Fig 2: TLR3 signal activation induces HAO1 expression in alveolar epithelial cells.A Effect of 67NR and 4T1 exosomes treatment on HAO1 expression in alveolar epithelial cells by Western blot analysis. B Effect of transfection of RNA isolated from 67NR and 4T1 exosomes on HAO1 expression in alveolar epithelial cells by Western blot analysis. C Effect of poly (I:C) treatment on HAO1 expression in alveolar epithelial cells by Western blot analysis. D Effect of poly (I:C) inhalation on HAO1 expression in mice lung by Western blot analysis. E Detection of oxalate production in alveolar epithelial cells treated with poly(I:C) or poly(I:C) + CCPST. Means ± s.e.m are provided (n = 3). F Detection of TLR3 expression in alveolar epithelial cells infected with sg-control or sg-Tlr3 lentivirus. G Detection of HAO1 expression in alveolar epithelial cells treated with 4T1 exosomes, 4T1 exosomes + CU CPT 4a or 4T1 exosomes + sg-Tlr3 lentivirus by Western blot analysis. H Detection of oxalate production in alveolar epithelial cells treated with 4T1 exosomes, 4T1 exosomes + CU CPT 4a or 4T1 exosomes + sg-Tlr3 lentivirus. I Effect of IRF3 over-expression on IRF3, P-IRF3 and HAO1 expression in alveolar epithelial cells by Western blot analysis. J Diagram of the HAO1 promoter showing the location of IRF3 binding sites. K Luciferase activity of HAO1-promoter construct after transfection of IRF3 plasmid in alveolar epithelial cells. L Detection of IRF3, P-IRF3 and HAO1 expression in alveolar epithelial cells treated with 4T1 exosomes or 4T1 exosomes + shIRF3 by Western blot analysis. M Detection of oxalate production in alveolar epithelial cells treated with 4T1 exosomes or 4T1 exosomes + shIRF3. Means ± s.e.m are provided (n = 3). N Schematic diagram of the role of HAO1-mediated oxalate metabolism at pre-metastatic stage and metastatic stage. ***P < 0.001, ****P < 0.0001 according to two-tailed Student’s t test.

Fig 3: Schematic model of HSP27 negatively regulating cGAS-STING-mediated IFN-ß expression upon PRV proliferation. cGAS recognizes the dsDNA of PRV and then activates cGAS-STING-mediated IFN-ß signaling pathway. HSP27 directly binds to cGAS, promotes cGAS degradation through ubiquitination, attenuates cGAS-STING axis signaling transduction, and inhibits IRF3 phosphorylation and IFN-ß expression, thereby promoting PRV infection.

Fig 4: (A) PK15 cells were transfected with shRNA targeting US3 (0.5 µg) or control shRNA (0.5 µg) for 24 h. Then cells were infected with MOI of 1 PRV for 12 h. Cells were collected for cellular RNA extraction. cDNA was transcripted for IFN-ß mRNA detection. Data were listed as mean ± SD from three independent experiments. Comparison between two groups were examined by unpaired Student’s t test. ** p < 0.01. (B) PK15 cells were transfected with shRNA-US3 (1 µg) or shRNA-control (1 µg) for 24h followed with Myc-US3 (0.5 µg) plasmid transfection. 24 h post transfection, cells were collected and lysed for IRF3 and Myc tagged US3 expression using specific antibodies. GAPDH served as loading control. (C) PK15 cells were transfected with shRNA-US3 (1 µg) or shRNA-control (1 µg) for 24 h followed with ISD (2 µg/mL) plasmid transfection. 12 h post transfection, above cells were uninfected or infected with MOI of 3 PRV for another 12 h. Cells were collected for total RNA extraction. 1 µg RNA was transcripted into cDNA for IFN-ß mRNA detection. Data were expressed as mean ± SD from three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001. (D) PK15 cells were infected with PRV Bartha strain (WT) and PRV-shRNA-US3 at 3 MOI. Endogenous IRF3 expression level was determined by western blotting at 6 h and 12 h post infection. GAPDH served as loading control.

Fig 5: HSP27 enhances encephalomyocarditis virus (EMCV)-triggered RIG-I-like receptor (RLR)/melanoma differentiation-associated gene 5 (MDA5) signaling pathway. (A) A549 cells were transfected with either 150 nmol of siHSP27-Mix or siNC for 36 h before infecting with EMCV at an multiplicity of infection (MOI) of 0.01 for 36 h. The protein expression of MDA5, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, HSP27, and VP1 were analyzed by immunoblotting. GAPDH was used as a loading control. (B) A549 cells were transfected with pCMV-Myc or Myc-HSP27 (0.5 µg) for 24 h before infecting with EMCV at an MOI of 0.01 for 36 h. The protein expression of MDA5, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, Myc, and VP1 were analyzed by immunoblotting. GAPDH was used as a loading control.