Journal: Molecular and Cellular Biology

Article Title: Deficiency of a Lipid Droplet Protein, Perilipin 5, Suppresses Myocardial Lipid Accumulation, Thereby Preventing Type 1 Diabetes-Induced Heart Malfunction

doi: 10.1128/MCB.00133-14

Figure Lengend Snippet: Levels of Plin and lipase proteins in the heart. (A) Images of immunoblotting of Plin proteins and lipases using total protein extracts from the heart (15 μg of total protein per lane). Coomassie brilliant blue (CBB) staining of the gel was used as a loading control. (B to F) The graphs show the levels of Plin5, Plin2, Plin3, ATGL, and HSL relative to those in saline-treated control WT mice (n = 3 per group). Open bars, WT mice; filled bars, Plin5−/− mice; ND, not detectable. Data are shown as means ± SEM (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Article Snippet: Proteins were probed using the following antibodies: Plin5 (developed in-house [ 8 ] and by Progen [GP31]); Plin2 (GP40; Progen); Plin3 (3883; Prosci); ATGL (2138), phospho-p38 (4511), and p38 (9212) (Cell signaling); pan-cadherin (C3678), PKC (P5704), and p47 phox (SAB4502810) (Sigma); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-25778) and β-tubulin (sc-9104) (Santa Cruz); hormone-sensitive lipase (HSL) (ab45422) and uncoupling protein 3 (UCP3) (ab3477) (Abcam); p67 phox (610912; BD Bioscience); and acyl coenzyme A (CoA) oxidase (Acox), very long-chain acyl-CoA dehydrogenase (VLCAD), and medium-chain acyl-CoA dehydrogenase (MCAD) (gifts from T. Hashimoto and S. Yamaguchi).

Techniques: Western Blot, Staining, Control, Saline

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: a , b , Levels of glycerol and NEFAs in starvation medium released by iBAs in basal state ( n = 4; F = 6.491 ( a ) and 6.884 ( b )). c , IB of HSL Ser660ph , HSL, ATGL, CGI-58, PLIN1 and γ-tubulin in iBAs 72 h after Pex2 knockdown ( n = 6 in Pex2 and 12 in Nc siRNA, F = 27.6). d , Representative immunofluorescence (IF) result of ATGL in iBAs 72 h after Pex2 knockdown. ATGL in red, LDs in green and nuclei in blue. Scale bar, 20 μm. Experiments were repeated four times. e , IB of ATGL and γ-tubulin in HepG2 cells 48 h after PEX2 knockdown ( n = 6 in Pex2 and 10 in Nc siRNA, F = 23.94). f , g , IB of endogenous ATGL or ectopically expressed ATGL–FLAG and γ-tubulin in HEK293T cells 48 h after PEX2 knockdown ( n = 6 in Pex2 and 12 in Nc siRNA; F = 17.66 ( f ) and 20.9 ( g )). Results are shown as the mean ± s.e.m. and analysed using ANOVA with Dunnett correction for multiple comparisons between control and other groups. Statistical differences are indicated by exact P values.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Immunofluorescence

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a-b) IB of endogenous HSL Ser660ph , HSL, ATGL, CGI-58, PLIN1 and γ-tubulin in iBAs 72 h after knockdown of Pex10/12 (N = 6 in Pex10/12 and 12 in Nc siRNA, F = 36.91 in a and 42.4 in b). (c) Representative IF of ATGL in iBAs 72 h after knockdown of Pex10 / 12 . ATGL in red, LDs in green and nuclei in blue. Scale bar, 20 μm. Repeated 4 times. (d) IBAs were transfected with siRNAs to knock down Pex2 . After 72 h, ATGL lipase activity assay was conducted using the WCE (N = 4). (e) IBAs were transfected with siRNAs to knock down Pex2/10/12 . After 72 h, qPCR was conducted to check Atgl transcript (N = 4 in Pex2/10/12 and 5 in Nc siRNA, F = 3.853). (f) IBAs were transfected with Pex5 and Pex19 siRNAs. After 72 h, IB was conducted to check protein levels by indicated antibodies. (g) Human PEX2 knockdown efficiency was validated by qPCR 48 h after HepG2 cells were transfected with PEX2 siRNAs (N = 3, F = 94.61). (h-i) HepG2 cells were transfected with PEX10 / 12 siRNAs for human PEX10 and PEX12 antibodies validation by IB (N = 4). (j) IB of ATGL and γ-tubulin in HepG2 cells 48 h after knockdown of PEX10/12 (N = 6 in PEX10/12 and 12 in Nc siRNA, F = 26.32). (k) HepG2 cells were transfected with PEX5 and PEX19 siRNAs. After 72 h, IB was conducted to check protein levels by indicated antibodies. (l) HepG2 cells were transfected with siRNAs to knock down PEX2/10/12 . After 48 h, qPCR was conducted to quantify ATGL transcript (N = 3, F = 0.1052). (m) IB of ATGL and γ-tubulin in HEK293T cells 48 h after knockdown of PEX10/12 (N = 4 in PEX10/12 and 6 in Nc siRNA, F = 0.3977). (n) IB of ectopically expressed ATGL-FLAG and γ-tubulin in HEK293T cells 48 h after knockdown of PEX10/12 (N = 4 in PEX10/12 and 8 in Nc siRNA, F = 0.6143). (o) PEX2/10/12 knockdown efficiency was determined by qPCR 48 h after transfecting HEK293T cells by siRNAs (N = 3). (p) HEK293T cells were transfected with siRNAs to knock down PEX2 . After 72 h, cells were fixed for LDs staining. LDs are marked in green and nuclei in blue. Scale bar, 20 μm. Repeated 4 times. Results are shown as mean ± SEM and analyzed using Student’s two-sided t test (d, h, i) and an ANOVA test with Dunnett correction for multiple comparisons between control and other groups (a, b, e, g, j-o).

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Transfection, Activity Assay, Staining

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: a , Co-IP conducted in HEK293T whole cell extract (WCE) via FLAG antibody 48 h after expressing PEX2–FLAG. Co-IP was analysed by IB using the indicated antibodies. Experiments were repeated three times. b , Representative images of wild-type ATGL–EGFP, ATGLΔHD–EGFP and ATGL3KR–EGFP distribution in HEK293T cells treated with 400 μM oleic acid (OA). LDs were stained by LipidTOX Deep Red dye and nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. Experiments were repeated three times. c , d , IB of ectopically expressed ATGL3KR–EGFP and ATGLΔHD–EGFP in HEK293T cells 48 h after PEX2 siRNA transfection ( n = 8 in PEX2 and 16 in Nc siRNA, F = 3.644 ( c ); n = 4 in PEX2 and 8 in Nc siRNA, F = 52.67 ( d )). e , HEK293T cells were co-transfected by plasmids to express ATGL–FLAG and HA–Ub, followed by siRNA transfection. After 48 h, IP and IB were conducted to detect the ubiquitination pattern. Experiments were repeated three times. f , HEK293T cells were transfected by the ATGL–FLAG plasmid, followed by IP to enrich ATGL–FLAG for poly-ubiquitination type analysis via K48- or K63-linkage poly-ubiquitination antibodies. Experiments were repeated three times. g , h , HEK293T cells were transfected by ATGL–FLAG ( g ) and ATGLK92–FLAG ( h ) plasmids, followed by siRNA transfection. After 48 h, IP was conducted to enrich ATGL–FLAG or ATGLK92–FLAG for K48-linkage poly-ubiquitination pattern detection. Experiments were repeated three times. Results are shown as the mean ± s.e.m. and analysed using ANOVA with Dunnett correction for multiple comparisons between control and other groups.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Co-Immunoprecipitation Assay, Expressing, Staining, Transfection, Plasmid Preparation

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a) Co-IP conducted in HEK293T WCE via FLAG antibody 48 h after expressing PEX2-FLAG. Co-IP was analyzed by IB using indicated antibodies. Repeated 3 times. (b) Co-IP conducted in HEK293T WCE via FLAG antibody 48 h after expressing indicated constructs. Co-IP was analyzed by IB using indicated antibodies. Repeated 3 times. (c) Direct interaction of ATGL and PEX2 in PEX2-FLAG-EGFP expressing iBAs was checked via proximity ligation assay (PLA) at different conditions. Upper panel represents the PEX2 and ATGL interaction. Lower panel is the nuclei (blue) merged with interaction intensity between PEX2 and ATGL. Repeated 3 times. (d) Positions of lysine residues and hydrophobic domain (HD) in human ATGL protein. Three lysine residues with strikethrough are not conserved in murine ATGL.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Co-Immunoprecipitation Assay, Expressing, Construct, Proximity Ligation Assay

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a) ATGL lysine-only mutants were overexpressed in HEK293T cells. After 36 h, cells were treated by 10 μM MG132 for 8 h. IB was conducted to analyze protein levels after blocking proteasome dependent degradation. Repeated 2 times. (b) ATGL lysine-only mutants were overexpressed in HEK293T cells, followed by PEX2 siRNA transfection. After 48 h, IB was conducted to determine protein levels. Repeated 2 times. (c) HEK293T cells were transfected by ATGLK0-FLAG or ATGLK92-FLAG plasmids, followed by siRNA transfection for 48 h. IB was conducted to analyze protein levels of ATGLK0-FLAG and ATGLK92-FLAG (ATGLK0-FLAG, N = 4 in PEX2 and 8 in Nc siRNA, F = 0.9678; ATGLK92-FLAG, N = 5 in PEX2 and 10 in Nc siRNA, F = 15.26). (d) ATGL ubiquitination was analyzed via IP and IB after stimulating ATGL-FLAG expressing iBAs with Iso. Repeated 3 times. (e) IBAs were fractionated to collect different fractions for protein analysis using indicated antibodies. Repeated 3 times. (f) ATGL-FLAG expressing iBAs were fractionated to collect LDs for IP and IB by indicated antibody. The K48-linkage poly-ubiquitination level of LD associated ATGL was analyzed from iBAs at both basal state and activated state induced by 1 μM Iso. Repeated 3 times. (g) ATGL-FLAG expressing iBAs were fractionated to collect cytosol fraction for IP and IB by indicated antibody. Repeated 3 times. (h) HEK293T cells were transfected by ADRP-EGFP plasmid, followed by PEX2 siRNA transfection. After 48 h, IP and IB were conducted by indicated antibody to check K48-linkage poly-ubiquitination level. Repeated 3 times. Results are shown as mean ± SEM and analyzed using an ANOVA test with Dunnett correction for multiple comparisons between control and other groups.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Blocking Assay, Transfection, Expressing, Plasmid Preparation

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a) IBAs expressing PEX2-FLAG-EGFP were collected for IP and IB by indicated antibodies. Repeated 3 times. (b) Human PEX2 was overexpressed in HEK293T cells. After treatment by 10 μM MG132 for 6 h, IP and IB were conducted to analyze PEX2 oligomerization pattern by indicated antibodies. Repeated 3 times. (c) Single cysteine to glycine mutants of human PEX2 were overexpressed in HEK293T cells. IP and IB were conducted to analyze their oligomerization patterns by indicated antibodies. Repeated 2 times. (d) Single cysteine to glycine mutants of human PEX2 were overexpressed in HEK293T cells. Cells were treated with 0.5 mM H 2 O 2 for 2 h. IP and IB were conducted to analyze PEX2-FLAG-Myc protein by indicated antibodies. Repeated 2 times. (e-f) Wild type PEX2 or PEX2 mutants (siRNA resistant) were overexpressed in HEK293T cells or co-expressed with ATGL-FLAG, followed by siRNAs transfection. IP and IB were conducted using the indicated antibodies. Repeated 3 times. (g) PEX2 and PEX2C1-7G mutant were overexpressed in HEK293T cells, followed by 0.5 mM H 2 O 2 treatment for 2 h. IP and IB were conducted to check K48-linkage poly-ubiquitination level. Repeated 3 times. (h-i) IBAs were transfected with Cop1 siRNAs or treated by 0.5 mM H 2 O 2 and harvested at the indicated time points. IB was conducted using the indicated antibodies (h, N = 4 in Cop1 and 10 in Nc siRNA, F = 2.544; i, N = 3 in H 2 O 2 treatment and 9 in control, F = 0.9765). Results are shown as mean ± SEM and analyzed using an ANOVA test with Dunnett correction for multiple comparisons between control and other groups.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Expressing, Transfection, Mutagenesis

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a) 72 h after knocking down Cat and Acox1 in iBAs, ATGL lipase activity assays were conducted using the WCE (N = 4, F = 70.21). (b) IBAs were treated with 0.5 mM H 2 O 2 for 9 h or 2 mM NAC for 24 h. ATGL lipase activity assays were conducted using the WCE (N = 4, F = 43.33). (c-g) IBAs were transfected with Nc and Pex2 siRNAs. Under this background, cells were treated with Cat siRNA, Acox1 siRNA, 100 μM C26:0 and C24:0, 0.5 mM H 2 O 2 or 2 mM NAC, as indicated. IB was conducted to check protein levels by indicated antibodies (f, N = 3 in H 2 O 2 treatment and 6 in control; N = 4 in c, d, e, g; F = 13.29 in Nc and 0.08457 in Pex2 siRNA in c; F = 119.5 in Nc and 0.06723 in Pex2 siRNA in d; F = 16.26 in Nc and 0.214 in Pex2 siRNA in e). (h) PEX2-FLAG and ATGL-FLAG were co-transfected in HEK293T cells. After 48 h, cells were treated by 0.5 mM H 2 O 2 and harvested at indicated time points. IB was conducted by indicated antibodies. Repeated 3 times. (i) Wild type PEX2 and PEX2C1-7G (siRNA resistant) were expressed in HEK293T cell under the background of endogenous PEX2 depletion. After 48 h, cells were treated by 0.5 mM H 2 O 2 and harvested at indicated time points. IB was conducted by indicated antibodies. Repeated 3 times. (j) IBAs were transfected with Cpt1b and Cpt2 siRNAs to inhibit mitochondrial fatty acid oxidation. After 72 h, IB was conducted to check protein levels by indicated antibodies (N = 4 in Cpt1b&Cpt2 and 8 in Nc siRNA, F = 0.125). (k) IBAs were treated with mitochondrial fatty acid oxidation inhibitor etomoxir (ETO) at indicated time points. IB was conducted to check protein levels by indicated antibodies (N = 3 in ETO treatment and 9 in control, F = 1.016). (l) HepG2 cells were transfected with CPT1A and CPT2 siRNAs to inhibit mitochondrial fatty acid oxidation. After 72 h, IB was conducted to check protein levels by indicated antibodies (N = 4 in CPT1A&CPT2 and 8 in Nc siRNA, F = 0.3906). (m) HepG2 cells were treated with mitochondrial fatty acid oxidation inhibitor ETO at indicated time points. IB was conducted to check protein levels by indicated antibodies (N = 3 in ETO treatment and 9 in control, F = 0.3629). (n) IBAs were treated with mitochondria targeted antioxidant MitoQ at indicated doses for 24 h. IB was conducted to check protein levels by indicated antibodies (N = 4, F = 0.7356). Results are shown as mean ± SEM and analyzed using Student’s two-sided t test (g) and ANOVA method with Dunnett correction for multiple comparisons between control and other groups (a-f, j-n).

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Activity Assay, Transfection

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: (a-b) Peroxisomal H 2 O 2 levels in HepG2 cells and differentiated iBAs were quantified by HyPer3-PTS1 after ATGL depletion by siRNA (a, cell number = 37 in ATGL and 47 in Nc siRNA; b, cell number = 18 in Atgl and 25 in Nc siRNA). (c) IBAs with PEX2-FLAG-EGFP expression were treated with Atgl siRNA and harvested 72 hours after transfection. IP and IB were conducted using the indicated antibodies (N = 4). (d) Peroxisomal H 2 O 2 levels in differentiated iBAs were quantified by HyPer3-PTS1 6 hours after Iso treatment (cell number = 22). (e) IBAs were stimulated with Iso for 24 h at indicated doses. Atgl transcript was quantified by qPCR (N = 4, F = 7.57). (f) Peroxisomal H 2 O 2 levels in differentiated iBAs were quantified by HyPer3-PTS1 6 hours after Iso treatment in the presence of 5% BSA (cell number = 16 in Iso treatment and 17 in control). (g) IBAs with PEX2-FLAG-EGFP expression were treated with 0.1 μM Iso in the presence of 5% BSA and harvested at the indicated time points. IP and IB were conducted using the indicated antibodies (N = 4). (h) IBAs were treated with 0.1 μM Iso in the presence of 5% BSA and harvested at indicated time points. IB were conducted using the indicated antibodies (N = 4). (i) Pulse-chase experiments to monitor NBD-C12 (green) trafficking from LD to peroxisome in iBAs. IBAs transfected with mCherry-PTS1 were pulsed with 10 μM NBD-C12 for 24 h, followed by lipolysis inhibitor treatment (BAY) after washing. 24 h later, cells were fixed for imaging or collected for fractionation and fluorescence measurement (N = 5). LDs were labelled by LipidTOX Deep Red dye. Scale bar, 5μm. Repeated 3 times. (j-l) IBAs were treated by 100 μM C26:0 and C24:0 for 48 h in the presence of lipolysis inhibitor (BAY) or DGAT1/2 inhibitors. Peroxisomal ROS levels were measured (cell number = 58, F = 15.71) or IB was conducted to check protein levels by indicated antibodies (N = 4; F = 15.59 in DMSO and 7.561 in BAY treatment of k; F = 16.68 in DMSO and 9.005 in DGAT1/2i treatment of l). Results are shown as mean ± SEM and analyzed using Student’s two-sided t test (a-d, f-i) or an ANOVA test with Dunnett correction for multiple comparisons between control and other groups (e, j-l).

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Expressing, Transfection, Pulse Chase, Imaging, Fractionation, Fluorescence

Journal: Nature Metabolism

Article Title: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

doi: 10.1038/s42255-021-00489-2

Figure Lengend Snippet: a , Two weeks after Pex2 knockout induction, livers were collected and hepatic proteins were analyzed by IB, as indicated (wild-type n = 7; Pex2KO n = 9). b , PEX2–FLAG was expressed for 2 weeks before liver collection and homogenization. IP and IB were conducted using the indicated antibodies. Experiments were repeated four times. c , d , PEX2–FLAG was expressed in the liver of CatKO or Acox1KO mice. IP and IB were conducted using the indicated antibodies ( n = 9 ( c ); n = 4 ( d )). e , PEX2–FLAG was expressed in the liver of wild-type mice. After 2 weeks, NAC was administered to the mice via intraperitoneal injection at a dose of 500 mg kg −1 body weight for 24 h. IP and IB were conducted using the indicated antibodies ( n = 8). f , NAC was administered to the Pex2KO KO mice for 24 h. Livers were collected and hepatic proteins were analysed by IB ( n = 5 for Pex2KO for both conditions). g , ATGL levels in human liver biopsies were analysed by IB and the relative ATGL levels are presented (50 human samples). P = 0.0372 using a Spearman test. h , Wild-type mice with PEX2–FLAG expression in the liver were challenged with HFD for 16 weeks. IP and IB were conducted using the indicated antibodies ( n = 5). i , Wild-type and AtglKO mice were fed with NCD, HFD and HFD plus NAC (40 mM) in drinking water for 8 weeks. Liver lipids were extracted for TAG level determination (for wild-type mice, NCD n = 6, HFD n = 7, HFD + NAC n = 7; for AtglKO mice, n = 7). NCD, normal chow diet. j , Representative hematoxylin and eosin staining images of livers from wild-type or AtglKO mice fed on HFD and NAC-containing water for 8 weeks. Scale bar, 100 μm. Experiments were repeated three times. k , Working model illustrating the whole pathway in which peroxisomal β-oxidation generates H 2 O 2 to stabilize PEX2 protein, resulting in increased ATGL degradation and decreased lipolysis in turn. Results are shown as the mean ± s.e.m. analysed using a two-sided Student’s t -test.

Article Snippet: Human ATGL–FLAG (catalogue no. RC205708) and PEX2–FLAG–Myc (catalogue no. RC218196) expression plasmids were obtained from OriGene.

Techniques: Knock-Out, Homogenization, Injection, Expressing, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Hormone-sensitive lipase drives pro-resolving macrophage polarization and enhances efferocytosis

doi: 10.1186/s12964-025-02631-z

Figure Lengend Snippet: HSL is a critical regulator of efferocytosis and the pro-resolving phenotype in M2 macrophages. (A) Total lipase activity in cell lysates from M1 and M2 polarized macrophages. ( n = 4 independent donors). ( B ) Efferocytosis efficiency in M2 macrophages with a pan-lipase inhibitor, Orlistat (50 and 100 µM). ( n = 4 independent donors). (C) Schematic of intracellular triglyceride (TAG) metabolism, illustrating pathways for lipid storage via DGAT and lipolysis via cytosolic lipases (ATGL, HSL, MAGL) or lysosomal lipophagy (LAL). (D) Efferocytosis efficiency in M2 macrophages following treatment with specific inhibitors for ATGL (ATGLi/Atglistatin, 20 µM), HSL (HSLi/ HSL-IN-1, 5 µM), or MAGL (MAGLi, 5 µM). ( n = 4–5 independent donors). (E) Efferocytosis efficiency in M1 macrophages following treatment with the same specific lipase inhibitors. ( n = 6 independent donors). (F-G) Efferocytosis efficiency in M2 macrophages after inhibition of (F) triglyceride synthesis with a DGAT1 inhibitor (DGATi/ T863, 50 µM) or (G) lysosomal acid lipase with Lalistat 2 (LALi, 5 µM). ( n = 4 independent donors). (H) M2 macrophages treated with M2 and HSLi (HSL-IN-1, 5 µM) were stained for LDs using PLIN2 and imaged with confocal microscopy. Total LD volume per cell was measured with Imaris. (n = cells from 3 independent donors). ( I ) Representative high-resolution confirmation of the LD phenotype by transmission electron microscopy (TEM) of M1, M2, and HSL-inhibited M2 (M2 + HSLi) macrophages. Lower panels show magnified views of the boxed areas. Red arrows indicate LDs. Scale bars = 2 μm (top), 0.5 μm (bottom). (J) Quantification of LD size (left) and density (right) in M1, M2, and M2 + HSLi macrophages was conducted using ImageJ software from a single donor with triplicate measurements per condition. Each dot represents a single LD, and they are not used for statistical comparison between groups. (K) Surface expression of CD163 and CD36 on M2 macrophages with or without HSL inhibition, measured by flow cytometry. ( n = 4–6 independent donors). (L) Secreted levels of the pro-resolving mediator Annexin A1 and the pro-inflammatory cytokine TNFα from M2 macrophages with or without HSL inhibition, measured by ELISA. ( n = 5–6 independent donors). (M) Efferocytosis efficiency in murine bone marrow-derived macrophages (BMDMs) polarized to M1 or M2 phenotypes, with or without HSL inhibition. ( n = 6–10 biological replicates). For panels A, F, G, H, K, L, a paired t -test was used. For panels B, D, E, and M, a one-way ANOVA with Tukey’s multiple comparisons test was used. Data in A shows paired individual donors. Panels B, D-G and K-M are presented as box-and-whisker plots showing median with all points. Data in J are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant

Article Snippet: The cells were transfected with either a PNPLA2 -encoding plasmid (ATGL, GeneCopoeia, Rockville, MD), a LIPE -encoding plasmid (HSL, GeneCopoeia), or a control plasmid (GeneCopoeia).

Techniques: Activity Assay, Inhibition, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Software, Comparison, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Whisker Assay

Journal: Journal of Lipid Research

Article Title: PNPLA2 influences secretion of triglyceride-rich lipoproteins by human hepatoma cells

doi: 10.1194/jlr.m090928

Figure Lengend Snippet: Figure 5A, no evidence was found that a combination of the two PNPLA2 inhibition methods leads to

Article Snippet: The cells were subsequently incubated for one hour with 1:50 diluted PNPLA2 monoclonal antibody (Novus Biologicals) conjugated with Alexa Fluor 488 using the Zenon Alexa Fluor 488 Mouse IgG2b Labeling Kit (Thermo Fisher Scientific) at RT, followed by overnight incubation with either 1:100 diluted PDI (Enzo) or 1:100 diluted PLIN2 (R&D Systems) monoclonal antibodies.

Techniques: Inhibition