Fig. 6: Reelin activated Notch1/Srebp2 mediated cholesterol metabolism.

A The expression of Dab1 and phosphorated Dab1 (p-Dab1) in TSPCs treated with or without 1 µg/mL Reelin, as assessed by western blotting. B, C The Sirius Red staining and Masson’s trichrome staining of TSPCs in vehicle, Reelin and Dab1 siRNA with Reelin treated groups. D The heatmap indicated the expression levels of genes related with Notch signaling in Vldlr⁺ and Vldlr⁻ TSPCs, analyzed by bulk RNA-seq. E The expression of NICD and Notch1 in Vldlr⁺ and Vldlr⁻ TSPCs, analyzed by western blotting. F The expression of Notch1 and NICD in cytoplasm and nucleus of control and Reelin treated groups, as assessed by western blotting. G The expression of NICD and Notch1 of TSPCs in vehicle, Reelin and Dab1 siRNA with Reelin treated groups, as analyzed by western blotting. H The localization of Notch1 in vehicle, Reelin and Dab1 siRNA with Reelin treated groups, as assessed by immunofluorescence staining. I Co-IP of Dab1 with NICD in vehicle, Reelin and Dab1 siRNA with Reelin treated TSPCs groups. J Co-IP of Dab1 with NICD in HEK-293 cells, which were transfected with indicated wild type (WT) or site mutated plasmids. K Co-IP evaluated the protein level of ubiquitinated NICD in HEK-293 cells, which were transfected with the indicated WT or site mutated plasmids and treated with MG132 (20 μmol/L) for 6 h before cell harvested. L The cholesterol level in control and Reelin treated TSPC groups. M The mRNA level of Srebp2 in vehicle, Reelin and DAPT with Reelin treated groups, analyzed by RT-qPCR. N The expression of SREBP2 of TSPCs in vehicle, Reelin and DAPT with Reelin treated groups. O The cholesterol level in vehicle, Reelin and DAPT with Reelin treated groups. Each dot represents one individual replicate (n = 3 biological replicates in C, L, M, O). Statistical significance was analyzed by One-way ANOVA with Tukey’s multiple comparisons test (C, M, O). Statistical significance was analyzed by unpaired Student’s t test (L). For the western blot analysis, similar results were obtained in three independent experiments.