Figure 4

GNMT coordinates with NPC2 and influences hepatic cholesterol homeostasis after LDL and progesterone treatment. CHO cells were cultured in normal medium (panel 1) and transfected with either NPC2-DsRed (A) or GNMT-GFP and NPC2-DsRed (B) for 24 h. After reseeding, CHO cells were cultured in LPDS (panel 2), LDL (panel 3) or LDL plus progesterone (panel 4) medium for 24 h. After washing out progesterone at 6, 12, 18 or 24 h (panels 5–8), confocal microscopy was used to detect colocalization between NPC2-DsRed (red) and GNMT-GFP (green). NPC2-GNMT colocalization percentages are shown in B. Scale bar, 20 µm. (C) SK-Hep1 cells were infected with Ad-GNMT for 24 h, followed by LDL or LDL plus progesterone treatment for 24 h. After washing out progesterone at 6 or 18 h, cells were harvested for cytosol-lysosome fractionation experiments. LAMP1 and α-tubulin are indicated as lysosome and cytosol markers, respectively. (D) Ad-GNMT and Ad-vector-infected SK-Hep1 cells were cultured in normal and LPDS media, followed by LDL or LDL plus progesterone treatment for 24 h. After washing out progesterone at 6 or 18 h, cells were harvested to determine cholesterol concentrations. *P < 0.05. −■−, Ad-GNMT; −□−, Ad-vector.