Fig 1.
In panel, images from confocal scanning microscopy.
Spheroid no differentiated in the first column (NDIF) and differentiated spheroids in subsequent columns (WA and WA-TNFα). The cell’s nucleus was stained with DAPI, lipid droplets were stained with LipidSpot and both staining were merged. Almost the whole space of adipocytes is occupied by lipid droplets after 14 days exposed to differentiated cocktail (WA and WA-TNFα) when compared with undifferentiated condition (NDIF). Confocal laser scan microscopy was applied to detect DAPI and LipidSpot. B) ATP quantification to analysis of spheroids viability through the time. C) Accompaniment of spheroid size through during the differentiation process. Statistical significance (One-Way ANOVA with Tukey post-test, where ***p<0,0005, **p<0,005 and *p<0,05). Error bars represent standard deviation of means from at least 7 replicates for size measurements and 12 replicates for viability.
Fig 2.
A) Glucose uptake assay of a metabolically healthy and insulin resistance in vitro model. B) Quantification of interleukin IFNγ secreted by the spheroids. Influence of differentiation coktail on the expression of adipocyte markers. Graphs display the mRNA expression profile, by qRT-PCR, from C) Pparg, D) Lpl, E) Lep and F) Adipoq, of differentiated 3T3-L1 spheroids (WA) and treated with TNF-α. Error bars represent standard deviation of means from at least 3 replicates; the measurement was performed after 14 days of differentiation.
Fig 3.
Pipeline of mass spectrometry-based analysis of adipose tissue from lean and obese mice (WAT and WAT-HDF) and from adipose tissue models (WA and WA-TNF); as a representative result, the Venn diagram of mass spectrometry-based proteomics of adipose spheroids (WA and WA-TNF-α) and white adipose tissue from mice (WAT and WAT-HDF).
The samples were submitted to protein extraction; proteolytic digestion followed by MS analysis. Venn diagram of identified proteins, WA had 842 proteins identified, WA-TNF-α 967 proteins, WAT had 1315 and WAT-HFD 1330. WA has 11 exclusive proteins, WA-TNF-α has 15, WAT 19 and WAT-HDF 33 exclusive proteins identified.
Fig 4.
A) Heat map of mass spectrometry-based proteomics of adipose spheroids showing a comparative proteomic profile among adipose spheroids and adipose tissue from mice. B) Joint pathway analysis, the graph shows the pathways that are enriched. C) top 10 metabolic process networks; in red the process from WA and in dark blue from WAT. D) top 10 metabolic process networks; in green the process from WA-TNF-α and in dark blue from WAT-HDF.
Fig 5.
Regulation of metabolism, the role of adiponectin in regulation of metabolism.
1) WA, 2) WAT, 3) WA-TNF-α and 4) WAT-HDF. The pathway images were generated by GeneGo Metacore™ enrichment analysis. Well-characterized proteins or protein complexes are shown as individual symbols within the image; experimental data from all the records are connected and depicted as thermometer-like figures on the maps. Upward-facing thermometers are shown in red and indicate more abundant proteins. The linkage of proteins by arrows depicts the stimulatory and inhibitory effects or interaction of the encoded protein on the desired protein. Further explanations are provided at https://portal.genego.com/help/MC_legend.pdf.
Fig 6.
The map illustrates the process of glycogenolysis involving distinct components where 1) WA, 2) WAT, 3) WA-TNFα, and 4) WAT-HDF.
These visual representations of pathways were created using GeneGo Metacore™ enrichment analysis. The image depicts well-defined proteins or protein complexes as separate symbols within the pathway. Experimental data from various records are represented as thermometer-like figures connected throughout the maps.