Fig 1: Human plasma exosomes from T2D subjects induce EMT genes in prostate cancer cell lines. The DU145 human prostate cancer cell line was treated with either ND or T2D plasma exosomes (109) for 2 days. (A) Plasma exosomes from four, independent T2D subjects (?) increased SNAI1 mRNA expression after normalizing to ACTB of the respective sample and compared to the control cells that were treated with growth media exosomes. (?). Exosomes from four, independent ND subjects (?) did not increase SNAI1 mRNA expression relative to ACTB control. TGF-ß (TGFB, 5 ng/mL) was used as a positive control (?) for induction of pro-EMT gene transcription or repression of pro-MET gene transcription. (B) Plasma exosomes from T2D subjects did not induce CDH1 expression. ND plasma exosomes increased CDH1 mRNA expression. Data in A and B were obtained from four biological replicates of ND and T2D, and each biological replicate was conducted in three technical replicates, that are averaged in the graph. Data were analyzed by two-way ANOVA with statistical significance presented as: *, P = 0.0244; and ****, P < 0.0001; ns, not significant. (C) Expression of selected EMT genes were analyzed by commercial PCR array from Qiagen. Relative expression of significantly differentially expressed genes in three independent, T2D exosome-treated samples was compared to three independent, ND exosome-treated samples. Equal numbers of exosomes (109) from each sample were used. The heatmap of the PCR array result was calculated by hierarchical clustering. Scale bar (right) shows fold change, with red color to identify upregulation and blue color to identify downregulation. (ND, non-diabetic; T2D, Type 2 diabetic; TGFB, TGF-ß positive control). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: T2D plasma exosomes have distinct miRNA profile compared to ND plasma exosomes. (A) Heatmap of differentially expressed miRNAs in plasma exosomes of two T2D subjects compared to plasma exosomes of ND subjects. Total RNA was isolated from plasma exosomes of T2D and ND subjects, and miRNAs were profiled by a commercial PCR array. DU145 cells were transfected with individual miRNAs from (A) (25 nM), selected based on their functional significance for EMT and immune exhaustion. The mRNA expression of SNAI1 (B), CD274 (C) and CDH1 (D) was tested and expression relative to ß-actin (ACTB) is shown. Scale bar (A, right) shows fold change, with red color to identify upregulation and blue color to identify downregulation. Data were analyzed by one-way ANOVA with statistical significance presented as: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (Control, media-only exosomes; TGFB, TGF-ß positive control at 5 ng/mL for SNAI1 induction; IFNgamma, interferon-? positive control at 5 ng/mL for CD274 induction). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: T2D plasma exosomes require BRD4 to upregulate EMT genes and PD-L1 expression. (A) Expression of SNAI1 and CD274 genes in DU145 cells measured by RT-PCR of cellular RNA after exosome treatment. T2D exosomes (109; T2D Exo) were compared to media-only control exosomes (Control), or T2D Exo + JQ1 or MZ1. (JQ1 is a pan-BET inhibitor (400 nM) and MZ-1 is a BRD4-selective degrader (50 nM)). (B) Expression of PD-L1 was measured by flow cytometry after treatments. One million events were analyzed by Flow-Jo and presented as overlaid histograms (control, red trace; experimental, black trace). (C) Flow cytometry data of B were quantified with PD-L1 as percent of parent population. The experiment was conducted in triplicate with differences between means represented as bar graphs. Data were analyzed by two-way ANOVA with statistical significance presented as: ****, P < 0.0001 (TGFB, TGF-ß positive control at 5 ng/mL for SNAI1 induction; IFNgamma, interferon-? positive control at 5 ng/mL for CD274 induction). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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