Fig 1: Fusion can be triggered in 3T3-L1 adipocytes.(a) 3T3-L1 adipocytes were replated and imaged in real-time using bright field microscopy for a total of 30 min. No LD fusion events were observed when cells were treated with vehicle (DMSO). However, addition of 50 µM SR 59230A, 50 µM H-89 or 50 µM ML-7 triggered fusion of LDs (arrows in blow-up) and cell rounding. Bar = 20 µm. (b) Perilipin A-YFP was transiently expressed in 3T3-L1 adipocytes and imaged by real-time microscopy in the absence or presence of 200 µM propranolol. Z-stack confocal microscopy images were acquired every 30 s over 45 min and rendered to produce a 3D image of the cell. There was no significant motility or detectable fusion of labelled LDs in control cells. Following treatment with 200 µM propranolol, multiple LD fusion events were observed in over 80% of the cells expressing perilipin A-YFP. Many LDs underwent multiple fusion events, highlighted in the sequential fusion of three LDs (insert). Bar = 10 µm, insert = 2 µm. (c) 3T3-L1 cells transiently expressing Plin A-YFP were imaged in a single plane during treatment with 50 µM SR 59230A. The membranes of the two fusing LDs became continuous within 10 s and the LD cores had merged within 30 s. Bar = 20 µm, blow-up = 10 µm.
Fig 2: Lipid droplet fusion can be triggered specifically by chemicals.(a) Representative images NIH-3T3 cells treated with a variety of chemical reagents and stained with Bodipy493/503. Bar = 10 µm, N = nucleus. The chemical structure of each reagent appears beside the image. (b) Random micrographs from each reagent treatment were analysed for both LD size and number. Fusogenic reagents caused a decrease in LD number concurrent with an increase in LD radius. Error bars represent the S.E.M of at least 12 cells from 3 or more replicates, *p<0.0005, **p<0.005, ***p<0.05. (c) Prolonged imaging of LDs using Bodipy493/503 in NIH-3T3 cells failed to detect LD fusion. Bar = 5 µm, N = nucleus (d) Time-lapse imaging of NIH-3T3 cells stained with Bodipy493/503 demonstrated that multiple fusion events were triggered by addition of 50 µM H-89. Coloured arrows indicate fusing pairs of LDs. Bar = 10 µm (e) Plin A-YFP was expressed in NIH-3T3 cells and the LDs detected using Nile Red. Bar = 10 µm.
Fig 3: Lipid droplet fusion occurs in two stages and the lipid droplet surface is disrupted upon treatment with fusogenic reagents.(a) 3T3-L1 adipocytes expressing perilipin A-YFP were imaged in real-time for a total of 30 min. Analysis of single fusion events in 3T3-L1 adipocytes treated with 50 µM SR 59230A showed that the initial fusion (defined by the continuity of the LD membranes) was completed within 1 frame (30 s) whereas the reformation of a spherical structure could take several minutes. Bar = 10 µm. (b) H-89 (50 µM final concentration) was added directly to the medium whilst imaging (Bar = 20 µm). Prior to any LD fusion being observed, Plin A-YFP redistributed into dense patches on the LD surface (arrows). Bar = 10 µm. (c) In adipocytes treated with SR 59230A the directional loss of Plin A-YFP across the surface of the LDs was observed. Bar = 10 µm. (d) An example of the appearance of a discrete, intensely fluorescent structure at the site of LD fusion. Bar = 10 µm. (e) 3D rendering of z-stack images show the LD core (stained with Nile Red) remains spherical although the surface has been disrupted (as seen by Plin A-YFP labelling). Bar = 5 µm.
Fig 4: Dysregulation of H19X-encoded miRNAs in APs affects their differentiation into beige adipocytes. A, B Cells were cultured in a differentiation medium for beige fat lineage and harvested for analysis at several time points (n = 3). A Mature miRNAs, miR-322-5p and miR-542-3p, were downregulated in differentiated beige adipocytes. B The primary transcripts of miR-503 and miR-542 were downregulated immediately upon the differentiation of adipocytes. The let-7b primary transcript was used as a control. C-E APs were transiently transfected with miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) for 24 h and stimulated with an adipogenic cocktail for the differentiation. Cells were harvested for analysis at several time points after the induction of differentiation (day 0, 2, 4, 6, and 8) (C) The representative images of Oil Red O staining in differentiated beige adipocytes at day 8 (left panel). The quantified intensities of Oil Red O staining are shown (right panel). The pretreatment of miRNA mimic mix reduced the number of differentiated adipocytes. Scale bar, 100 µm (D) The mRNA levels of Ucp1 and Ap2 were analyzed during beige adipocyte differentiation (n = 2). The pretreatment of miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) attenuated the expression of Ucp1 and Ap2. E The protein levels of CCND1 and PLIN1 were examined by western blotting during beige adipocyte differentiation. miRNA mimic mix downregulated CCND1 protein levels and delayed the expression of PLIN1 protein. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05
Fig 5: NTN1 supplementation rejuvenates the aged BM niche.a Representative immunofluorescence images of trabecular regions of femurs showing an improvement of aging-related BM niche defects following NTN1 treatment. b, c Analysis of BM niche function following NTN1 treatment of aged mice by determining vascular leakiness (10 kDa dextran extravasation), vascular perfusion (Hypoxyprobe staining), and BM adiposity (PLIN1 + adipocytes/high power field) in both trabecular b, and diaphyseal c regions of femoral BM, as compared to young controls (N = 5 mice/group). Data is presented as the mean ± standard error of the mean (SEM). Statistical significance determined using One-Way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. ns denotes statistically not significant.
Supplier Page from MilliporeSigma for Anti-Perilipin A antibody produced in rabbit