Fig 2: ATGL is highly expressed in human HCC tissues and predicts poor prognosis. a Real-time PCR analysis of ATGL expression in 40 pairs of HCC and matched non-tumor tissues. b Western blot analysis of ATGL expression in HCC and matched non-tumor tissues C: cancer tissue; P: para-cancer tissue. c Representative immunohistochemical detection of ATGL in normal liver tissue and HCC tissues. d Kaplan-Meier analysis of overall survival in 40 patients indicated that high expression of ATGL predicts poor prognosis. The cutoff lines to divide into high and low group was median value. e Real-time PCR analysis of ATGL expression in HCC cell lines. f Western blot analysis of ATGL expression in HCC cell lines. g Relative levels of DAG and FFA in 40 pairs of HCC and matched non-tumorous tissues. Data are expressed as mean ± SD of three independent experiments. Statistical significance was concluded at *P < 0.05, **P < 0.01

Fig 3: Inhibition of DGAT1 suppresses tumor growth in vivo. Grafts containing PC-3 cells with or without DGAT1 inhibitor were implanted under the kidney capsule of SCID mice, and, after 2 weeks, the kidneys were harvested. (A) Tumor volume was calculated in the treated and untreated tumors. (B) H&E stain was performed in kidney sections of treated or untreated tumors using 20x and 40x objectives (I: invasion; N: necrosis). Size bars: 20 µm (C) ATGL-positive LD density in treated vs untreated tumor tissues was analyzed (n = 50). (D) PC-3 cell proliferation in vivo was analyzed using BrdU staining (n = 50). (E) Immunohistochemical stains were performed for BrdU and GM130 to analyze cell proliferation and intracellular GM130 protein, respectively. Size bars: 20 µm. Data are presented as mean ± SEM. Student’s unpaired t test. ****P < 0.0001.

Fig 4: PEDF acts as an inhibitor of DGAT1 in aggressive prostate cancer cells. (A) ATGL (56 kD), perilipin 2 (PLIN2) (51 kD), DGAT1 (55 kD), and PEDF (50 kD) levels were analyzed by western blot in PC-3 control (CTR) and treated with 1 µM DGAT1 inhibitor or 10 nM PEDF. (B) ATGL, (C) PLIN2, (D) DGAT1, and (E) PEDF densities were normalized by GAPDH. (F) PC-3 cells were treated with 10 nM PEDF for 48 h, and, after fixation, the cells were stained with rabbit anti-DGAT1 antibody (red), BODIPY (green) and DAPI (blue) to localize DGAT1 protein, LDs and nucleus, respectively. Size bar: 10 µm. (G) PC-3 cells were treated with 10 nM PEDF for 48 h, and, after fixation, the cells were stained with rabbit anti-PEDF antibody (red), BODIPY (green) and DAPI (blue) to localize PEDF protein, LDs and nucleus, respectively. Size bar: 10 µm. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Nat8l deficiency impairs lipid turnover in white adipocytes. A) Expression of DNL protein ACC in sWAT of 10-wk-old male WT and Nat8l-ko mice on CD (n = 3). B) Quantification of Western blot signal from A using ImageJ, (n = 3). C–F) sWACs from WT and Nat8l-ko mice were differentiated to adipocytes for 7 d. C) Protein expression of Asp-NAT and DNL-proteins phospho-ACC, ACC, and FAS (n = 4). D) 14C-glucose incorporation into FAs, MGs, DGs, TGs, cholesteryl esters (CEs), and phospholipids (PLs) (n = 8). E) Protein expression of key lipases for lipolysis, phospho-HSL, HSL, and ATGL (n = 4). F) FFA content in the supernatant after 4 h serum starvation with or without isoproterenol treatment (10 µM) (n = 4). G) FA release from sWAT with or without isoproterenol treatment (10 µM) for 1 h; isolated from 9-mo-old CD-fed male WT and Nat8l-ko mice (n = 7). H) Relative Nat8l mRNA expression in sWAT and eWAT from 13-wk-old C57BL/6 mice injected with either 1 mg/kg saline or CL compound for 7 consecutive days (n = 4/5). I, J) Flox2 and Nat8l-ako mice at the age of 10 wk were injected with CL compound (1 mg/kg BW) and plasma FFAs (I), and blood glucose levels (J) were measured at the indicated time points (n = 6/5). K) Flox2 and Nat8l-ako mice at the age of 15 wk were kept at 4°C without access to food for 5 h. Body temperature was measured at indicated time points (n = 5/4). Data are shown as means ± sd. *P < 0.05, **P < 0.01 vs. control (Student’s t test; B, D, H); *P < 0.05, ***P < 0.001 vs. WT (1-way ANOVA), ###P < 0.001, ####P < 0.0001 vs. untreated (Tukey’s multiple comparisons test; F, G); *P < 0.05, **P < 0.01, ***P < 0.001 vs. flox2 (2-way ANOVA), #P < 0.05 vs. flox2 (Sidak’s multiple comparisons test for the individual time points; I, K).