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. 2020 Jul 23;21(15):5230. doi: 10.3390/ijms21155230

Figure 5.

Figure 5

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.