Fig 1: Comparative expression of fatty acid metabolism regulating genes in LNCaP and PBMCs. a Screening for fatty acid metabolism genes which are differentially expressed in LNCaP and PBMCs was done using western blot analysis; the genes differentially expressed are shown. GAPDH is used as an internal control. The experiment was repeated three times and a representative experiment is shown. HeLa and differentiated OP9 cell extracts have been used as positive control for DGAT2, perilipin and HSL expression. b Histogram plot showing comparative fold expressions of ABHD, ACAT, ATGL and DGAT1 between LNCaP and PBMCs. The fold change is calculated by taking base levels expressed in LNCaP cell line as 1 after GAPDH calibration and is an average of three separate experiments, (*)-(P ≤ 0.05). c Effect of plasma treatment on the expression levels of ABHD5, ACAT1, ATGL and ABHD5. Western blots were performed after 50% plasma treatment for 12 h. on both PBMCs and LNCaP cells. Out of the three experimental repeats a representative experiment is shown. Fold changes mentioned in the results section are an average of all the experiments
Fig 2: Cyclin D1 inhibits lipolysis and LAL expression. (A‐C) Rat hepatocytes and (D,E) AML12 cells were cultured as in Fig. 1. (A) Lipolysis as measured by TG turnover in rat hepatocytes. (B) Expression of lipolysis‐associated mRNA in rat hepatocytes. (C) Representative western blot of protein expression in rat hepatocytes. (D) mRNA expression in AML12 cells (n = 3). (E) Western blot of AML12 cells. The graph shows the expression of these proteins normalized to actin (n = 3). Data represent mean ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. Abbreviations: Abhd5, abhydrolase domain containing 5; FA, fatty acid.
Fig 3: Blocking of ABHD5 and DGAT induces apoptosis and autophagy respectively in LNCaP cells: a Immunofluorescent image showing staining with apopxin (green) and 7AAD (red) after siRNA treatment (72 h). Green only-early apoptotic cells, red only-dead cells and red and green both - late apoptotic or cells with damaged plasma membrane. b Apoptosis assay showing increased cell population of apopxin positive cells with ABHD5 siRNA as compared to NT siRNA using flow method. Black-NT siRNA, Red- DGAT1 and ABHD5 siRNA. Apopxin peak (x axis) at 106 represents the apoptotic cell population. c Immunofluorescent image showing staining with apopxin (green) and Lyso tracker (red) after siRNA treatment (72 h). All immunofluorescent experiments have been performed three times and a representative experiment has been shown. For each experiment 4–5 xy planes were randomly selected for microscopy. Maximum intensity image has been shown in the figures which either included all the z-stacks (a) or some selected z-stack (c - apopxin staining) to depict localization. Bar is 50 μm
Fig 4: DZA activates ATGL in 3T3-L1 adipocytes. Immunofluorescence images showing localization of (A) ATGL, (B) pATGL-Ser(406), (C) CGI-58 and (D) GOS2 in BODIPY 493/503 and DAPI stained control and DZA-treated 3T3-L1 adipocytes (Scale bar—20 µm) with a magnified images of a representative 100 µM DZA-treated cell showing localization of pATGL-Ser(406) and CGI-58 on the LD surface. (E) The fluorescence intensity of ATGL, (F) pATGL-Ser(406), (G) CGI-58 and (H) GOS2 expression in each group of cells normalized to % of control cells, (n = 4) experiments; confocal images of 31 random cells from CGI-58 mRNA expression in control and DZA-treated 3T3-L1 adipocytes, n = 5. Data are presented as the mean ± SEM; values not sharing a common letter significantly differ from each other at p ≤ 0.05.
Fig 5: Schematic representation of possible apoptosis and autophagy triggering pathways after ABHD5 and DGAT1 siRNA treatment respectively. ABHD5 (alpha/beta- hydrolase domain containing protein 5), DGAT1 (diglyceride acyltransferase), AMPK (AMP activated protein kinase), ACC (acetyl CoA carboxylase), pP70S6/P70S6 (ribosomal protein S6 kinase), PARP (Poly ADP-ribose polymerase), raptor (regulatory-associated protein of mTOR), ULK (serine/threonine-protein kinase) and LC3B (microtubule-associated protein 1A/1B–light chain 3)
Supplier Page from Abcam for Anti-Abhd5/CGI-58 antibody [EPR12621]