Figure 1.

ADORA2A siRNAs decrease ADORA2A mRNA and A_2AR protein expression

(A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3⁺ or CD8⁺ T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A_2AR protein expression in activated HD CD3⁺ or CD8⁺ T cells 24 h (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples), 48 h (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples), or 72 h (n = 4; 2 CD3⁺ T cell samples, 2 CD8⁺ T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A_2AR expression showing the highest degree of A_2AR protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A_2AR expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A_2AR expression. The unstained sample is indicated in orange in the histogram while the A_2AR expression of cells treated with scr-RNAs is indicated in blue and the A_2AR expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A_2AR antibody specificity where CD3⁺ T cells were stained with A_2AR antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A_2AR antibody + peptide is indicated in red and A_2AR antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.