Fig 2: Neratinib inhibited fibrogenesis by interrupting the FGF2 signaling pathway. (A) Neratinib inhibited the induction of the MAPK/ERK signaling pathway in activated hpHSCs. (B) The effect of neratinib on FGF2 mRNA level in activated hpHSCs was measured. (C) hpHSCs were serum-starved overnight before exposure to neratinib (250 nM) in the absence or presence of FGF2 (10 ng/mL). Western blotting for p-FGFR1, p-MEK1/2, MEK1/2, p-ERK, ERK, and p-CREB was performed. GAPDH was used as the loading control. (D) The effects of neratinib on FGF2 mRNA expression and (E) serum FGF2 levels were analyzed in the early fibrosis mouse model. ##P < 0.01, ###P < 0.001 versus the non-treated group. *P < 0.05, **P < 0.01 versus the activated group. (F) Scheme of the proposed working model of neratinib in liver fibrosis. Data are expressed as the mean ± SEM. Abbreviations: qHSCs, quiescent HSCs; aHSCs, activated HSCs; TßRI, TGF-ß receptor 1; TßRII, TGF-ß receptor 2; Veh, vehicle; Ner, neratinib.

Fig 3: HSV-2 infection up-regulated cell membrane-associated TLR4 expression. a The distribution of TLR4 in cytoplasm and membrane of U937 cells. U937 cells were harvested and cytoplasmic or membrane-associated total proteins were exacted as described. TLR4 expression in each component was determined via western blot. MEK1/2 was set as the control for monitoring cytoplasm/membrane fractionation quality. b HSV-2 infection increased membrane-anchoring TLR4 content. HEC-1-A cells were mock-infected or infected with HSV-2 (moi = 1). Cells were harvested and cytoplasmic or membrane-associated total proteins were exacted as describe 24 h p.i. TLR4 expressions were examined via western blot. c LPS could enhance HSV-2-induced AP-1 activation. HEC-1-A cells were transfected with AP-1-luc plasmid. Cells were mock-infected or infected with HSV-2 (moi = 1) in the absence or presence of serial concentrations of LPS 24 h post-tranfection. Relative luminescence units were determined as described. All experiments were performed twice, and the representative results were shown

Fig 4: MI Exo promotes ADMSCs proliferation in vitro partly via ERK1/2 activation. (A) Inverted contrast phase microscopy showed that the number of ADMSCs was increased after treatment with MI Exo for 24 h compared with the results for control and Con Exo. Scale bar, 80 µm. (B) The proliferation rate of ADMSCs was measured using CCK-8 assay after treatment with Con Exo and MI Exo for 24 h. Data are shown as mean ± SD. *, significantly different from control; *, p < 0.05. #, significantly different from Con Exo; #, p < 0.05. Three independent experiments were performed. (C) EdU incorporation analysis of ADMSCs after treatment with Con Exo and MI Exo for 24 h. The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst33342-positive cells. Scale bar, 200 µm. Data are shown as mean ± SD. *, significantly different from control; **, p < 0.01. #, significantly different from Con Exo; #, p < 0.05. Two independent experiments were performed. (D) Western blot analysis of p-Akt and Akt; p-MEK1/2 and MEK1/2; p-ERK1/2 and ERK1/2 after treatment with Con Exo and MI Exo for 24 h. Representative images of three independent experiments are shown. p-, phosphorylated. Data are shown as mean ± SD. *, significantly different from control; **, p < 0.01. #, significantly different from Con Exo; #, p < 0.05. After being treated with MI Exo and 10 µmol/L U0126 for 24 h, the expression of p-ERK1/2 and ERK1/2 of ADMSCs was determined using western blotting (E), and the proliferation rate was measured using CCK-8 assay (F). For E, representative images of three independent experiments are shown. For F, two independent experiments were performed. Data are shown as mean ± SD. *, significantly different from MI Exo; *, p < 0.05; **, p < 0.01. One-way ANOVA followed by Tukey's post-test was performed (B-D). Unpaired Student's t-test was performed (E and F).

Fig 5: CHIR and PD activate Wnt and MEK signaling pathways. (A) Relative protein expression levels of p-MEK1/2, p-ERK1/2, ß-catenin and p-GSK3 in human mammary epithelial cells cells following treatment with CHIR and PD detected by western blot analysis. (B) Relative quantification of western blot analysis is presented in bar charts. All assays were performed in duplicates and data are presented as the mean ± standard deviation. *P<0.05 vs. control group. PD, PD184352; CHIR, CHIR99021; MEK, mitogen-activated kinase kinase; ERK, extracellular-signal regulated kinase; GSK, glycogen synthase kinase 3; p-, phosphorylated.