Effect of type 2 diabetes on the inducible degrader of LDL receptor

Figures

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  Fig. 1. Representative figures of all flow cytometry measurements (A) and Western blots of IDOL (B) in type 2 diabetes mellitus and control.

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  Fig. 2. Pearson's correlation analysis of the association between HbA1c and IDOL expression in CD14+ monocytes in type 2 diabetes mellitus (A) and control (B).

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  Fig. 3. Pearson's correlation analysis of the association between FGF21 and IDOL expression in CD14+ monocytes in type 2 diabetes mellitus (A) and control (B).

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  Fig. 4. Effect of glucose (A) and FGF21 (B) on IDOL mRNA and protein expression in human monocyte-derived macrophages. Peripheral blood mononuclear cells were allowed to differentiate into monocyte-derived macrophages using RPMI 1640 medium with 10% autologous serum and 5 mM glucose. Cells were then treated for 24 h with different concentrations of added glucose and FGF21, respectively, and IDOL was determined. Data represent the mean ± SD of four experiments. ∗P < 0.05; ∗∗P < 0.01 for treated versus control cells. The statistical significance of differences was determined using ANOVA followed by Bonferroni’s comparison.

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  Fig. 5. Human monocyte-derived macrophages from four healthy subjects were transfected with Si-SR309214A or Si-scramble control SiRNA, and quantitative mRNA levels (A) and Western blot (B) confirmed the efficiency of IDOL knockdown 72 h after transfection. Macrophages incubated with 10 μg/ml LDL for 6 h and stained with Oil Red O to visualize lipid accumulation showed an increase in intracellular lipid accumulation in IDOL knockdown macrophages (C) and an increase in cellular cholesterol levels (D).

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  Fig. 6. Cytokine production of TNFα (A), IL-6 (B), IL-1β (C), and activation of NF-kB p65 (D) in IDOL knockdown and control macrophages. Data represent the mean ± SD of four experiments. ∗P < 0.05, ∗∗P < 0.01 versus Si-scramble control macrophages. The statistical significance of differences was determined using Student’s t-test.

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  Supplemental Figure S1. The specificity of anti-ABCG1 antibody was confirmed in ABCG1 knockdown human cells. Human monocyte-derived macrophages from four healthy subjects were transfected with Silencer Select (Si-S18484 or Si-scramble control SiRNA primers, Thermo Fisher Scientific, MA) to knockdown ABCG1 expression. Reduced ABCG1 protein expression was demonstrated by Western blot (A) and by flow cytometry (B).

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  Supplemental Figure S2. Gating strategy for identification of double positive CD14+/IDOL monocytes using fluorescence minus one (FMO) as gating control. Monocytes were first prestained with CD14+ fluorescein isothiocyanate (FITC) and separated from neutrophil, granulocytes and lymphocytes based on size (forward-scattered light, FSC) (A, B), and granularity/internal complexity (side-scattered light, SSC). An acquisition threshold was set to eliminate platelets, dead cells, and debris. Doublets were further excluded based on FSC area/FSC height (FSC-A/FSC-H) and SSC area/SSC height (SSC-A/SSC-H) (C, D). An allophycocyanin (APC) FMO control was used as gating control (E). Mouse IgG2a Kappa APC-conjugated antibody was used as isotype control to help to differentiate non-specific background signal from positive IDOL-APC specific antibody signal (F). Double positive staining of CD14+/IDOL expression is shown in figure (G). An isotype control was run for each datapoint and the value was subtracted from the measurement.

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  Supplemental Figure S3. To control for osmotic effect on IDOL expression, human monocyte-derived macrophages were treated for 24 h with or without 25 mM mannitol added to the culture medium. Data represent the mean ± SD of four experiments.

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  Supplemental Figure S4. Pearson's correlation analysis of the association between intracellular lipid accumulation and IDOL expression (A) and ABCG1 expression (B) inCD14+ monocytesin type 2 DM.