Journal: bioRxiv

Article Title: Primer- and template-independent RNA polymerization by terminal nucleotidyltransferase TENT4B

doi: 10.64898/2026.03.05.709691

Figure Lengend Snippet: A, Extension of a 20-mer RNA substrate oligo (oligo 1) using rTENT4B and ATP analogs demonstrating differential incorporation. (AMP: Adenosine-5’-monophosphate; ADP: Adenosine-5’-diphosphate; ATP: Adenosine-5’-triphosphate. n=3 replicates, one representative shown. B, Utilization of nucleotide diphosphates (NDPs) by rTENT4B. rTENT4B along with a 20-mer RNA-substrate oligo (oligo 2) was incubated in the presence of four canonical NDPs, in RNA-substrate extension assays, and reaction products were resolved using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. C , rTENT4B was incubated with four canonical NDPs plus 5’-Cy5-labeled ATP as substrate. NDP incorporation was confirmed via detecting generation of Cy5-labeled RNA chains after 30 min in no RNA-substrate added reactions. D , De novo ADP polymerization using rTENT4B. rTENT4B along with increasing concentrations of ADP was subjected to RNA-substrate extension assay in the absence of an RNA-substrate, and generation of de novo synthesized RNA products were detected using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. E , Consumption of ADP by rTENT4B via analysis of Pi yield, in the presence/ absence of inorganic pyrophosphatase (PPA) as a measure of ATP contamination (ADP + Pi), in the presence/absence of an RNA-substrate oligo, compared to that of rTENT4B alone versus rTENT4B plus ATP. n=4 biological replicates. Mean + S.D. shown, 2-way ANOVA, ns- not significant, ****p<0.001.

Article Snippet: ATP- Adenosine-5’-triphosphate (N0450, New England Biolabs) ADP- Adenosine-5’-diphosphate (NU-1198, Jena Bioscience) AMP- Adenosine-5’-monophosphate (A1752, Sigma) GTP- Guanosine-5’-triphosphate (N0450, New England Biolabs) GDP- Guanosine-5’-diphosphate (G7127, Sigma) CTP- Cytidine-5’-triphosphate (N0450, New England Biolabs) CDP- Cytidine-5’-diphosphate (C9755, Sigma) UTP- Uridine-5’-triphosphate (N0450, New England Biolabs) UDP- Uridine-5’-diphosphate (94330, Sigma) Cy5-ATP- γ-(6-Aminohexyl)-ATP-Cy5- (NU-833-CY5, Jena Bioscience) Cy5-GTP- γ-(6-Aminohexyl)-GTP-Cy5- (NU-834-CY5, Jena Bioscience)

Techniques: Incubation, Acrylamide Gel Assay, Staining, Labeling, Synthesized

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

doi: 10.4049/jimmunol.0803383

Figure Lengend Snippet: FIGURE 4. The enhanced inhibitory effect of NECA is mediated by the enhanced contribution of A2AR-mediated signaling and the decreased contribution of A3R-mediated signaling. Unstimulated and activated microglia were exposed to 100 ng LPS/ml in the presence or absence of 10 M NECA in combination with A1, A2A, A2B, and A3 receptor antagonists. After 16 h, TNF- (A) and IL-12p40/p70 (B) levels were measured and expressed as relative values of exposure to 100 ng LPS/ml alone. DPCPX was used as an A1R, SCH58261 as an A2AR, alloxazine as an A2BR and VUF5574 as an A3R antagonist. All antagonists were used at 10 M concentrations. Solid lines in the graph indicate relative cytokine production levels of exposure to 100 ng LPS/ml 10 M NECA without antagonists of unstimulated microglia, the dashed lines indicate activated microglia. , p 0.05; Student’s t test. TNF- (C) and IL-12p40/p70 (D) production after exposure to 100 ng LPS/ml in the presence or absence of 10 M CGS21680, an A2AR-selective agonist, and/or of 10 M VUF5574, an A3R antagonist. Cytokine production is expressed relative to production after exposure to 100 ng LPS/ml alone for unstimulated and activated microglia. , p 0.05; Student’s t test. TNF- (E) and IL-12p40/p70 (F) production after exposure to 100 ng LPS/ml different concentrations of CGS21680, in the presence or absence of A3R antagonist, is expressed relative to production after exposure to 100 ng LPS/ml alone for unstimulated and activated microglia. All data are representative for three independent experiments using different donors.

Article Snippet: Cells were washed and incubated for 30 min with anti-adenosine A3 receptor Abs (SP055P, Acris Antibodies) or total rabbit IgG (AbD Serotec) as an isotype control.

Techniques:

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Stimulation of the A 2B Adenosine Receptor Subtype Enhances Connexin26 Hemichannel Activity in Small Airway Epithelial Cells.

doi: 10.33594/000000160

Figure Lengend Snippet: Fig. 4. Expression of adenosine receptor subtypes in Calu-3 cells. (A) Increase in the dye uptake rate induced by NECA (10 µM, 24 h) as quantified from dye uptake experiments. (B) RT-PCR analysis and western blot experiments showed expression of the adenosine receptor subtypes A2A and A2B. qRT-PCR analysis showed a remarkably higher expression of the A2B adenosine receptor subtype. The results are given as average ± SEM from three cell culture passages. (C) NECA increased the intracellular cAMP concentration. The results are given as average ± SEM from three cell culture passages. (D) The increase in the dye uptake rate was attenuated by the AC inhibitor SQ22536 (SQ, 400 µM) and the PKA inhibitor Rp-cAMPs (Rp-c, 200 µM). (E) The NECA-induced increased dye uptake rate was not significantly affected by the A2A adenosine receptor antagonist SCH58261 (SCH, 0.5 μM). The A2B adenosine receptor antagonist MRS1754 (MRS, 0.5 μM) blocked the NECA-induced increase in the dye uptake rate. The A2A adenosine receptor-specific agonist CGS21680 (CGS, 50 nM) did not change the dye uptake rate, while the A2B adenosine receptor-specific agonist BAY60- 6583 (BAY, 100 nM) significantly increased the dye uptake rate. (F) siRNA-knockdown of the A2B adenosine receptor subtype blocked the NECA-induced increase in the dye uptake rate. The dye uptake rates are given as average ± SEM from respectively six experiments with three cell culture passages. The data were statistically compared to the control using student’s t test (P<0.05 *, P<0.01 **, P<0.001 ***).

Article Snippet: Anti-β-tubulin antibody as loading control (1:4,000, Sigma-Aldrich, T4026), anti-Cx26 antibody (0.5 μg/ml, Merck, MABT198), anti-Cx43 antibody (0.19 μg/ml, Sigma-Aldrich, C6219), anti-Cx45 antibody (1 μg/ml, Thermo Fisher Scientific, 41-5800), anti-claudin1 antibody (0.54 μg/ml, Thermo Fisher Scientific, 51-9,000), anti-claudin3 antibody (0.5 μg/ml, Thermo Fisher Scientific, 34-1700), anti-claudin4 antibody (1 μg/ml, Thermo Fisher Scientific, 32-9400), anti-claudin7 antibody (1.3 μg/ml, Sigma-Aldrich, SAB4500436), anti-A2A adenosine receptor antibody (4 μg/ml, alomone labs, AAR-007), and anti-A2B adenosine receptor antibody (3.75 μg/ml, alomone labs, AAR-003) were diluted in PBS-T and applied to the membranes at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Cell Culture, Concentration Assay, Knockdown, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by 1-way (A, B) or 2-way ANOVA (D, E) . Data are represented as the mean ± SD from a representative experiment of n = 3 experiments (A, B, D, E) .

Article Snippet: After blocking with 5% milk for 1 h, the membranes were incubated with anti-adenosine A2b receptor (AAR-003, alomone labs) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies at 4°C overnight.

Techniques: Blocking Assay, Co-Culture Assay, Lysis, Expressing, Knock-Out, Western Blot, Cell Culture, Control