Fig 2: Tpl2 is required for VEGF-A-stimulated ATF-2-dependent VCAM-1 expression. (A) Control, scrambled, Tpl2 or ATF-2-specific siRNA-treated endothelial cells were stimulated (25 ng/ml) with either VEGF-A165 or VEGF-A121 (negative control) for 4 or 8 h, prior to immunoblot analysis. (B–E) Quantification of protein levels by immunoblot analyses (see the Materials and Methods) of VEGF-A (25 ng/ml) stimulated control, scrambled, Tpl2-depleted or ATF-2 depleted endothelial cells for protein levels of (B) VCAM-1, (C) Tpl2, (D) ATF-2-pT71 and (E) ATF-2. Error bars indicate ±s.e.m. (n=3). In panels B–E, statistical tests=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 3: H-KI20 inhibited VEGF-stimulated phosphorylation of ATF2 and JNK. HRECs were pretreated with H-KI20 for 24 hours before stimulation with or without VEGF for 15 minutes. Protein expression of ATF2 and p-ATF2 (A) and JNK and p-JNK (B) were analyzed by Western blotting. Densitometric analysis of Western blot images of p-ATF2 over ATF2 and p-JNK over JNK was performed. Three independent experiments were performed, and data were expressed as means± SEM when compared to untreated cells, *P < 0.05, **P < 0.01.

Fig 4: PZP promotes UCP1 expression via a P38 MAPK‐ATF2 pathway. A) Immunoblots of cell lysate from differentiated BA which were cultured in refeeding medium supplemented with PZP protein (10, 100, 500 ng mL−1) using indicated antibodies which showed PZP significantly increased the phosphorylation of p38 MAPK in condition of refeeding. B) Immunoblots of cell lysate from differentiated BA which were cultured supplemented with or without thapsgargin (600 µm) and PZP protein (100 ng mL−1) using indicated antibodies. C) Immunoblots of BAT lysates from control mice and PZP KO mice when mice were fed upon HFD and IF after 2 weeks without difference in body weight between groups using indicated antibodies which showed phosphorylation of p38 MAPK and ATF2 were decreased in PZP KO mice. The samples were collected when mice were refed for 6 h after 24‐h fast. D) Immunoblots of cell lysate from siGRP78‐transfected BA and controls which were cultured in refeeding medium supplemented with or without PZP protein (100 ng mL−1) using indicated antibodies, which showed UCP1 expression and phosphorylation of p38 MAPK and ATF2 upon PZP treatment were significantly inhibited by GRP78 siRNA. E) Immunoblots of cell lysate from differentiated BA which were cultured in refeeding medium supplemented with or without SB203580 (10 µm) and PZP protein (100 ng mL−1) using indicated antibodies, which showed PZP‐induced UCP1 expression under refeeding condition was significantly inhibited by SB203580 treatment. F) Immunoblots of cell lysate from differentiated BA which were cultured supplemented with or without SB203580 (10 µm) and PZP protein (100 ng mL−1) after thapsigargin (600 µm) stimulation using indicated antibodies, which showed PZP induced UCP1 expression under thapsigargin treatment was significantly inhibited by SB203580 treatment. Data are shown as mean ± SEM. One‐way (A–C) or two‐way ANOVA (D–F) with multiple comparisons and Tukey's post‐test were performed; *** p <0.001, ** p < 0.01, and * p < 0.05 were considered to be significant.

Fig 5: JNK signaling pathway is involved in LH-induced apoptosis in HSC-3 cells. (A) mRNA expression of c-Jun was detected via reverse transcription-quantitative polymerase chain reaction analysis. *P<0.05 was considered to indicate a statistically significant difference using Dunnett's test for multiple group comparisons with the control group (0 µM LH) and Tukey's test for comparisons between group differences (groups, 20 and 20 + NAC). (B) Expression levels of the JNK signaling pathway-related apoptotic proteins were detected via western blot analysis. HSC-3 cells were treated with LH for 24 h, harvested and total protein lysate was subjected to western blot analysis using antibodies against MKK4, JNK, c-Jun, ATF2 and their phosphorylation products. (C and D) Apoptotic protein expression was evaluated via histogram analyses. *P<0.05 was considered to indicate a statistically significant difference using Dunnett's test for multiple group comparisons with the control group (0 µM LH). LH, lycorine hydrochloride; p-, phosphorylation levels.