Fig 2: ERK1/2 phosphorylation may be one of the downstream mediators for S1PR2-induced cholangiocyte-reactive phenotype. Extrahepatic cholangiocyte organoids were exposed to either Veh or 10 µM S1P with or without costimulation of ERK phosphorylation inhibitor 2 µM U0126 for 24 hours. (A) S1PR2 and (B) COX2 gene expression analyzed by quantitative real-time PCR relative to Veh; (C) % phosphorylated ERK1/2 relative to total ERK1/2 detected by ELISAs, (D) gene expression of reactive phenotype markers analyzed by quantitative real-time PCR relative to Veh; (E) % LDH activity in cell culture media quantified using LDH assay. Inhibiting ERK1/2 phosphorylation downstream of S1PR2 can suppress S1PR2 pathway mediators, cholangiocyte-reactive phenotype marker upregulation, and LDH activity. N = 4–6, error bars represent SD, p<0.05, * compared to Veh, # S1P+U0126 compared to S1P alone. Abbreviations: COX2, cyclooxygenase-2; ERK1/2, extracellular signal-regulated kinase 1/2; LDH, lactate dehydrogenase; S1P, sphingosine-1-phosphate; S1PR2, sphingosine-1-phosphate receptor 2; Vim, vimentin.

Fig 3: The ERK-CREB signalling pathway is involved in PA-mediated hepatocyte apoptosis. A–B. TUNEL assay was used to observe the apoptotic index of hepatocytes with ERK inhibition using PD98059. The number of apoptotic cells was recorded. C–F. Western blotting for the inflammatory response via measuring the levels of inflammatory factors. G. ATP production in Sirt3-overexpressed cells with ERK inhibition. H–I. ROS production was measured via flow cytometry. The data represent the mean ± SEM (n = 4 mice per group). *P < 0.05.

Fig 4: MiR-183-5p directly targeted FOXP1. (A) Online database StarBase showed sequence alignment between miR-183-5p and FOXP1. (B) The luciferase reporter gene assay validated the binding of miR-183-5p and FOXP1. Firefly and Renilla luciferase activities were determined. (C) Expression of FOXP1 in different cell lines was tested by using western blot analysis. (D) The quantification of band intensity relative to β-actin intensity of (C) was quantified by MBF ImageJ software. (E) Expression of FOXP1 in human neuroblastoma tissues (n = 30) and normal tissues (n = 30). β-actin was used as an internal reference. (F) Effect of miR-183-5p on FOXP1 in SK-N-SH or (G) SH-SY5Y neuroblastoma cells and the quantification of band intensity relative to β-actin intensity of (F, G) was quantified by MBF ImageJ software. (H) Expression of FOXP1 in different cell lines was tested by using western blot analysis. (I) Western blot detected the phosphorylation of ERK, AKT, and pS6 in neuroblastoma cells. (J–L) The quantification of band intensity relative to β-actin intensity of (I) was quantified by MBF ImageJ software. Statistical significance was determined using an independent-sample t-test. Values were expressed as mean ± standard error of mean, n = 3. *P < 0.05 and **P < 0.01 vs normal or control.

Fig 5: Gas6-dependent AXL receptor activation enhances GnRH receptor signaling processes. A Proportion of phosphorylated ERK relative to total ERK (pERK/ERK) in αT3-1 and LβT2 cells under control conditions and after incubation with Gas6 (100 nM for 3 h); n = 3 for each condition. B Time course of Gas6-dependent changes (100 nM) in pERK/ERK in LβT2 cells (from left); effect of incubating LβT2 cells with GnRH (10 nM) and GnRH plus Gas6 (100 nM) for 5 min on pERK/ERK (at right); n = 3 for each condition. C Time course of Gas6-dependent (100 nM) changes on Egr-1 transcript abundance in αT3-1 and LβT2 cells; n = 3. D LHβ protein abundance in the culture media and lysates of LβT2 cells under control conditions and after 2 h incubations with Gas6 (100 nM) and GnRH (10 nM); n = 3. € LHβ protein abundance in LβT2 culture media under control conditions and after 2 h incubations with Gas6 (100 nM), GnRH (10 nM), the GnRH receptor antagonist cetrorelix (2 nM), the AXL receptor antagonist R428 (50 µM), and the MEK inhibitor U0126 (10 µM); n = 3 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; * P < 0.05