treated with 1 µM erastin or incubated under hypoxic conditions. Western blotting was performed using an antibody against GPX4. G In cultured HK-2 cells, a co-IP assay was performed using an antibody against legumain or control IgG and western blotting for legumain, GPX4, Lamp-2a, HSC70, and HSP90 was performed. H HK-2 cells were incubated under normoxic or hypoxic conditions for 24 h. Co-IP assays were performed using an antibody against legumain or control IgG and then western blotting for legumain, GPX4, Lamp-2a, HSC70, and HSP90 was performed.