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: 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: 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: