Fig. 3: Progesterone receptor membrane-associated component 1 (PGRMC1) contributed to low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) uptake by regulating the translocation of LDL-R and VLDL-R.

a, b Analyses of the effect to LDL uptake by PGRMC1. a Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with Alexa Fluor 488-acetylated LDL (green) and DAPI (blue) are shown (scale bar: 10 μm). b Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with Alexa Fluor 488-acetylated LDL for 60 min. The graph shows the mean of fluorescence intensities (per 10,000 cells) (n = 4). c, d Analyses of the effect to VLDL uptake by PGRMC1. c Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with DiI–VLDL (red) and DAPI (blue) are shown (scale bar: 10 μm). d Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with DiI–VLDL for 60 min. (n = 4). e Analyses of regulation of the LDL-R and VLDL-R localization by PGRMC1. Protein expressions in whole-cell lysates were detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, GAPDH, or PGRMC1. f After plasma membrane fractions of 3T3L1 cells (control and PGRMC1 KD) were extracted, protein expression in the plasma membrane was detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, Na-K ATPase α1, or PGRMC1. f, g Co-immunoprecipitation assay for interaction between PGRMC1 and LDL-R, or PGRMC1 and VLDL-R. FLAG-PGRMC1 WT or Y113F was overexpressed in 3T3L1 cells, and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1, anti-LDL-R antibody, or anti-VLDL-R antibody. h Co-immunoprecipitation assay for interaction between endogenous PGRMC1 and LDL-R. 3T3L1 cell lysate was incubated with anti-PGRMC1 antibody or normal rabbit IgG, and then incubated with 10 µl protein A-sepharose beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-LDL-R antibody. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test (b, d). ^*P < 0.05.