Fig. 5: CER and S1P promote apoptosis and proliferation in post-hepatectomy hepatocytes, respectively.
Primary hepatocytes were isolated from the remnant liver at 2 days after hepatectomy and incubated with CM from M0, M1, or M2 macrophages, or treated with 10 μM d18:1/C16:0-CER, 500 nM d18:1-S1P, 100 ng/ml LPS, and 10 ng/ml IL-4 for 48 h, then subjected to examination of proliferation and apoptosis. A The viability of hepatocytes treated with CM of BMDMs was measured by MTT assay. B Hepatocytes treated with CM of BMDMs were fixed and stained with proliferation marker PCNA and apoptosis maker C-Caspase 3. Top: representative images of immunofluorescent staining of PCNA (green). Bottom: representative images of immunofluorescent staining of C-Caspase 3 (green). Nuclei were counterstained with DAPI (blue). C, D PCNA-positive hepatocytes and C-Caspase 3-positive hepatocytes were enumerated to evaluate the proliferation (C) and apoptosis (D) in hepatocytes. E, F Hepatocytes treated with CM of BMDMs were collected to measure the levels of CER metabolites, including d18:1-CER (E), d18:1-SPH, and d18:1-S1P (F) by targeted UHPLC-ESI-MS/MS. G Primary hepatocytes isolated from post-hepatectomy mice were treated with d18:1/C16:0-CER (10 μm) and d18:1-S1P (500 nm) for 48 h, then the viability of treated cells was examined by MTT assays. H Protein levels of apoptosis marker C-caspase 3 and proliferation marker PCNA in d18:1/C16:0-CER (10 μm) and d18:1-S1P (500 nm) treated hepatocytes were measured by immunoblotting. Images in B and H represent results from three independent experiments. Data in A and C–G were demonstrated as mean ± SD, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.
