Fig 2: Tunicamycin incubation for 48 and 72 h did induce hepatocyte apoptosis. (A) Semi-quantitative PCR showing unspliced (Xbp1u) and spliced Xbp1 (Xbp1s) mRNA after 24 and 48 h tunicamycin (TM) incubation. The levels of Xbp1s mRNA became almost level in all types of hepatocytes at 48 h. (B) Western blots of UPR signaling proteins BiP, GRP94, IRE1α, ATF6, PERK, p-eIF2α, eIF2α, ATF4, and CHOP. High amounts of CHOP in Lcn2−/− declined comparable to wild type levels at 72 h. Accumulation of glycosylated and non-glycosylated LCN2 proteins in wild type showed significant uptick compared to Chop−/− hepatocytes in 48 and 72 h TM incubation, with GAPDH as loading control. (C) Depicting TM induction of p65 NF-κB, TRAF2, JNK, and p38 MAPK activations. Phosphorylation of JNK/c-Jun and p38 was markedly increased in Chop−/− hepatocytes, while the signals in wild type and Lcn2−/− already declined. The ATF2 phosphorylation remained persistent in all types of hepatocytes, but were slightly lower in Chop−/− at 72 h. (D) Bcl2 family protein expression showed decreased Bcl2 and Bcl-xL levels in all types of hepatocytes, while Bax was upregulated in Lcn2−/−. BH3-only proteins BIM and PUMA increased with highest levels in Lcn2−/− hepatocytes well in correspondence to the levels of cleaved Caspase-9 and 3, but minimal in Chop−/− hepatocytes. GAPDH served as loading control.

Fig 3: Chronic CCl4-induced hepatocyte apoptosis via JNK/c-Jun and mitochondrial pathways. (A) Western blot analysis and (B) quantification of p65 NF-κB, TRAF2, p-JNK, JNK, p-c-Jun, and c-Jun signaling cascades with GAPDH as a loading control, showing significant JNK/c-Jun activation in CCl4-treated mice. (C) Western blot analysis and (D) quantification showing Bcl2 family proteins Bcl-xL, Bcl2, Bax, and PUMA. Bax levels were higher in both types of CCl4-treated mice, with lower levels of Bcl2 in CCl4-treated Lcn2−/− livers, with fluctuating levels of PUMA. Cleaved Caspase-9 levels were higher in oil- and CCl4-treated Lcn2−/− mice, but TRB3 and cleaved Caspase-3 levels were increased in CCl4 administered livers, with GAPDH loading controls. (E) Immunohistochemistry staining of cleaved caspase-3 depicted positive signals distributed homogenously in non-parenchymal liver cells after mineral oil administration. In contrast, CCl4-treated wild type livers showed caspase-3 staining in infiltrating inflammatory macrophages around hepatic central veins, while in Lcn2−/− mice cleaved caspase-3 was detected in both inflammatory cells and surrounding hepatocytes. (F) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-stained paraffin slides showing TUNEL-positive infiltrating inflammatory cells around hepatic central veins of CCl4-treated wild type livers. Lcn2−/− livers showing TUNEL-positive cells scattered in hepatocytes zone 3 with minimal TUNEL-positive cells after mineral oil administration. (G) Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were significantly higher in CCl4-treated Lcn2−/− mice, while serum albumin levels showed decreases in CCl4-treated mice. Details about the statistics are given in Supplementary Tables S2–S4.

Fig 4: SC inhibits the NF-κB signalling pathway by promoting TRAF2 expression. A, B The interaction between TRAF2 and P65 and between TRAF2 and IκBα was predicted by the HDOCK server online. C, D Marc-145 cells were seeded in 12-well plates and transfected with siTRAF2 (50 nM), siNC (negative control), pXJ40-HA-TRAF2, or vector. Cells were harvested at 36 hpi after transfection to detect the expression levels of TRAF2, P65, p-P65, IκBα, p-IκBα, P100 and P52. E Marc-145 cells were seeded in 12-well plates and cultured for 12 h. The cells were infected with PRRSV SD-YL1712 (MOI = 1) and collected at 24 and 36 hpi for Western blotting to determine the expression levels of TRAF2 and p-TRAF2. F Marc-145 cells in 12-well plates were infected with PRRSV SD-YL1712 (MOI = 1) and then treated with 10 µM SC. Cells were harvested at 36 hpi for Western blotting to determine the expression levels of TRAF2, p-TRAF2, and PRRSV N.

Fig 5: PRRSV Nsp10 interacts with TRAF2 and decreases its expression. A CRL-2843 cells were transfected with pEGFP-C-PRRSV nonstructural proteins for 36 h and then harvested to determine the expression of pEGFP-C-Nsps and TRAF2. B HEK-293T cells were cotransfected with p3×Flag-Nsp10 and pXJ40-HA-TRAF2. After 36 h, the cells were collected, and Co-IP was used to determine the interaction between the Nsp10 protein and TRAF2. C HEK-293T cells were transfected with pXJ40-HA-TRAF2 and p3×Flag-Nsp10. After 36 h, the cells were collected to determine the reverse Co-IP interaction between TRAF2 and Nsp10. D HEK-293T cells were cotransfected with the p3×Flag-Nsp10 and pXJ40-HA-TRAF2 or p3×Flag-Nsp11 and pXJ40-HA-TRAF2 proteins. After 24 h, the cells were stained with anti-Flag and anti-HA antibodies to determine the position of Nsp10 (green) relative to TRAF2 (red), and images were captured by confocal microscopy.