Journal: bioRxiv

Article Title: miRNA Sensor HuR Compartmentalizes Ago2-Uncoupled miRNAs to Lipid Droplets to Buffer miRNA Activity in Mammalian Cells

doi: 10.64898/2026.01.10.698809

Figure Lengend Snippet: A-B. Effect of miR-122 on the number of lipid droplets in HeLa cells. Image of the Cy-3 labeled miR-122 (red) used for transfection (50nm; A) and (200 nM; B), and lipid droplets visualized with BODIPY 493/503 dye (green). DAPI stains the nucleus. Scale bars are 10μm and 2 μm (zoomed panel). Merged 1 and 2 showing different cells in 200nM miR-122 mimic transfection conditions. In Merged 2, the a and b parts marked are zoomed to show colocalization of miRNA and LD. A. Effect of miR-122 mimic transfection ( 50 or 200 nM) on lipid droplet (LD) number. Quantification of LDs number (>100nm) observed in confocal images of the cells stained for BODIPY 493/503 dye. A minimum of 20 cells was counted for each set. D-E. Effect of Perilipin 2 depletion by siRNAs in HeLa cells on LD (D) and miRNA levels (E). The SiRNA mixture targeting Perilipin 2 was used to deplete LDs in HeLa cells, and miR-122 levels were quantified in control and GW4869-treated HeLa cells, normalized to U6 RNA (n=3). Values with siControl were considered as units. Scale bar 10 μm. F. The effect of SiRNAs against perilipin 2 (siPlin2) on LD number in HeLa cells co-transfected with miR-122 mimic (200nM) (n=10). Data suggest that miRNA levels are lower with fewer LDs. G-H. Effect of expression GFP-Perilipin 2 on LDs (G) and level of cellular miRNAs (H) in HeLA cells transfected with GFP-Perilipin or PCI-neo along with 200nM miR-122 mimic. RNA values quantified by qRT-PCR were normalized to U6 RNA (n=3). Values with pCIneo control were considered as units. Scale bar 10 μm. I. Effect of GFP-Perilin2 expression on LD number determined in HeLa cells having LD with a diameter of more than 100nM diameter. More than 10 cells were analyzed in each case. J-K. Effect of the ATGL inhibitor Atglistatin or ATGLi (100 nM, 8h) on lipid droplets (K) and cellular miRNA content (L) in HeLa cells transfected with 200 nM miR-122 mimic. RNA was quantified by qRT-PCR, and relative levels were normalized to U6 RNA. Scale bar, 10 μm. L. Effect of ATGL inhibitor on LD numbers in HeLa cells. Cells were treated with 100nM ATGLi Atglistatin for 8 h. Data are presented as SEM ± SD, and P values are reported in the respective panels tested for statistical significance. P-values were calculated by a two-tailed paired t-test in most of the experiments unless mentioned otherwise. The fold change of miRNA was calculated by 2 −ΔCt method.

Article Snippet: Plasmid GFP-perilipin 2 was purchased from Origene.

Techniques: Labeling, Transfection, Staining, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

doi: 10.1194/jlr.m008771

Figure Lengend Snippet: Fig. 2. TNF- as well as IL-1 and IFN- but not IL-6 decrease FSP27 levels and increase glycerol release in 3T3-L1 adipocytes. A–C: Cells treated with 10 ng/ml TNF- for 16 h. A: Quantitative RT-PCR of Fsp27 and perilipin mRNA levels; B) representative immunoblot of FSP27, perilipin, and actin (loading control); C: densitometric quantitation of immunoblots of FSP27 and perilipin protein levels. A–C, Data are expressed as means ± SEM for six different experiments; P values were calculated using Student’s t -test; *, P < 0.05; **, P < 0.0005. A.U., arbitrary units. D: 3T3-L1 adipocytes were treated with IL-6 at the indicated concentrations and times. D, left panel, glycerol release; D, right panel, quantitative RT-PCR of FSP27 mRNA levels. E, F: Cells treated with IL1- or IFN- , respectively, at the indicated concentrations.

Article Snippet: Polyclonal perilipin antibody was purchased from Fitzgerald (product no. 20R-PP004).

Techniques: Quantitative RT-PCR, Western Blot, Control, Quantitation Assay

Journal: Journal of Lipid Research

Article Title: Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

doi: 10.1194/jlr.m008771

Figure Lengend Snippet: Fig. 4. TNF- treatment of 3T3-L1 adipocytes increases lipolysis that is enhanced by further depletion of FSP27 using siRNA. A: Glycerol released in medium was measured in 3T3-L1 adipocytes electroporated with scrambled (Scr) or FSP27 siRNA and treated with 10 ng/ml TNF- for 16 h. A.U., arbitrary units. B: Western blot analysis using FSP27, perilipin (PLIN), and actin antibodies showing FSP27 and PLIN protein levels in control and under TNF- -treated conditions. MW, molecular weight. C: Densitometric analysis of FSP27 and PLIN protein levels in siRNA or TNF- -treated cells. Data are expressed as means ± SEM for six different experiments. KD, knock-down. P values were calculated using ANOVA; *, P < 0.05; **, P < 0.0005.

Article Snippet: Polyclonal perilipin antibody was purchased from Fitzgerald (product no. 20R-PP004).

Techniques: Western Blot, Control, Molecular Weight, Knockdown

Journal: Journal of Lipid Research

Article Title: Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

doi: 10.1194/jlr.m008771

Figure Lengend Snippet: Fig. 6. Adenoviral expression of FSP27 protects against TNF- -mediated lipolysis and lipid droplet size diminution. 3T3-L1 adipocytes were infected with control virus (Ad-GFP) or HA-FSP27- expressing virus (Ad-HA-FSP27) on the fourth day of differentia- tion. On the fi fth day, cells were serum starved overnight, treated with 10 ng/ml TNF- for 24 h, and analyzed for protein, glycerol release, and lipid droplet morphology. A: Western blot analysis us- ing FSP27, HA, GFP, perilipin (PLIN), actin, and Vti1a antibodies show maintenance of HA-tagged FSP27 protein levels even after treatment of TNF- . Actin is the loading control for HA, GFP, and PLIN, and Vti1a is the loading control for FSP27. B: Glycerol re- leased into the medium in 24 h with or without TNF- treatment. A. U., arbitrary units. C: Confocal microscopy image showing lipid droplets stained with Oil Red O (red), nuclei stained with DAPI (blue), and GFP expression (green) in 3T3-L1 adipocytes express- ing Ad-GFP or Ad-HA-FSP27 in the presence or absence of TNF- . Data are expressed as means ± SEM for fi ve individual experiments. P values were calculated using ANOVA; *, P < 0.05; **, P < 0.0005.

Article Snippet: Polyclonal perilipin antibody was purchased from Fitzgerald (product no. 20R-PP004).

Techniques: Expressing, Infection, Control, Virus, Western Blot, Confocal Microscopy, Staining

Journal: Journal of Lipid Research

Article Title: Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

doi: 10.1194/jlr.m008771

Figure Lengend Snippet: Fig. 7. Isoproterenol delays degradation of FSP27 to increase protein level in 3T3-L1 adipocytes. A: Lysates from adipocytes treated with 10 µM isoproterenol (Iso) for 15, 30, 60, 120, or 180 min were immunoblotted for FSP27, perilipin (PLIN), or Vti1a (loading control). B: Densitometry of immunoblot shows increased FSP27 protein level after 180 min of 10 µM isoproterenol treatment of mature adipocytes. A.U., arbitrary units; Con, control. C: Mature adipocytes treated with 10 µM isoproterenol, pulsed with 5 µg/ml cyclohex- imide (CHX), and chased for 15, 30, and 60 min show delayed degradation of FSP27 in the presence of isoproterenol compared with that of control (0). D: Densitometry of FSP27 level in the presence or absence of 10 µM isoproterenol shows delayed FSP27 degradation in the presence of isoproterenol. E: Adipocytes transfected with either Scr or PKA siRNA and treated with or without 10 µM isopro terenol. The effect of iso- proterenol on FSP27 is abrogated when PKA is knocked down (KD). The absence of perilipin phosphorylation despite isoproterenol treatment in adipocytes treated with PKA siRNA is used as the positive control. F: Mature adipocytes were treated with 10 µM isoproterenol, 5 µM forskolin (For), 1 mM 8-bromo-cAMP (B-cAMP) plus IBMX, or 80 µM KT5720 plus 10 µM isoproterenol. The effect of isoproterenol on FSP27 is mimicked by forskolin and 8-bromo-cAMP and inhibited by KT5720. G: Mature adipocytes treated with 10 µM isoprot- erenol were harvested for RNA and analyzed for FSP27 mRNA. It shows no change in FSP27 transcript with isoproterenol treatment. Data are expressed as means ± SEM for six individual experiments.

Article Snippet: Polyclonal perilipin antibody was purchased from Fitzgerald (product no. 20R-PP004).

Techniques: Control, Western Blot, Transfection, Phospho-proteomics, Positive Control

Journal: Obesity (Silver Spring, Md.)

Article Title: Intramyocellular Lipid Droplet Size Rather Than Total Lipid Content Is Related to Insulin Sensitivity After 8-Weeks of Overfeeding

doi: 10.1002/oby.21980

Figure Lengend Snippet: A) Perilipin 3 (PLIN3) protein content decreased with overfeeding (p=0.04, N=27). B) Baseline human primary myotubes treated with 200μM of palmitate showed that PLIN2 is expressed later over a time course of 2 hours. C) Changes in PLIN2 protein content correlated inversely with glucose disposal rate (GDR, r=-0.43, p=0.03, N=25). Graphs represent mean±S.E.M, *p<0.05.

Article Snippet: Antibodies against Total Akt (Cat no. 9272), p-S473 Akt (Cat no. 9271), Total IRS1 (Cat no. 2382S), p-S1101 IRS1 (Cat no. 2385), and mTOR (Cat no. 4517) were obtained from Cell Signaling Technology (Danvers, MA) The antibodies for PLIN2 (Cat no. NB110-40877) and PLIN3 (Cat no. NB110-40764) were obtained from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Cancers

Article Title: Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

doi: 10.3390/cancers11030385

Figure Lengend Snippet: Expression of the perilipin family in liver and hepatocellular carcinoma (HCC). ( A ) Quantitative real-time PCR analysis of hepatic Plin2 , Plin3 , Plin4 and Plin5 expression in healthy mice ( n = 4), non-tumoral liver ( n = 8) and tumoral liver extracts ( n = 8) of HCC mice. The quantity of mRNA in healthy mouse livers was set to 1, and expression levels in the HCC groups were expressed as relative values. All measurements were normalized to β-Actin expression. Primers are listed in Table 2. Statistical analysis was performed by Student t-test and standard deviations below 0.05 were considered significant. ( B ) Confirmation of differential expression of the perilipin proteins by Western blot analysis. Western blots were performed to detect PLIN2, PLIN3, PLIN4 and PLIN5 proteins in liver protein extracts from healthy mice ( n = 3) and mice with HCC on tumoral and non-tumoral liver extracts ( n = 8 animals/group). An extract of primary mouse hepatocytes (marked with asterisk), transient overexpressing PLIN5, was used to define the protein band of PLIN5 as it is lowly expressed in liver. Glycerinaldehyd-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The antibodies used are listed in Table 1.

Article Snippet: PLIN3 , NB-110-40765 , poly, rabbit , Novus Biologicals , h, m.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Western Blot, Control

Journal: Cancers

Article Title: Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

doi: 10.3390/cancers11030385

Figure Lengend Snippet: Antibodies used for immunostaining and Western blot analysis.

Article Snippet: PLIN3 , NB-110-40765 , poly, rabbit , Novus Biologicals , h, m.

Techniques: Immunostaining, Western Blot

Journal: Cancers

Article Title: Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

doi: 10.3390/cancers11030385

Figure Lengend Snippet: Primers used for quantitative real time PCR.

Article Snippet: PLIN3 , NB-110-40765 , poly, rabbit , Novus Biologicals , h, m.

Techniques:

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