Journal: Cell Metabolism

Article Title: Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca 2+ homeostasis with adipose tissue lipolysis

doi: 10.1016/j.cmet.2021.07.008

Figure Lengend Snippet: Impaired lipolysis upon fasting in POMCOpa1KO mice precedes the onset of obesity (A) Body weight (n = 9–11/genotype/nutritional status; pooled from two independent experiments). (B) pgWAT, scWAT, and BAT mass (n = 9–11/genotype/nutritional status; pooled from two independent experiments). (C) Plasma FFA levels (n = 9–10/genotype/nutritional status). (D) Gene expression of lipolytic enzymes in pgWAT (n = 7–8/genotype/nutritional status). (E and F) Representative immunoblot images (E) and densitometric quantification (F) of pHSL (normalized by total HSL protein) and perilipin A (normalized by tubulin) levels in pgWAT (n = 4/genotype/nutritional status). (G) Epinephrine and norepinephrine content in pgWAT (n = 4/genotype/nutritional status). All studies were conducted in 5- to 6-week-old male control and POMCOpa1KO mice under fed or overnight (16 h) fasting conditions. Data are expressed as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. ns, not significant. See also Figure S4 .

Article Snippet: Perilipin , Cell Signaling , Cat# 3467; RRID: AB_2167270.

Techniques: Clinical Proteomics, Gene Expression, Western Blot, Control

Journal: Cell Metabolism

Article Title: Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca 2+ homeostasis with adipose tissue lipolysis

doi: 10.1016/j.cmet.2021.07.008

Figure Lengend Snippet: Modulation of POMC neuron activity influences lipolysis (A) Schematic of viral activatory DREADD injection. (B) Fasting-induced FFA increase after CNO-mediated stimulation of POMC neuronal activity (n = 9–13/group). (C) Fasting-induced pgWAT mass after CNO-mediated stimulation of POMC neuronal activity (n = 12–13/group). (D) Fasting-induced body weight change after CNO-mediated stimulation of POMC neuronal activity (n = 13/group). (E) Schematic of viral inhibitory DREADD injection. (F) Fasting-induced FFA increase after CNO-mediated inhibition of POMC neuronal activity (n = 8–13/group). (G) Fasting-induced pgWAT mass after CNO-mediated inhibition of POMC neuronal activity (n = 8–13/group). (H) Fasting-induced body weight change after CNO-mediated inhibition of POMC neuronal activity (n = 8–13/group). (I) Immunoblot images and quantification of fasting-induced changes of lipolytic enzymes in pgWAT after CNO-mediated inhibition of POMC neuronal activity (n = 8/group). Phosphorylated HSL was normalized by total HSL and perilipin A was normalized by actin. All studies were conducted in 14- to 16-week-old male POMC Cre/+ mice or POMC +/+ . Data are expressed as mean ± SEM. ∗ p < 0.05. See also Figure S5 .

Article Snippet: Perilipin , Cell Signaling , Cat# 3467; RRID: AB_2167270.

Techniques: Activity Assay, Injection, Inhibition, Western Blot

Journal: Cell Metabolism

Article Title: Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca 2+ homeostasis with adipose tissue lipolysis

doi: 10.1016/j.cmet.2021.07.008

Figure Lengend Snippet: Mitochondrial Ca 2+ dyshomeostasis in POMC neurons from POMCOpa1KO mice underlies defective lipolysis (A) Gene expression of MCU complex subunits in ARC microdissections from control (n = 5) and POMCOpa1KO mice (n = 11). (B and C) Recordings of mitochondrial (B) and cytosolic (C) Ca 2+ normalized fluorescence signal from POMC neurons in fasted mice. Food presentation response is marked with a dotted frame. Inset represents the area under the curve (AUC) quantification of the fluorescence increase over baseline during the acquisition period. Mitochondrial Ca 2+ measurements: control (n = 6) and POMCOpa1KO (n = 8) mice. Cytosolic Ca 2+ measurements: control (n = 5) and POMCOpa1KO (n = 3) mice. (D and E) Representative mitochondrial Ca 2+ fluorescence trace of POMC neurons from control and POMCOpa1KO fasted mice after i.c.v. injection of either vehicle (Veh) or Ru265. (E) AUC quantification of the fluorescence increase over baseline during the acquisition period. n = 3/genotype. (F) Schematic of acute i.c.v. injection setup. (G) Fasting-induced increase of plasma FFA after vehicle (Veh) or Ru360 i.c.v. injection in control and POMCOpa1KO mice. n = 7–12 genotype/treatment. (H and I) Representative immunoblot images (H) and densitometric quantification (I) of lipolytic markers (HSL-pS660/HSL, HSL-pS563/HSL, and perilipin/tubulin) in pgWAT from control and POMCOpa1KO mice after i.c.v. treatment with vehicle (Veh) or Ru360 (n = 4/genotype/treatment). (J) Epinephrine and norepinephrine content in pgWAT from control and POMCOpa1KO mice after i.c.v. treatment with vehicle (Veh) or Ru360. n = 9–11/genotype/treatment. (K) Fasting-induced increase of plasma FFA levels in control and POMCOpa1KO mice after vehicle (Veh) or Ru360 i.c.v. injection together with i.p. administration of saline (Sal) or β 3 -adrenergic blocker SR59230A (SR). n = 10–12/genotype/treatment. All studies were conducted in 12- to 16-week-old male control and POMCOpa1KO mice under fed or overnight (16 h) fasting conditions. Data are expressed as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. ns, not significant. See also Figure S6 .

Article Snippet: Perilipin , Cell Signaling , Cat# 3467; RRID: AB_2167270.

Techniques: Gene Expression, Control, Fluorescence, Injection, Clinical Proteomics, Western Blot, Saline

Journal: Cell Metabolism

Article Title: Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca 2+ homeostasis with adipose tissue lipolysis

doi: 10.1016/j.cmet.2021.07.008

Figure Lengend Snippet:

Article Snippet: Perilipin , Cell Signaling , Cat# 3467; RRID: AB_2167270.

Techniques: Virus, Plasmid Preparation, Recombinant, Electron Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay, Reverse Transcription, RNAscope, Multiplex Assay, Hybridization, Software, Microscopy

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: (A) Radioligand binding assay for detection of anti-PLIN1 antibodies. Radiolabeled PLIN1 protein was incubated with sera from healthy controls (HC, n = 54) or from APS1 patients with or without lipodystrophy (APS1 +/- LD, n = 1/68). Dotted line indicates mean + 3SD of healthy controls. (B) Validation of autoantibodies to PLIN1 in orthogonal cell-based assay. Fixed stomach tissue of mice was mounted and immunostained with sera from Case report patient 1 and commercial antibody to PLIN1. A mix of secondary antibodies anti-Human IgG-alexa547 and anti-Rabbit alexa488 at 1:2000 were used to visualize Human IgG and PLIN1 antibodies, respectively. A Merge is provided on the right of individual images. Images taken at 20X magnification. Note yellow in Merge indicating colocalization of PLIN1 (Green) and Human IgG (RED). DAPI is blue and indicates nuclei. Scale bar indicates 100 microns.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Radio Ligand Binding Assay, Incubation, Cell Based Assay

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Autoantibodies to PLIN1 in sera from a patient with autoimmune AGL following cancer immunotherapy. (A) Radioligand binding assays screening for PLIN1 antibodies in sera from all time points from index patient 2 as well as checkpoint-treated control patients without lipodystrophy (n=7), as done for case report 1. Dotted line indicates mean + 3 std. dev. of healthy controls (n = 11). Numbers to the right of circles indicate the time point series, for reference in panel B. The left panel describes the clinical timeline corresponding to timepoints T0-T4. CPI was discontinued at 34 cycles (16 months) due to progressive weight loss and elevated LFTs, with futher work-up over subsequent months. (B) Validation of autoantibodies to PLIN1 using immunohistochemistry on mouse enteric tissue, as done in . Sera was used from either a control (immunotherapy but no AGL) or case report 2 (Immunotherapy with AGL) with various time points. Each column represents an individual sample, from left to right ; sera from checkpoint control pre-treatment, sera from checkpoint control post treatment with no autoimmunity, Case report 1 pre-therapy, Case report 1 post-therapy-1, Case report 1 post-therapy-3, Case report 1 post-therapy-4. Images are 600×600 pixel insets from original 40X image. Scale bar represents 100 microns. CP = Checkpoint patient controls.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Binding Assay, Immunohistochemistry

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Discovery and validation of autoantibodies to Perilipin-1 in Aire -/- mouse sera. (A) PhIP-Seq analysis in Aire -/- (“KO”) and Aire +/+ (“WT”) mice. Aggregated Plin1 PhIP-Seq data from Aire -/- (KO, n=4) and Aire +/+ (WT, n = 3) mice. (B) Whole cell lysates generated from 293T cells expressing full-length mouse PLIN1 were incubated with sera from Aire -/- (KO, n=4) or Aire +/+ (WT, n=3) mice. Antibodies were immunoprecipitated using AG beads and IP elutions were subject to SDS-PAGE immunoblotting. AG lane indicates an IP using AG beads only, no sera. Input lane indicates loading of whole cell lysate with and without (-) transfection of PLIN1-myc-flag plasmid. IP elutions and Input were immunostained with either anti-Flag IgG to identify positive anti-Plin1 signal, or anti-mouse IgG to show qualitative capture of IgG from sera. (C) Representative image of immunohistochemistry on mouse enteric tissue showing positive colocalization of antibodies from Aire -/- sera (KO) and commercial anti-Plin1 IgG. Primary antibodies from Aire -/- mouse (KO) sera and commercial antibody to PLIN1 were visualized with secondaries to Mouse IgG (Alexa-567, RED) and Rabbit IgG (Alexa-488, GREEN), respectively. DAPI (BLUE) stains nuclei.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Generated, Expressing, Incubation, Immunoprecipitation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Immunohistochemistry

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: A, A current model for the hormonal regulation of lipolysis initiation is shown. Proteins depicted include perilipin 1A (Peri), Hormone Sensitive Lipase (HSL), Adipocyte Triglyceride Lipase (ATGL), CGI-58, and PKA. Lipid species depicted include triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), and fatty acid (FA). Under basal conditions, perilipin and HSL are unphosphorylated and HSL is found throughout the cytoplasm. Stimulation of lipolysis involves activation of PKA, phosphorylation of perilipin 1A and HSL, release of CGI-58 from perilipin, binding of CGI-58 to ATGL, and translocation of HSL to perilipin. TAG is sequentially processed to DAG by ATGL and to MAG by HSL with FA released at each step. B, Amino acid sequences are shown for perilipin 1A PKA site 5, and PKA site 6, and for HSL serine 563 and serine 660. The target serine in each sequence is underlined.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Translocation Assay, Sequencing

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: Hela cells were transfected separately with plasmids encoding wild-type (GFP, green) or mutant (mCherry, red) perilipin 1A ( PLIN1 ) plasmids. Oleic acid was added for 24 h, followed by 7 min treatment with 24 µM forskolin plus 125 µM IBMX and the cells labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. For each condition, images are shown for nuclei (DAPI), fluorescent protein (either GFP or m-Cherry), or for antibody labeling (the far red fluorescence channel). A, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 5. B, Cells expressing mCh-perilipin 1A S497A, which did not label for anti-pPeri-site 5. C, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 6. D, Cells expressing mCh-perilipin 1A S522A, which did not label with anti-pPeri-site 6.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Transfection, Mutagenesis, Labeling, Antibody Labeling, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: Preadipocytes were transfected with either control or perilipin 1A siRNA (0 to 50 nM), exposed to differentiation medium for 6 days, treated with 6 µM FSK for 20 minutes, then fixed and labeled for nuclei (blue), lipid (green), and either anti-pPeri-site 5 or anti-pPeri-site 6 (red) plus GP29 (yellow). A, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 5 plus GP29. B, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 6 plus GP29. C, D, and E are mean values for Area of the Protein mask (Area Pm), for GP29, anti-pPeri-site 5, and anti-pPeri-site 6, respectively. Data are normalized to the 0 siRNA control, and each bar represents the mean ± SD, for n = 6 wells. *** p<0.001 for perilipin vs. control siRNA at each concentration (Student’s t-test).

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Transfection, Control, Labeling, Concentration Assay

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: Human subcutaneous adipocytes were exposed to either control medium, or medium supplemented with 10 µM FSK and 500 µM IBMX for 2 minutes, then fixed and labeled for nuclei, lipid droplets, and with either anti-pPeri-site 5 or anti-pPeri-site 6. A goat-anti-mouse secondary antibody coupled to Texas Red was used to visualize the phospho-perilipin antibodies in the red fluorescent channel. A, Control adipocytes are shown visualized for nuclei (blue) and lipid droplets (green). B, the same field is shown visualized for anti-pPeri-site 5 (red). C, FSK/IBMX-treated adipocytes are shown visualized for nuclei and lipid droplets. D, The same field is shown visualized for anti-pPeri-site 5. E, Control adipocytes are shown visualized for nuclei and lipid droplets. F, The same field is shown visualized for anti-pPeri-site 6. G, FSK/IBMX-treated adipocytes are shown visualized for nuclei and lipid droplets. H, The same field is shown, visualized for anti-pPeri-site 6. Scale bar = 50 µm.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Control, Labeling

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 3T3L1 adipocytes were exposed to either FSK (6 µM) or L-γ-MSH (100 nM) for 5 minutes, then fixed and labeled for nuclei (blue), lipid droplets (green), and phosphorylated perilipin (red). The cells were then imaged (20Xobjective, 4 images/well) and the images analyzed utilizing the Colocalization algorithm. A, B, and C, Images are shown of adipocytes exposed to control, FSK, or LγMSH, respectively, labeled with anti-pPeri-site 5. D, E, and F, Images are shown of adipocytes exposed to control, FSK, LγMSH, respectively, labeled with anti-pPeri-site 6. G and H represent Area Pm for pPeri-site 5 and for pPeri-site 6, respectively. Each bar represents the mean SD for n = 3 wells/condition (an average of 532 cells/well) for G, or n = 8 well/condition (an average of 272 cells/well) for H. ** p<0.01 vs. Con (ANOVA followed by Tukey’s test). *** P<0.001 vs. Con. ### p<0.001 vs. FSK. Scale bar = 50 µm.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Labeling, Control

Journal: PLoS ONE

Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

doi: 10.1371/journal.pone.0055511

Figure Lengend Snippet: 3T3L1 adipocytes were exposed to either 1 µM isoproterenol or 100 nM L-γ-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (µg) of blocking peptides corresponding to pPeri-site 5, pPeri-site 6, or pHSL-serine 660. Anti-phospho-perilipin 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C, Results are shown cells treated with L-γ-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A, each bar represents a single well; for B and C, each bar represents the mean ± SD for n = 3 wells.

Article Snippet: The cDNA sequence encoding human perilipin 1A was purchased from Origene (Rockville, MD).

Techniques: Labeling, Blocking Assay, Fluorescence

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 1. Anti-PLIN1-autoAbs in patients with autoimmune endocrine diseases. Anti-PLIN 1-autoAbs indices in APS-1, AAD/APS-2, IAD, GD, T1D, POI and blood donor groups. Mean index values for each patient group are marked by horizontal lines. Threshold for positivity (horizontal dashed red line) has been calculated for the PLIN1 autoantigen based on the mean of indices of blood donors + 3SD (index ≤122). Thirteen dominant negative mutations in AIRE patients (gray dots) are included in the APS-1 patient subgroup. APS-1: autoimmune polyendocrine syndrome type 1; AAD: autoimmune Addison’s disease; APS-2: autoimmune poly endocrine syndrome type 2; IAD: idiopathic Addison’s disease; GD: graves’ disease; T1D: type 1 diabetes; POI: primary ovarian insufficiency; SS: Sjögren’s syndrome.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques: Dominant Negative Mutation

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 2. A longitudinal study of PLIN1-autoAbs for an APS-1 patient (patient #27, Table 2). PLIN1 indices of five serial samples from the same patient at five different time points in the period 2009–2021, and 21OH autoAbs indices plotted at the same time spanning. The thresholds for positivity to PLIN1-autoAbs and 21OH autoAbs are shown with horizontal dashed red and black lines, being 122, and 100 respectively.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques:

Journal: Autoimmunity

Article Title: Anti-perilipin-1 autoantibodies in autoimmune Addison's disease and related endocrine disorders.

doi: 10.1080/08916934.2025.2461703

Figure Lengend Snippet: Figure 3. Analysis of anti-PLIN1 IgM autoantibodies using ELISA. Sera samples of anti-PLIN1-autoAbs positive patients in radioimmunoassay were used to detect frequencies of IgM PLIN1-autoAbs through ELISA. (A) Frequency of anti-PLIN1 IgM autoAbs compared to control. (B) Serial sera samples from one patient with APS-1 (patient #27) analyzed for PLIN1- IgM autoAbs in ELISA. A commercial monoclonal antibody against PLIN1 was used as positive control. In (A) the blue dot depicts one blood donor scoring PLIN1 autoAbs borderline positive in RIA. In (A and B) the violet dot corresponds to the identical sample that has also been plotted in the PLIN+ in both the RIA and longitudinal groups.

Article Snippet: For coating of wells, 50 μL recombinant PLIN1 protein (Cat: TP306292, Origene) at 2.0 μg/ mL in 1X PBS (Cat: 524650, EMD Millipore,) was incubated overnight at 4 °C in a 96-well ELISA plate (Nunc MaxiSorpTM Flat-bottom, Cat: 439454, Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, RIA Assay, Control, Positive Control