Journal: Gene Expression

Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

doi: 10.3727/105221617X15042723767876

Figure Lengend Snippet: Adenosine induced interleukin-6 (IL-6) expression and secretion via the A2b adenosine receptor (A2bAR) in H69 cholangiocyte cells. (A) Reverse transcription polymerase chain reaction (RT-PCR) experiments were performed on complementary DNA (cDNA) extracted from H69 cholangiocyte cells using specific primers for each adenosine receptor isoforms (as listed in Table 1). Agarose gel (2%) electrophoresis with ethidium bromide staining was used to visualize the PCR products. Human liver cDNA was used as a positive control. The expected molecular weight PCR products were observed in the positive control. (B) Immunofluorescence staining of H69 cells with human A2bAR-specific antibody (anti-A2bAR) or anti-rabbit secondary antibody as negative control (anti-rabbit) confirmed that H69 cells express A2bAR at the protein level (green). Nuclei were stained with DAPI (blue). (C, D) H69 cells (150,000 cells per well in six-well plates) were stimulated with 100 μM adenosine for 2 h in complete media (C) in the presence of A2bAR antagonist MRS-1754 (1 μM; n = 6) or (D) 72 h after transfection with A2bAR-specific siRNA (n = 5). RNA was extracted, and IL-6 expression was assessed by qPCR using a specific probe for human IL-6 gene. β-2-microglobulin was used as a reference gene. #Significant when compared to the control without agonist; *significant when compared to vehicle (C) or nontarget siRNA (D). (E) H69 cells (30,000 cells per well in 48-well plates) were stimulated with 100 μM ADO for 4 h in 48-well plates in complete media 72 h after transfection with an A2bAR-specific siRNA. Supernatants were collected, centrifuged, and analyzed for IL-6 contents by ELISA. #Significant compared to the media alone; *significant compared to nontarget siRNA (n = 4, performed in triplicate). (F) H69 cells were transfected with nontarget siRNA or A2bAR-specific siRNA with Lipofectamine 2000 for 72 h. RNA was extracted, and A2bAR expression was analyzed by qPCR using a human ADORA2b-specific probe. β-2-microglobulin was used as the gene of reference. *Significant when compared to nontarget siRNA (n = 8).

Article Snippet: Following fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked in a solution of 7% (v/v) goat serum and 0.5% (w/v) bovine serum albumin (BSA) in 1× phosphate-buffered saline (PBS) buffer and incubated overnight with a polyclonal rabbit anti-human A2bAR antibody (dilution 1:100 in blocking solution; Alomone Laboratories) followed by a 1-h incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:1,000; Life Technologies).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Staining, Positive Control, Molecular Weight, Immunofluorescence, Negative Control, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Gene Expression

Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

doi: 10.3727/105221617X15042723767876

Figure Lengend Snippet: Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA (n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation (n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA (n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA (n = 3, performed in triplicate).

Article Snippet: Following fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked in a solution of 7% (v/v) goat serum and 0.5% (w/v) bovine serum albumin (BSA) in 1× phosphate-buffered saline (PBS) buffer and incubated overnight with a polyclonal rabbit anti-human A2bAR antibody (dilution 1:100 in blocking solution; Alomone Laboratories) followed by a 1-h incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:1,000; Life Technologies).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy, Positive Control, Fluorescence, Microscopy, Expressing

Journal: Gene Expression

Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

doi: 10.3727/105221617X15042723767876

Figure Lengend Snippet: Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR−/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only (n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR−/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 (n = 9 and 7, respectively) or A2bAR−/− mice (n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H&E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH-specific probe was used as control. C57BL/6 and A2bAR−/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

Article Snippet: Following fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked in a solution of 7% (v/v) goat serum and 0.5% (w/v) bovine serum albumin (BSA) in 1× phosphate-buffered saline (PBS) buffer and incubated overnight with a polyclonal rabbit anti-human A2bAR antibody (dilution 1:100 in blocking solution; Alomone Laboratories) followed by a 1-h incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:1,000; Life Technologies).

Techniques: Knock-Out, Ligation, Injection, Recombinant, Staining, Enzyme-linked Immunosorbent Assay

Journal: Gene Expression

Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

doi: 10.3727/105221617X15042723767876

Figure Lengend Snippet: Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19+ duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR−/− sham (7), A2bAR−/− sham + IL-6 (3), A2bAR−/− BDL (13), and A2bAR−/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR−/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67+ pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

Article Snippet: Following fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked in a solution of 7% (v/v) goat serum and 0.5% (w/v) bovine serum albumin (BSA) in 1× phosphate-buffered saline (PBS) buffer and incubated overnight with a polyclonal rabbit anti-human A2bAR antibody (dilution 1:100 in blocking solution; Alomone Laboratories) followed by a 1-h incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:1,000; Life Technologies).

Techniques: Staining, Recombinant, Injection

Journal: Purinergic Signalling

Article Title: Adenosine enhances cisplatin sensitivity in human ovarian cancer cells

doi: 10.1007/s11302-018-9622-7

Figure Lengend Snippet: Expression and functional activity of adenosine receptors in A2780, A2780CisR, and HEY cells. a Gene expression of adenosine receptors using RT-PCR. b Protein expression of adenosine receptors using Western blot. c Concentration-dependent effect of adenosine on cAMP-related luminescence in A2780 cells. d–f Concentration-dependent effect of SLV320, PSB603, and SCH 442416 (selective A1, A2B, and A2A antagonists, respectively) on 200 μM adenosine-induced luminescence in A2780 cells

Article Snippet: Rabbit anti-A1, A2A, A2B, and A3 adenosine receptor antibodies were obtained from Alomone labs (Israel).

Techniques: Expressing, Functional Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy.

doi: 10.3390/cells14120890

Figure Lengend Snippet: Figure 2. Immunohistochemical analysis of glomerular alterations in diabetic rats. (A) The panels show representative immunofluorescent images of the detection of A2BAR, α-SMA, ZO-1, nephrin, and Snail in renal tissue from control rats, diabetic nephropathy (DN) rats, and diabetic rats treated with the A2BAR antagonist MRS1754 (DN + MRS1754). The images emphasize changes in the levels of these proteins within the glomerulus. Nuclear staining with DAPI is in blue. The bar indicates 50 µm. (B) The graphs correspond to the means ± SD of relative fluorescent units (RFUs) of a quantitative analysis of A2BAR, α-SMA, ZO-1, nephrin, and Snail in glomeruli from the control (white bar), diabetic (black bar), and MRS1754-treated diabetic (grey bar) rat groups. The means in the control group were normalized to 1. Images of 12 glomeruli from 2 control, 4 diabetic, and 4 treated diabetic rats were analyzed. *, p < 0.05 vs. control; #, p < 0.05 vs. diabetes.

Article Snippet: The samples were incubated with an antibody against A2BAR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Immunohistochemical staining, Control, Staining

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy.

doi: 10.3390/cells14120890

Figure Lengend Snippet: Figure 3. A2BAR antagonism prevents the loss of podocyte epithelial markers in diabetic glomeru- lopathy. (A) Representative Western blot analysis showing the levels of epithelial ZO-1 and nephrin, as well as mesenchymal α-SMA and Snail, in purified glomeruli from the different rat groups. (B) The graphs represent the means and SD of the levels of epithelial and mesenchymal markers relative to β-actin. The means in the control group were normalized to 1. (C) Representative Western blot analysis of the Snail distribution in cell fractions purified from the kidneys of experimental rat groups. (D) The graph represents the ratio of the Snail subcellular distribution. The mean in the control was normalized to 1. The number of animals analyzed in each experimental group was 5 in (B) and 2 in (D). *, p < 0.05 vs. control; #, p < 0.05 vs. DN; &, p < 0.05 vs. control.

Article Snippet: The samples were incubated with an antibody against A2BAR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Western Blot, Purification, Control