Fig 2: Increased GALR1 promotes SREBP1 expression via NR1D1, thus inducing lipid accumulation in HepG2 cells.mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, and RXR after NR1D1 knockdown or NR1D2 knockdown (a, b) as well as NR1D1 overexpression or NR1D2 overexpression (c, d) in HepG2 cells. a, c Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified. b, d mRNA abundance detected by qPCR was presented. e mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after galanin treatment (#1179, Tocris Bioscience, Bristol, UK) at the concentration of 0, 50, 150, and 300 pg/mL for 24 h in HepG2 cells. Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified (above); mRNA abundance detected by qPCR was presented (below). f mRNA and protein abundances of GALR1, GALR2, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT and T-AKT after GALR1 knockdown or GALR2 knockdown and further treatment with 300 pg/mL galanin for 24 h in HepG2 cells. g mRNA and protein abundances of GALR1, GALR2, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after GALR1 overexpression or GALR2 overexpression in HepG2 cells. h mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after treatment with(out) 300 pg/mL galanin, with(out) 10 µM LY294002 (#19-142, Sigma-Aldrich, St. Louis, USA) and with(out) 20 µM PD98059 (#19-143, Sigma-Aldrich). i mRNA and protein abundances of GALR1, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-AKT, and T-AKT after GALR1 overexpression and further treatment with 10 µM LY294002 in HepG2 cells. GAPDH or ß-ACTIN were used as loading controls for western blot and qPCR analyses. Representative Oil Red O staining and Nile Red staining after the induction of oleic acid and palmitic acid (OPA) and treatment with 300 pg/mL galanin for 24 h (j) or NR1D1 siRNA (k) in HepG2 cells. Left, representative images were shown. Right, intensity was quantified. Blots and images are representative. Statistical analysis was performed with unpaired Student’s t-test or one-way ANOVA followed by Newman–Keuls multiple comparison test. Data present means ± SEM from 3 to 5 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 against si-NC cells or against Vec-NC cells or against control cells; #P < 0.05, ##P < 0.01 against si-GALR2 cells or against PD98059 cells or against Vec-GALR1 cells.