Journal: Scientific Reports

Article Title: Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein

doi: 10.1038/srep26835

Figure Lengend Snippet: BMSCs were incubated in osteogenic media for 21 days and then transferred to low pH (pH = 7.0) basal media for different times (t = 0, 5, 10, 20, or 40 min). The phosphorylation of MYPT1 and CREB was assessed by western blotting (A) . BMSCs were incubated in osteogenic media at different pHs containing a cAMP activator (forskolin) or a PKA inhibitor (H 89 2 HCl) for 21 days. The calcium deposits in the differentiated BMSCs were evaluated by Alizarin red S staining (B) , and the expressions of IBSP and BGLAP were examined by qRT-PCR analysis (C). * P < 0.05 compared with the pH = 7.0 group; # P < 0.05 compared with the pH = 7.4 group. (D,E) The pH-dependent regulation of the nucleo-cytoplasmic translocation of YAP and phosphorylation of YAP and LATS1 was regulated by Forskolin and H 89 2HCl, as assessed by western blotting. (F) YAP localization was assessed by immunofluorescence assay after Forskolin or H 89 2HCl treatment. Scale bar: 50 μm.

Article Snippet: Forskolin and H 89 2HCl (H89) were obtained from Selleck (Houston, TX, USA), and recombinant human CTGF protein was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA).

Techniques: Incubation, Phospho-proteomics, Western Blot, Staining, Quantitative RT-PCR, Translocation Assay, Immunofluorescence

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

Journal: eLife

Article Title: Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide

doi: 10.7554/eLife.80944

Figure Lengend Snippet: ( A ) Immunoblot of pS6, S6, pCREB, and CREB and quantification of pS6 expression (pS6 /S6) in CHO-K1 cells transfected with the a vector expressing the hGLP1R and treated with DMSO or the mTORC1 inhibitor rapamycin (100 nM) for 30 min followed by treatment with 1 X PBS (Veh) or liraglutide (Lira, 10 nM) for 1 hr (Interaction: F (1, 8) = 6.926, p=0.0301; Inhibitor: F (1, 8) = 6.926, p=0.0301; Treatment: F (1, 8) = 22.50, p=0.0015). ( B–C ) Immunoblot of pS6, S6, pCREB, and CREB and quantification of pS6 expression (pS6/S6) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO (Veh) or either of the PKA inhibitors, ( B ) H89 (10 µM) or ( C ) KT5720 (10 µM or 50 μM), for 30 min followed by treatment with vehicle (PBS), Lira (10 nM), forskolin (Fsk, 10 µM), or insulin (Ins, 10 mg/mL) for 1 hr (H89: Interaction: F (1, 12) = 4.782, p=0.0493; Inhibitor: F (1, 12) = 4.782, p=0.0493; Treatment: F (1, 12) = 12.50, p=0.0041). KT5720: (Interaction: F (2, 18) = 15.32, p=0.0001; Inhibitor: F (2, 18) = 15.32, p=0.0001; Treatment: F (1, 18) = 36.14, p<0.0001). Quantification of pCREB expression (pCREB/CREB) is shown in . ( D ) Immunoblot of pS6, S6, pAkt, and Akt and quantification of pS6 expression (pS6/S6) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO or the pan-Akt inhibitor MK-2206 (5 μM or 20 µM) for 30 min followed by treatment with vehicle (PBS), Lira (10 nM), Fsk (10 µM), or Ins (10 mg/mL) for 1 hr (Interaction: F (1, 12) = 0.02486, p=0.8773; Inhibitor: F (1, 12) = 0.02486, p=0.8773; Treatment: F (1, 12) = 110.2, p<0.0001). Quantification of pAkt expression (pAkt/Akt) is shown in . Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are normalized to the respective control (Veh or Veh and inhibitor). Absolute data are shown in . All data are shown as mean ± SEM, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=3–4 biological replicates. Figure 1—source data 1. Graphs of absolute quantification of data from and raw data in Excel spreadsheet.

Article Snippet: Cells were washed twice with serum-free Ham’s F12 (CHO-GLP-1R) or DMEM (β-TC3) media containing 5.5 mM glucose for 3 hr and were treated with vehicle (1 X PBS) or liraglutide (Victoza, Novo Nordisk) for 1 hr with or without 30 min pre-treatment with vehicle (DMSO), PKA inhibitors H89 (Cat#2910, Tocris Bioscience) or KT5720 (Cat#1288, Tocris Bioscience), or the Akt inhibitor MK-2207 (Cat#S1078, Selleck Chemicals LLC) at concentrations stated in the figure legends.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Stable Transfection, Control

Journal: eLife

Article Title: Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide

doi: 10.7554/eLife.80944

Figure Lengend Snippet: (Resubmission Rev 2 ). ( A ) Quantification of pS6 expression (pS6 /S6) in CHO-K1 cells transfected with the a vector expressing the hGLP1R and treated with DMSO or the mTORC1 inhibitor rapamycin (100 nM) for 30 min followed by treatment with 1 X PBS (Veh) or liraglutide (Lira, 10 nM) for 1 hr (Interaction: F (1, 8) = 31.10, p=0.0005; Inhibitor: F (1, 8) = 50.10, p=0.0001; Treatment: F (1, 8) = 39.17, p=0.0002). ( B–C ) Quantification of pS6 (pS6/S6) and pCREB (pCREB/CREB) expression in CHO-K1 cells stably expressing the hGLP1R treated with DMSO (Veh) or either of the PKA inhibitors, ( B ) H89 (10 µM) or ( C ) KT5720 (10 µM or 50 μM), for 30 min followed by treatment with vehicle (PBS) or Lira (10 nM) for 1 hr (H89 pS6/S6: Interaction: F (1, 12) = 13.40, p=0.0033; Inhibitor: F (1, 12) = 21.00, p=0.0006; Treatment: F (1, 12) = 18.40, p=0.0011. H89 pCREB/CREB: Interaction: F (1, 8) = 356.7, p<0.0001; Inhibitor: F (1, 8) = 578.5, p<0.0001; Treatment: F (1, 8) = 361.3, p<0.0001. KT5720 pS6/S6: Interaction: F (2, 18) = 15.32, p=0.0001; Inhibitor: F (2, 18) = 15.32, p=0.0001; Treatment: F (1, 18) = 36.14, p<0.0001. KT5720 pCREB/CREB: Interaction: F (2, 12) = 30.76, p<0.0001; Inhibitor: F (2, 12) = 43.93, p<0.0001; Treatment: F (1, 12) = 63.66, p<0.0001). Quantification of pS6 and pAkt (pAkt/Akt) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO or the pan-Akt inhibitor MK-2206 (5 μM or 20 µM) for 30 min followed by treatment with vehicle (PBS), Lira (10 nM) or Ins (10 mg/mL) for 1 hr (pS6/S6: Interaction: F (1, 12) = 17.67, p=0.0012; Inhibitor: F (1, 12) = 31.37, p=0.0001; Treatment: F (1, 12) = 181.6, p<0.0001. pAkt/Akt: Interaction: F (2, 12) = 60.57, p<0.0001; Inhibitor: F (1, 12) = 62.79, p<0.0001; Treatment: F (2, 12) = 61.73, p<0.0001). Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are presented as absolute densitometry values and are shown as mean ± SEM, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=3–4 biological replicates. Figure 1—figure supplement 1—source data 1. Quantification of absolute values from data in and raw data.

Article Snippet: Cells were washed twice with serum-free Ham’s F12 (CHO-GLP-1R) or DMEM (β-TC3) media containing 5.5 mM glucose for 3 hr and were treated with vehicle (1 X PBS) or liraglutide (Victoza, Novo Nordisk) for 1 hr with or without 30 min pre-treatment with vehicle (DMSO), PKA inhibitors H89 (Cat#2910, Tocris Bioscience) or KT5720 (Cat#1288, Tocris Bioscience), or the Akt inhibitor MK-2207 (Cat#S1078, Selleck Chemicals LLC) at concentrations stated in the figure legends.

Techniques: Expressing, Transfection, Plasmid Preparation, Stable Transfection

Journal: eLife

Article Title: Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide

doi: 10.7554/eLife.80944

Figure Lengend Snippet: ( A ) Immunoblot and quantification of phospho-PKA substrate (pRRXS*/T*) and Myc in CHO-K1 cells stably expressing the hGLP1R transfected with Myc-wild-type (WT) Raptor or Myc-Ser 791 Ala Raptor and treated with vehicle (DMSO) or H89 (10 µM) for 30 min followed by treatment with Lira (10 nM), forskolin (Fsk, 10 μM), or insulin (Ins, 10 mg/mL) for 1 hr (Interaction: F (4, 21) = 3.695, p=0.0071; Genotype: F (2, 21) = 10.51, p=0.0007; Treatment: F (2, 21) = 6.328, p=0.0071). ( B ) Immunoblot and quantification of phospho-PKA substrate and Myc in CHO-K1 cells stably expressing the hGLP1R transfected with Myc-wild-type (WT) Raptor and treated with vehicle (DMSO) or MK-2206 (20 μM) for 30 min followed by treatment with Lira (10 nM), Fsk (10 μM), or Ins (10 mg/mL) for 1 hr (Interaction: F (3, 16) = 0.4599, p=0.7141; Inhibitor: F (1, 16) = 0.04511, p=0.8345; Treatment: F (3, 16) = 44.26, p<0.0001). ( C ) Immunoblot and quantification of phospho-PKA substrate and Myc in β-TC3 cells transfected with Myc-wild-type (WT) Raptor or Myc-Ser 791 Ala Raptor and treated with vehicle (DMSO) or MK-2206 (10 µM) for 30 min followed by treatment with Lira (10 nM), forskolin (Fsk, 10 μM), or insulin (Ins, 10 mg/mL) for 1 hr (Interaction: F (3, 16) = 7.345, p=0.0026; Genotype: F (1, 16) = 22.18, p=0.0002; Treatment: F (3, 16) = 10.93, p=0.0004). Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are shown as mean ± SEM, no symbol = not significant, * p<0.05, ** p<0.01, *** p<0.001, n=3–4 biological replicates. Figure 2—source data 1. , raw blots from Figure 2, and raw data for .

Article Snippet: Cells were washed twice with serum-free Ham’s F12 (CHO-GLP-1R) or DMEM (β-TC3) media containing 5.5 mM glucose for 3 hr and were treated with vehicle (1 X PBS) or liraglutide (Victoza, Novo Nordisk) for 1 hr with or without 30 min pre-treatment with vehicle (DMSO), PKA inhibitors H89 (Cat#2910, Tocris Bioscience) or KT5720 (Cat#1288, Tocris Bioscience), or the Akt inhibitor MK-2207 (Cat#S1078, Selleck Chemicals LLC) at concentrations stated in the figure legends.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection