Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: GnT-V expression is associated with oxaliplatin chemosensitivity in CRC cells. (A) The mRNA expression of MGAT5 was examined by reverse transcription-quantitative PCR. (B) Protein levels of GnT-V in CRC cell lines were measured by western blotting. (C) Cells were treated with the indicated concentrations of oxaliplatin for 48 h, and oxaliplatin sensitivity was determined based on cell death using the trypan blue method. * P<0.05 vs. HT29 group and # P<0.05 vs. HCT116 group. (D) Acute treatment with 1.5 µM oxaliplatin for 24 or 48 h did not lead to changes in GnT-V expression levels in CW-2 and HT29 cells. (E) CW-2 and HT29 cell lines were exposed to long-term oxaliplatin treatment to obtain stably resistant lines, named CW-2/R and HT29/R, respectively. Endogenous MGAT5 expression was increased in the stably resistant cell lines as compared with the parental cells at the mRNA level. (F) GnT-V expression and β-1,6-oligosaccharide branches were detected by western blot and lectin blot analyses, respectively, in wild-type and oxaliplatin-resistant cell lines. The graphs depict results from three independent experiments each performed in triplicate. Results are presented as the mean ± SEM. * P<0.05, # P<0.05 and ** P<0.01. GnT-V, N-acetylglucosaminyltransferase V; CRC, colorectal cancer; MGAT5, the gene encoding GnT-V; OXA, oxaliplatin; UT, untreated; PHA-L, phytohemagglutin-L.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection

Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: Cells with GnT-V knockdown exhibit enhanced survival and cell viability upon exposure to oxaliplatin. (A) and (B) shRNA mediated GnT-V knockdown and β-1,6-oligosaccharide reduction in (A) CW-2 and (B) CW-2/R cells as depicted by western blotting and lectin blotting, respectively, compared with the respective parental cell lines and NC cells. (C) Cells were exposed to indicated concentrations of oxaliplatin (0.25-16 µg/ml) for 48 h, and cell viabilities were determined by Cell Counting Kit-8 assay. Representative images of (D) wild-type and (E) drug-resistant cells showing that oxaliplatin-treated GnT-V knockdown cells had reduced chemosensitivity, resulting in an increased colony-forming potential compared with NC cells. Results are presented as the means ± SEM from three independent experiments. * P<0.05. GnT-V, N-acetylglucosaminyltransferase V; shRNA, short hairpin RNA; shRNA#1 and #2, shRNAs for knockdown of GnT-V; NC, negative control; OXA, oxaliplatin; PHA-L, phytohemagglutin-L; UT, untreated.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: shRNA, Western Blot, Cell Counting, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: OCT2 acts as a substrate of GnT-V and affects the cytotoxic response to oxaliplatin in CRC cells. (A and B) GnT-V knockdown did not lead to marked changes in OCT2 expression in CW-2 and CW-2/R cells as compared with the respective wild-type and NC cells. (C) Reduced cytotoxic responses to oxaliplatin were observed after treatment with 100 µM cimetidine. (D) Lectin precipitation was performed with PHA-L-bound agarose, followed by western blotting with an anti-OCT2 antibody. Data were obtained from triplicate experiments and are presented as the mean ± SEM. * P<0.05. OCT2, organic cation transporter member 2; GnT-V, N-acetylglucosaminyltransferase V; NC, negative control; shRNA#2, short hairpin RNA for knockdown of GnT-V; OXA, oxaliplatin; IP, lectin precipitate; IB, immunoblot; PHA-L, phytohemagglutin-L.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Negative Control, shRNA

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 1 VILI in mice gene-targeted for individual ARs. Previously characterized A1AR–/– (A; ref. 29), A2AAR–/– (B; ref. 30), A2BAR–/– (C), or A3AR–/– mice (D; ref. 22) or corresponding littermate controls were exposed to VILI, and survival times were determined during VILI. Mechanical ventilation was applied using pressure-con- trolled settings (inspiratory pressure of 35 mbar, inspired oxygen concentration 100%; respiratory rate and inspiratory/expiratory ratio were adjusted to maintain normal pH) until a cardiac standstill was observed in the surface electrocardio- gram. Note the significantly attenuated survival of A2BAR–/– mice (C; P < 0.001, n = 8). Albumin concentration in the BAL fluid was determined by ELISA. For this purpose, the mice were mechanically ventilated using pressure-controlled venti- lation with an inspired oxygen concentration of 100% for 180 minutes at 45 mbar. Note the significantly increased albumin concentration in the BAL fluid of A2BAR–/– mice (C; P < 0.001, n = 6).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 2 VILI in mice gene-targeted for the A2BAR. (A–H) A2BAR–/– mice or littermate controls (A2BAR+/+) were mechanically ventilated using pressure- controlled ventilation with an inspired oxygen concentration of 100% over 180 minutes at 45 mbar. (A) Following ventilation, lungs were excised en bloc and weighed. Lungs were lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Results are presented as mean ± SD (n = 6). (B) To assess pulmonary gas exchange, blood gas analyses were performed by obtaining arterial blood via cardiac puncture. Analysis was performed immediately, and the ratio of the arterial partial pressure of oxygen (PaO2) to the fraction of inspired oxygen (FiO2) was determined. Results are presented as mean ± SD (n = 6). (C) Pulmonary neutrophil accumulation was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD (ΔOD) over 5 minutes. Results are presented as mean ± SD (n = 6). (D–I) TNF-α, IL-6, KC, IL-10, NF-κB, and IκBα levels were evaluated in lung tissue homogenates using a mouse ELISA. Results are presented as mean ± SD (n = 6). (J) For quantification of histological tissue damage by VILI following 180 min ventilation, VILI scores were assessed in A2BAR–/– or corresponding littermate control mice. Results are displayed as median (midline within boxes) and range (bars above and below boxes) (n = 4). (K) One of 4 representative photomicrographs (original magnification, ×200) stained with hematoxylin and eosin is displayed.

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, MPO Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Staining

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 3 Transcriptional consequences of mechanical ventilation on AR expression. (A) C57BL/6 mice were mechanically ventilated (inspiratory pressure of 35 mbar, 100% oxygen). After the indicated time periods, lungs were harvested, total RNA was isolated, and A1AR, A2AAR, A2BAR, and A3AR mRNA levels were determined by real-time RT-PCR. Data were calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 min ventilation) ± SD at each indicated time (n = 4). Note selective induction of the A2BAR gene dur- ing high-pressure ventilation (10-fold, P < 0.01; n = 4). (B) Comparative gene expression of pulmonary ARs using real- time PCR. Relative expression levels in untreated controls or in mice after 180 min mechanical ventilation (inspiratory pressure of 35 mbar, 100% oxygen) are shown. Values are expressed as mean ± SEM (n = 4). *P < 0.05 compared with A2AAR. (C) Mice were mechanically ventilated (35 mbar inspiratory pressure, 100% oxygen), and lungs were har- vested at the indicated time points, shock frozen, and lysed, and proteins were resolved by SDS-PAGE. Resultant west- ern blots were probed with anti-A2BAR antibodies. To con- trol for loading conditions, blots were stripped and reprobed for actin expression. One representative experiment of 3 is shown. (D) To examine the influence of mechanical venti- lation on pulmonary A2BAR expression patterns, C57BL/6 mice were ventilated in a pressure-controlled setting over 0 h or 3 h (35 mbar inspiratory pressure, 100% inspired oxy- gen concentration). Lungs were stained with antibodies for A2BAR. IgG controls were used at identical concentrations and staining conditions as the target primary antibodies (original magnification, ×400; n = 4).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Gene Expression, Real-time Polymerase Chain Reaction, SDS Page, Concentration Assay, Staining

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 4 cAMP levels and PKA activity during VILI. (A and B) To assess cAMP levels and PKA activ- ity in pulmonary tissues during VILI, A2BAR–/– mice and littermate controls were mechani- cally ventilated using pressure-controlled ventilation with an inspired oxygen concentration of 100% over 180 minutes at 45 mbar. Animals were euthanized, and lungs were perfused with 5 ml of PBS through the right ventricle. Lungs were excised, shock-frozen utilizing liquid nitrogen, and mechanically homogenized. cAMP levels (A) and PKA activity (B) were determined by ELISA. Note that the increases in cAMP and PKA activity associated with mechanical ventilation were abolished in A2BAR–/– mice. (C) Treatment with the specific β2-adrenergic agonist zinterol during VILI. Control C57BL/6 mice were treated with 300 μl 10–7 M intratracheal zinterol, and baseline cAMP levels were determined in lung homogenates using a competitive immunoassay kit. (D) Control C57BL/6 mice were treated with 300 μl 10–7 M intratracheal zinterol or vehicle control, followed by mechanical ventila- tion using pressure-controlled settings at an inspired oxygen concentration of 100% and 45 mbar inspiratory pressure for 0 or 180 min. Albumin concentration in the BAL fluid was determined by murine ELISA. (E) Survival times during VILI after 300 μl 10–7 M intratracheal zinterol or vehicle treatment. Mechanical ventilation was applied using pressure-controlled settings (inspiratory pressure of 35 mbar, inspired oxygen concentration 100%, respiratory rate and inspiratory/expiratory ratio were adjusted to maintain normal pH) until a cardiac standstill was observed in the surface electrocardiogram.

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 5 A2BAR antagonist treatment PSB1115 during VILI. (A–C), A2BAR+/+, A2AAR–/–, and A2BAR–/– mice and their corresponding littermate controls were treated with 10 mg/kg PSB1115 or vehicle 30 minutes prior to induction of anesthesia. Mechanical ventilation was begun, and mice were ventilated using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration) until a cardiac standstill was observed in the surface electrocardiogram (P < 0.01, n = 8). (D) Mechanical ventilation was begun, and mice were ventilated for 0 or 180 minutes using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration). Albumin concentration in the BAL fluid was determined by ELISA (n = 6). (E) Pulmonary neutrophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as differ- ence in OD over 5 minutes (n = 6). (F–H) TNF-α, NF-κB, and IL-10 levels were evaluated in lung tissue homogenates using a murine ELISA (n = 6). (I) To assess pulmonary gas exchange, blood gas analyses were performed by obtaining arterial blood via cardiac puncture. Analysis was performed immediately, and the ratio of the arterial partial pressure of oxygen to the fraction of inspired oxygen was determined. Results are presented as mean ± SD (n = 6).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MPO Assay, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 6 A2BAR agonist (BAY 60- 6583) treatment. (A–C) A2BAR+/+, A2AAR–/–, and A2BAR–/– mice and their cor- responding littermate controls were treated with 2 mg/kg BAY 60-6583 or vehicle 30 minutes prior to induction of anesthe- sia. Mechanical ventilation was begun, and mice were ventilated using pressure- controlled settings (inspirato- ry pressure of 45 mbar, 100% inspired oxygen concentra- tion) until a cardiac standstill was observed in the surface electrocardiogram (P < 0.01, n = 8). In other studies, albu- min concentrations in the BAL fluid were determined by ELISA after mechanical ventilation using pressure- controlled settings with an inspired oxygen concentration of 100% for 180 minutes at 45 mbar. (D) Pulmonary neutrophil sequestration was quantified using a MPO assay (n = 6). (E–G) TNF-α, NF-κB, and IL-10 levels were evaluated in lung tissue homogenates using murine ELISA (n = 6). (H) To assess pulmonary gas exchange, blood gas analyses were per- formed by obtaining arterial blood via cardiac puncture. The ratio of the arterial partial pressure of oxygen to the frac- tion of inspired oxygen was determined. Results are pre- sented as mean ± SD (n = 6).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, MPO Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 7 A2BAR signaling during LPS-induced lung injury. (A and B) A2BAR–/– and A2BAR+/+ mice were exposed to LPS inhalation for 30 minutes. Twenty-four hours after LPS exposure, TNF-α in lung tis- sue homogenates and albumin concentration in the BAL fluid were determined using a murine ELISA (n = 6). (C and D) A2BAR+/+ mice received 2 mg/kg BAY 60-6583 i.p. or were treated with vehicle 30 minutes prior to LPS inhalation. (C) TNF-α levels in lung tissues and (D) albumin concentrations in the BAL fluid (n = 6).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 8 VILI in A2BAR bone marrow–chimeric mice. A2BAR bone marrow–chimeric mice were subjected to VILI using mechanical ventila- tion for 180 minutes at an inspiratory pres- sure of 45 mbar and 100% inspired oxygen concentration. (A) Albumin concentration in the BAL fluid was determined by ELISA. WT/WT, A2BAR+/+/A2BAR+/+; KO/WT, A2BAR–/–/A2BAR+/+; KO/KO, A2BAR–/–/ A2BAR–/–; WT/KO, A2BAR+/+/A2BAR–/–. (B) Following ventilation at the indicated settings, lungs were excised en bloc and weighed. Lungs were lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Note the increased albu- min concentration and lung water content in A2BAR–/–/A2BAR–/– and A2BAR+/+/A2BAR–/– mice compared with A2BAR+/+/A2BAR+/+ mice (P < 0.001). (C) Pulmonary neutrophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisi- dine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD over 5 minutes. (D–F) TNF-α, IL-6, and KC levels were evaluated in lung tissue homogenates using a mouse ELISA. Note the similar degree of pulmonary inflamma- tion in A2BAR–/–/A2BAR+/+ and A2BAR+/+/ A2BAR–/– mice compared with A2BAR+/+/ A2BAR+/+ mice. n = 6.

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MPO Assay, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 9 BAY 60-6583–dependent lung protection during VILI in A2BAR bone marrow–chimeric mice. (A–D) A2BAR bone marrow–chimeric mice were treated with BAY 60-6583 (2 mg/kg) 30 minutes prior to induction of anesthesia. Mechanical ventilation was begun, and mice were ven- tilated using an inspiratory pressure of 45 mbar, 100% inspired oxygen concentration, for 180 minutes. Results are presented as mean ± SD. n = 6. (A) Albumin concentration in the BAL fluid was determined by ELISA. (B) Following mechanical ventilation, lungs were excised en bloc and weighed. Lungs were then lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Capillary- alveolar barrier protection was observed only in A2BAR+/+/A2BAR+/+ and A2BAR–/–/A2BAR+/+ mice (P < 0.001; n = 6). (C) Pulmonary neu- trophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD over 5 minutes. (D) TNF-α levels were evaluated in lung tissue homogenates using a mouse ELISA. Note the similar lev- els of pulmonary inflammation in A2BAR–/–/A2BAR+/+ and A2BAR+/+/ A2BAR–/– mice.

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MPO Assay, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 10 Contribution of A2BAR signaling to AFC during VILI. (A) To determine whether A2BAR signaling affects pulmonary fluid transport, we measured AFC using a mechanically ventilated live mouse model. A2BAR–/– mice and littermate controls were mechanically ventilated in a pressure-controlled setting at 45 mbar for 0 to 180 minutes. AFC was measured by instilling 300 μl of iso-osmolar 0.9% NaCl solution with 5% BSA. Mechanical ventilation was continued for 30 minutes, and AFC was measured in the presence or absence of the ENaC inhibitor amiloride (1 mM). *P < 0.01 compared with no amiloride. n = 8. (B) Influence of A2BAR antagonist PSB1115 on AFC during VILI. Follow- ing induction of VILI, control mice received PSB1115 (1 μM) alone or in combination with amiloride (1 mM), and AFC was determined. *P < 0.01 compared with no amiloride. n = 8. (C) VILI was induced in A2BAR bone marrow–chimeric mice, and AFC was determined. *P < 0.001 compared with A2BAR+/+/A2BAR+/+. n = 8. (D) Influence of A2BAR agonist BAY 60-6583 on AFC during VILI. Following induction of VILI, control mice received BAY 60-6583 (1 μM) alone or in combination with amiloride (1 mM), and AFC was determined *P < 0.01 compared with no amiloride. n = 8. (E and F) Control mice were treated with intratracheal BAY 60-6583 (1 μM, 100 μl) and/or amiloride (1 mM) or vehicle following tracheotomy and initiation of mechanical ventilation. Mice were ventilated using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration) until a cardiac standstill was observed in the surface electrocardiogram. Note that the longer survival time during VILI with A2BAR agonist treatment was abolished following amiloride treatment (n = 8).

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Control, Concentration Assay

Journal: Journal of Clinical Investigation

Article Title: A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

doi: 10.1172/jci34203

Figure Lengend Snippet: Figure 11 Influence of β2-adrenergic and/or A2BAR signaling on AFC during VILI. (A) Epinephrine plasma levels in A2BAR–/– and A2BAR+/+ mice that were mechanically ventilated in a pressure-controlled setting at 45 mbar over 180 minutes. (B) Basal cAMP levels in lung tissue from A2BAR+/+ mice that were treated with zinterol and/or BAY 60-6583. (C and D) To determine β2-adrenergic and A2BAR signaling effects on pulmonary fluid trans- port, A2BAR–/– and A2BAR+/+ mice were mechanically ventilated in a pressure-controlled setting at 45 mbar for 0 to 180 minutes. AFC was measured by instilling 300 μl of iso-osmolar 0.9% NaCl solution with 5% BSA. Mechanical ventilation was continued for 30 minutes, and AFC was measured in the presence or absence of the nonselective β-adrenergic receptor antagonist pro- pranolol (intratracheal instillation of 10–4 M propranolol combined with 3 mg/kg i.p.) with or without BAY 60-6583 (10–3 M to the instilled fluid) or in the presence or absence of the β-adrenergic agonist zinterol (intratra- cheal, 10–7 M). *P < 0.01 compared with no propranolol (C) or zinterol (D), by ANOVA with Bonferroni post-hoc test. n = 8. In subsets of experiments, either proprano- lol or zinterol were added together with BAY 60-6583 10–3 M to the instilled fluid. §P < 0.01 compared with propranolol alone (C) or zinterol alone (D), by ANOVA with Bonferroni post-hoc test. n = 8.

Article Snippet: In subsets of experiments, mice were treated with the A2BAR antagonist PSB1115 (10 mg/kg i.p.; Tocris) or the A2BAR agonist BAY 60-6583 (2 mg/kg i.p.; Bayer Healthcare), the A1AR antagonist DPCPX (1 mg/kg i.p.; Tocris), the A3AR antagonist MRS1191 (1 mg/kg i.p.; Sigma-Aldrich), amiloride (10–3 M, 300 μl intratracheally; Sigma-Aldrich), or zinterol (10–7 M, 300 μl intratracheally; Tocris); or vehicle (13).

Techniques: Clinical Proteomics

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: Intestinal ischemia-reperfusion injury induced intestinal barrier disruption and epithelial cell injury. Mice were performed with a temporary occlusion of SMA with an atraumatic vascular for different period and then release the clamp to allow a 120-min reperfusion to build an I/R model. (A) The experimental design of the present work. To evaluate the intestinal injury of different ischemia period, the animals were divided into four groups: the different ischemia period (30,60 and 90 min) group and sham group(n = 6). To determine the protective effect of metformin, another set of animals were administrated with metformin (20 or 40 mg/kg) or saline immediately at the end of a 60-min ischemia period. CY-04 (20 mg/kg), a specific inhibitor of NLRP3 inflammasomes, was used as a positive control for metformin. The OGD/R model was launched in Caco-2 cells. si- Txnip/Gsdmd were applied to revealed the roles of pyroptosis and TXNIP/NLRP3/GSDMD axis in the protective effect of metformin. (B)Survival rates were calculated in different groups (n = 10). (C-D)The integrity of the intestinal barrier was evaluated with the serum I-FABP levels and TER. (E–F) the histopathological damage was estimated with the H&E staining and the Chiu's score classification of small intestine injury was applied to grade the histological score. (G–H) The expressions of the tight junction protein, ZO-1 and occludin, were analyzed by Western blot. Different OGD/R models were induced in Caco-2 cells as mentioned in Methods. (I)The releasing levels of LDH were detected. (J) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM in (C–J) (n = 6) and B(n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques: Disruption, Saline, Positive Control, Staining, Western Blot, CCK-8 Assay

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: Metformin protected against intestinal ischemia-reperfusion injury induced intestinal barrier disruption and cell injury. The animal models were launched with a 60-min ischemia period as mentioned. Different doses of metformin (20 or 40 mg/kg) were used to treat the mice as pervious mentioned. (A) The histological damage was evaluated with H&E staining. (B) The histological scores were calculated with the Chiu's score classification of small intestine injury . (C) The levels of I-FABP in serum were detected. (D) The TER of gut mucosae was measured. (E–F) The expressions of the tight junction protein, ZO-1 and occludin, were analyzed by Western blot. The cells were kept in a low-oxygen/glucose condition for 4 h then induced an OGD/R models as mentioned. Metformin were subjected at different dose (1 or 2 mM) (G) The releasing levels of LDH were detected. (H) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and # p < 0.05 compared with low dose group.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques: Disruption, Staining, Western Blot, CCK-8 Assay

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: Metformin protected against intestinal ischemia-reperfusion injury induced intestinal inflammation and pyroptosis. The I/R models were established as mentioned and the CY-09 was administered at 20 mg/kg as a positive control. (A–C) The inflammatory factors in intestinal tissues, including IL-6, IL-1β and TNF-α, were evaluated. (D–E) Pyroptosis-related proteins in intestinal tissue, including NLRP3, cleaved Caspase-1, IL-1β and IL-18, were examined by Western blot. (F–G) The expression of GSDMD in vivo was detected by Western blot. (H–I) The expression of NLRP3 was also detected in vitro by immunofluorescence. si- Gsdmd was used to inhibit the pyroptosis and revealed the relationship between metformin and pyroptosis (J) the releasing levels of LDH were detected (K) Cell viability was measured with CCK-8 assay. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and # p < 0.05 compared with metformin treatment group. $ p < 0.05 compared with si- Con group and & p < 0.05 compared with si- Con +OGD/R group.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques: Positive Control, Western Blot, Expressing, In Vivo, In Vitro, Immunofluorescence, CCK-8 Assay

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: Metformin protected against intestinal ischemia-reperfusion injury induced oxidative stress and TXNIP-NLRP3 interaction in vitro. Caco-2 cells were induced to an OGD/R model as mentioned. Metformin treated models at different dose (1 or 2 mM). (A–C) Oxidative stress was measured by SOD, GSH and MDA. (D–E) ROS was detected with a DHE staining and the ROS-positive cells were calculated by two random observers. (F–G) the expression of TXNIP was examined by Western blot. (H–I) The expression of TXNIP was also detected in vitro by immunofluorescence. (J) the co-localization of TXNIP and NLRP3 was showed by immunofluorescence. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with no-treatment group and # p < 0.05 compared with low dose group.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques: In Vitro, Staining, Expressing, Western Blot, Immunofluorescence

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: Knockdown of TXNIP abolished metformin-inhibited pyroptosis in vitro. Caco-2 cells were transfected with TXNIP-targeting siRNA or control siRNA. Then cells were induced as OGD/R models and received metformin treatment at different dose (1 or 2 mM). (A–B) The expression of TXNIP and pyroptosis-related proteins were detected by Western blot. (C) The levels of LDH releasing were detected. The values were showed as the mean ± SEM(n = 5–6). *p < 0.05 compared with si- Con group and #p < 0.05 compared with si- Con +OGD/R group.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques: Knockdown, In Vitro, Transfection, Control, Expressing, Western Blot

Journal: Redox Biology

Article Title: Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway

doi: 10.1016/j.redox.2020.101534

Figure Lengend Snippet: The mechanism map of the protective properties of metformin against intestinal ischemia-reperfusion injury via a TXNIP-NLRP3-GSDMD pathway.

Article Snippet: Thus, Metformin (TopScience, USA, T0740) was intraperitoneal injected at the beginning of reperfusion at 40 mg/kg or 20 mg/kg and then applied an optimum I/R injury model as previous mentioned in Metformin treatment group while saline would be intraperitoneal injected in control group.

Techniques:

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: Metformin-mediated activation of SIK1 protects against EV-D68-driven asthma exacerbation in house dust mite (HDM)-sensitized mice. (A) C57BL/6 mice (6–8 weeks) were administered metformin at doses of 100 mg/kg or 250 mg/kg once daily via intraperitoneal injection on day 1 and day 2. On day 3, lung tissues were collected, and the protein level of SIK1 was determined by western blotting analysis. (B) Experimental timeline. C57BL/6 mice (6–8 weeks) were intranasally sensitized with 250 μg kg −1 HDM extract on day 0 and challenged daily with the same dose on days 7–11. On days 12–13, animals received EV-D68 (1 × 10 6 PFU/kg) or DMEM (vehicle) intranasally. Metformin (100 mg/kg, intraperitoneal) was administered once daily on days 12–14. Airway hyper-responsiveness measurements and broncho-alveolar lavage fluid (BALF) collection were performed on day 15; lung tissue was used for quantitative PCR analyses. (C) Airway responsiveness to increasing doses of methacholine. (D) Differential cell counts of BALF by Wright-Giemsa staining. (E – H) The indicated genes were detected by quantitative PCR and normalized to GAPDH expression. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Viral exacerbation was induced on days 12–13 by intranasal instillation of EV-D68 (1 × 10 6 PFU/kg in 20 μL DMEM); animals assigned to the treatment cohort received metformin intraperitoneally at 250 mg kg −1 (MCE, USA) once daily from day 12 through day 14.

Techniques: Activation Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Staining, Expressing, Standard Deviation

Journal: iScience

Article Title: SUFU suppresses ferroptosis sensitivity in breast cancer cells via Hippo/YAP pathway

doi: 10.1016/j.isci.2022.104618

Figure Lengend Snippet:

Article Snippet: Metformin , TargetMol , T0740; CAS: 1115-70-4.

Techniques: Recombinant, Staining, Reporter Assay, Sequencing, Plasmid Preparation, Software