Journal: Cell reports

Article Title: Retrograde adenosine/A 2A receptor signaling facilitates excitatory synaptic transmission and seizures

doi: 10.1016/j.celrep.2024.114382

Figure Lengend Snippet: (A) Left, Diagram illustrating the recording configuration. MC and MPP EPSCs were recorded from the same GC and evoked with stimulation electrodes placed in the inner and middle molecular layer, respectively. Right, Current clamp recording showing GC theta-burst firing (GC TBF). LTP induction protocol (GC TBF) was composed of 10 bursts at 5 Hz of 5 action potentials at 50 Hz, repeated 4 times every 5 s. (B) Left, Representative traces before (1) and after (2) GC TBF delivery. Right, Time-course plot showing that GC TBF induced LTP at MC-GC but not at MPP-GC synapses. (C) GC TBF-induced LTP was associated with significant reduction in PPR and CV (n = 13 cells). ** p < 0.01, *** p < 0.001. (D) LTP was abolished when TrkB was conditionally knocked out from from GCs (Post TrkB cKO, TrkBfl/fl mice injected in the dorsal blade with AAV5.CaMKII.Cre.GFP). LTP was unaffected in control animals (Control, TrkBfl/fl mice injected in the dorsal blade with AAV5.CaMKII.eGFP). (E) LTP was normally induced when loading PKI6-22 (2.5 μM) in GCs via the recording pipette but completely blocked when the cell-permeable PKA inhibitor PKI14-22 myristoylated (1 μM) was bath applied. (F) Summary bar graph showing the magnitude of GC TBF-induced LTP in the presence of DGC-IV (1 μM), when TrkB was conditionally knocked out from MCs (Pre TrkB cKO), when loading the PKI6-22 (2.5 μM) in GCs, and in the presence of D-APV (50 μM). LTP was abolished in the presence of the TrkB antagonist ANA-12 (15 μM), when Botox (0.5 μM) was loaded postsynaptically, in postsynaptic BDNF and TrkB cKO mice, and during bath application of the PKA inhibitors H89 (10 μM) or myristoylated PKI14-22 μM). Time-course summary plots are shown in Figure S1. ** p < 0.01, *** p < 0.001. (G) Scheme illustrating the emerging model for the mechanism underlying GC TBF-LTP. GC TBF triggers postsynaptic BDNF release and subsequent TrkB activation in GCs (1). Presynaptic PKA is then engaged downstream of postsynaptic BDNF/TrkB signaling (2), suggesting the requirement of a retrograde signal. Lastly, presynaptic PKA activation resulted in a long-lasting increase in glutamate release (3). Numbers in parentheses indicate the number of cells. Data are presented as mean ± SEM.

Article Snippet: H89 dihydrochloride , Tocris Bioscience , Cat#2910.

Techniques: Injection, Control, Transferring, Activation Assay

Journal: Cell reports

Article Title: Retrograde adenosine/A 2A receptor signaling facilitates excitatory synaptic transmission and seizures

doi: 10.1016/j.celrep.2024.114382

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: H89 dihydrochloride , Tocris Bioscience , Cat#2910.

Techniques: Virus, Plasmid Preparation, Recombinant, Software

Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: PKA activity correlates with loss of GLP-1R from the cell surface . MIN6 cells were transfected with a GFP-tagged GLP-1R (GLP-1R-GFP) expressed under the control of the constitutive CMV promoter. (A) GLP-1R-GFP transfected MIN6 cells were cultured at low glucose (3 mM; LG) or high glucose (25 mM; HG) for 4 h in the presence or the absence of the PKA inhibitor, H89 (HG + H89). GFP was visualized in green, nuclei stained with dapi (blue) and β-catenin immune-stained to mark the plasma membrane (red). (B) GLP-1R-GFP transfected MIN6 cells were infected with a recombinant adenovirus expressing a constitutively active PKA catalytic subunit (caPKA). Cells were cultured at low glucose (LG), and the localization of the GLP-1R-GFP was determined by fluorescence microscopy. Cells infected with the caPKA adenovirus were identified using an antibody against the FLAG-tag of the caPKA (red). (C) GLP-1R-GFP transfected MIN6 cells were cultured for 4 h at high glucose (HG) with: forskolin (Fsk), to raise cAMP levels through the activation of adenylyl cyclases; H89, to inhibit PKA; or expression of a dominantly negative PKA regulatory subunit (dnPKA). GLP-1-GFP was visualized in green, nuclei by staining with dapi (blue), and the FLAG-tag epitope on the dnPKA in red.

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Activity Assay, Transfection, Control, Cell Culture, Staining, Clinical Proteomics, Membrane, Infection, Recombinant, Expressing, Fluorescence, Microscopy, FLAG-tag, Activation Assay

Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: Chronic exposure of β-cells to the GLP-1R agonist exendin-4 . (A) MIN6 cells were chronically cultured (20 h) at low glucose in the absence (LG) or the presence (LG + Ex4) of the GLP-1R agonist exendin-4, fixed and incubated with fluorescein-tagged exendin-4 to bind cell surface (but not internal) GLP-1R. (B) MIN6 cells transfected with GLP-1R-GFP were cultured for 4 h at high glucose with exendin-4 in the absence (HG + Ex) or the presence of H89 (HG + Ex + H89), and GLP-1R-GFP localization was determined by fluorescence microscopy (green). Cells were stained for β-catenin to mark membranes (red) and dapi to identify nuclei (blue). (C) To determine the in vivo effects of chronic exendin-4 exposure, male mice were administered exendin-4 at 4–6 h intervals for 24 h or were administered saline at each time-point (controls). Mice were then given a 5 μg/kg body exendin-4 dose and an hour later a 3 g/kg intraperitoneal glucose bolus. Plasma insulin levels were measured for 15 min following the glucose challenge (C) and blood glucose levels for 120 min (D). Data were analyzed by 2-way ANOVA with Bonferroni post hoc tests. *, P < 0.05; ***, P < 0.001. n = 6–8 mice for both C and D).

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Cell Culture, Incubation, Transfection, Fluorescence, Microscopy, Staining, In Vivo, Saline, Clinical Proteomics

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 reverses ABCB1-mediated MDR in CRC cells. ( A ) The chemical structure of H89. ( B , C ) Cells were treated with the indicated drugs for 72 h and detected by MTT assay. Representative cell viability curves are shown. ** p < 0.01 compared to the corresponding control groups. Data are presented as mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: MTT Assay, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: The combination of H89 with doxorubicin or vincristine induces cell cycle arrest. Cells were treated with the indicated reagents for 24 h, and the cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining. The concentrations of each agent were as follows: 10 μM H89, 0.03 μM doxorubicin and 0.003 μM vincristine for HCT-8 cells; 10 μM H89, 0.1 μM doxorubicin and 0.1 μM vincristine for HCT-8/V cells. ( A ) Representative histograms. Bright blue indicates Sub G1 period, and red indicates G0/G1-G2/M period. ( B ) Representative quantitative data. ** p < 0.01 compared to the corresponding control groups. Data are presented as the mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Flow Cytometry, Staining, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 enhances the intracellular accumulation of substrate drugs within HCT-8/V cells. After preincubatingHCT-8 and HCT-8/V cells with different concentrations of H89 or 10 μM verapamil for 1 h, 10 μM doxorubicin (red) or rhodamine 123 (green) was added and incubated together for 2 h. Subsequently, the cells were observed and imaged under a microscope, and quantitative analysis was performed using flow cytometry. Higher intracellular fluorescence intensity indicates a greater intracellular drug accumulation. Representative images ( A , D ), histograms ( B , E ), and quantitative data ( C , F ) are shown. The scale bar is 200 μm. “+” indicates the combination of two drugs. *** p < 0.001 compared to the corresponding control groups. Data are presented as the mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Incubation, Microscopy, Flow Cytometry, Fluorescence, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 inhibits the ATPase activity of ABCB1 and occupies its ATP-binding pocket. ( A ) ABCB1 expression levels in HCT-8/V cells treated with 10 μM and 30 μM H89 for the indicated time points were measured by Western blot. ( B ) The effect of H89 on the ATPase activity of ABCB1 was determined using the ABCB1 ATPase assay kit. Data are presented as the mean ± SD ( n = 3). ( C ) The best-scoring pose of H89 in the ATP-binding domain of ABCB1. ABCB1 is displayed as a silver ribbon, and H89 is shown as a sphere model, predominantly in yellow, where yellow represents C (carbon), blue represents N (nitrogen), bright red represents O (oxygen), dark red represents Br (bromine), and tan represents S (sulfur). ( D ) The best-scoring binding conformation of ABCB1 with ATP. ABCB1 is displayed as a silver ribbon, and ATP is shown as a sphere model, predominantly in orange, where orange represents S (sulfur), blue represents N (nitrogen), cyan represents C (carbon), and bright red represents O (oxygen). ( E ) Detailed illustration of the interactions between H89 and the ATP-binding pocket of ABCB1. ABCB1 is displayed as a translucent silver ribbon, and the key amino acids are shown as orange-red sticks. H89 is presented as yellow sticks. Hydrophobic interactions are indicated by gray dashed lines, π-π stacking interactions by gray solid lineswith bond lengths of 2.9 Å, and hydrogen bonds by blue solid lines. The bond lengths between H89 and GLY-521, LYS-532, LEU-475, and ASN-899 are 3.4 Å, 3.6 Å, 3.0 Å, and 3.1 Å, respectively. ( F ) Detailed illustration of the interaction between ABCB1 and ATP. ABCB1 is displayed as a translucent silver ribbon, with key amino acids shown as orange-red sticks. ATP is presented as bright blue sticks. Gray dashed lines indicate hydrophobic interactions; yellow dashed lines represent salt bridges; gray solid lines denote π-π stacking interactions with bond lengths of 3.9 Å and 4.2 Å; blue solid lines indicate hydrogen bonds. The bond lengths between ATP and GLY-521, GLN-526, GLU-522, LYS-532, LEU-527, and ALA-525 are 3.1 Å, 2.7 Å, 2.3 Å, 3.9 Å, 3.0 Å, and 2.8 Å, respectively.

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot, ATPase Assay