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. Author manuscript; available in PMC: 2017 Jul 23.
Published in final edited form as: Obesity (Silver Spring). 2017 Jan 23;25(3):527–538. doi: 10.1002/oby.21714

Figure 2.

Figure 2

Fold change in mRNA transcripts and protein levels for TREM-1 and TREM-2 relative to GAPDH in the study subjects. The expression of TREM-1, TREM-2 and TREM-1/TREM-2 ratio levels in biopsy samples and serum were compared between SO−D−, SO+D− and SO+D+ groups using One-way ANOVA for continuous variables. The TREM-1 and TREM-2 expression fold changes in SO+D− and SO+D+ were calculated, when compared to SO−D− fold ratio were stated as 1. Increased in folds of TREM-1 or TREM-2 expressions were regarded as up regulation, likewise decrease in folds as down regulation. To describe the balance between pro and anti-inflammatory state, we analysed TREM-1/TREM-2 ratio in all the study subjects. A) Gene Expression study in Liver biopsy sample (Aa) TREM-1; (Ab) TREM-2; (Ac) TREM-1/TREM-2 ratio. B) Gene Expression study in Omentum biopsy sample (Ba) TREM-1; (Bb) TREM-2; (Bc) TREM-1/TREM-2 ratio. C) Gene Expression study in Subcutaneous biopsy sample (Ca) TREM-1; (Cb) TREM-2; (Cc) TREM-1/TREM-2 ratio. The cDNA prepared from mRNA was subjected to Q-PCR with gene specific primers for TREM-1 and TREM-2 and the fold change relative to GAPDH as housekeeping gene are shown. Results were expressed as fold change in SO+D− and SO+D+ compared to SO−D− group. D) Quantification of sTREM-1 and sTREM-2 (in pg/ml) using ELISA in the study populations. Da) sTREM-1; Db) sTREM-2; Dc) serum TREM-1/TREM-2 ratio. E) Protein Expression study in Liver biopsy sample (Ea) TREM-1; (Eb) TREM-2; (Ec) TREM-1/TREM-2 ratio. F) Protein Expression study in Omentum biopsy sample (Fa) TREM-1; (Fb) TREM-2; (Fc) TREM-1/TREM-2 ratio. G) Protein Expression study in Subcutaneous biopsy sample (Ga) TREM-1; (Gb) TREM-2; (Gc) TREM-1/TREM-2 ratio. The protein samples were isolated from the biopsy tissues and quantified using Bicinchoninic acid assay. The protein samples (50μg/well) were electrophoresed by using SDS–PAGE and transferred to nitrocellulose membrane Immunoblot analysis was carried out using rabbit anti-human TREM-1 or TREM-2 and mouse anti-GAPDH as primary antibody and HRP conjugated goat anti-rabbit IgG and HRP conjugated rabbit anti-mouse IgG as secondary antibodies, respectively. The protein expression was detected by incubating the membrane to chemiluminescence solution. The exposure time was adjusted to keep the integrated optical densities within a linear and non-saturated range. The band intensity was measured by densitometric analysis using Image J software. GAPDH was used to normalize the target and the relative expressions were stated as fold ratio. Data were shown as mean ± SD (N= 5 SO− D−; 7 SO+D−; 15 SO+D+); *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: SO−D− = subjects without obesity and diabetes; SO+D− = subjects with obesity but not diabetes; SO+D+ = subjects with obesity and diabetes.