Journal: Cancer & Metabolism

Article Title: Glucagon signaling via supraphysiologic GCGR can reduce cell viability without stimulating gluconeogenic gene expression in liver cancer cells

doi: 10.1186/s40170-022-00280-1

Figure Lengend Snippet: GCGR overexpression partially sensitizes SNU398 to glucagon signaling. (a) qPCR mRNA levels of GCGR in SNU398, following lentiviral CMV-driven mammalian expression, compared to Primary Human Hepatocytes (PHH). Data represent a single experiment with 3 biological replicates (3 separate RNA samples). eGFP used as a control. b Protein assessment of GCGR and eGFP overexpression in SNU398. Lysate number denotes independent protein sample. c Quantification of cAMP in SNU398 expressing either eGFP or GCGR and treated with 100 nM glucagon (100 G). Data points represent a single experiment of 2 technical replicates. veh: vehicle (0.05 M acetic acid), 20F: 20uM forskolin (positive control). d Protein analysis of downstream effectors of cAMP signaling in SNU398 cells expressing either eGFP or GCGR and treated with 100 nM glucagon (+). (−): vehicle treated. e Protein localization of p-CREB following glucagon treatment of SNU398 cells expressing either eGFP or GCGR. f qPCR mRNA expression of G6PC in SNU398 cells expressing either eGFP or GCGR and treated with 100nM glucagon (100 G). Data represent a single experiment with 3 biological replicates (3 separate RNA samples). Error bars: ±SEM. veh: vehicle (0.05M acetic acid). ns: not significant, p > 0.05, ordinary one-way ANOVA with Tukey’s multiple comparisons test. g Glucose quantification in SNU398 expressing either eGFP or GCGR and cultured in no glucose media with 1 mM pyruvate for 1 day. 0mM and 25mM glucose media was used as control

Article Snippet: Forskolin (MedChem Express, catalog # HY-15371) was prepared in DMSO (Sigma-Aldrich, catalog # 2650) at a stock concentration of 10 mM.

Techniques: Over Expression, Expressing, Control, Positive Control, Cell Culture

Journal: Cancer & Metabolism

Article Title: Glucagon signaling via supraphysiologic GCGR can reduce cell viability without stimulating gluconeogenic gene expression in liver cancer cells

doi: 10.1186/s40170-022-00280-1

Figure Lengend Snippet: Glucagon treatment reduces in vitro cell viability of GCGR-overexpressing SNU398. a Protein assessment of apoptotic markers in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon. b Cell proliferation assay of SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100G). Vehicle, 100 G, and 20F were added daily in fresh media. Data represent a single experiment of 3 biological replicates. Error bars: ±SEM. ns: not significant, **** p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. 20F: 20 uM forskolin, 1P: 1 μM palbociclib (cell cycle inhibitor), 10B: 10 μg/ml blasticidin (cell death inducer). c Protein assessment of pCREB in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon or 20 μM Forskolin. d Crystal violet assay on SNU398 cells either expressing eGFP or GCGR and treated with 20 μM Forksolin and/or 1 μM CREB inhibitor, and/or 5uM PKA inhibitor. e (Upper panel) Histogram of flow cytometry analysis of gated, single cells staining positive for Annexin V/Propidium iodide in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100 G). Data represent a single, independent experiment of 3 biological replicates. Error bars: ±SEM. ns: not significant, ****: p < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. 10Blast: 10 μg/ml blasticidin (positive control). (Lower panel) Representative PI/Annexin V scatter plots for SNU398 GCGR with or without glucagon treatment. f Cell proliferation assay on SNU398 cells either expressing eGFP or GCGR and treated with glucagon. Data represent a single experiment with 3 biological replicates. Error bars: ± SEM. g Crystal violet assay on SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100 G)

Article Snippet: Forskolin (MedChem Express, catalog # HY-15371) was prepared in DMSO (Sigma-Aldrich, catalog # 2650) at a stock concentration of 10 mM.

Techniques: In Vitro, Expressing, Proliferation Assay, Crystal Violet Assay, Flow Cytometry, Staining, Positive Control

Journal: Physiological Reports

Article Title: Caffeine increases myoglobin expression via the cyclic AMP pathway in L6 myotubes

doi: 10.14814/phy2.14869

Figure Lengend Snippet: Effect of cyclic AMP (cAMP) and calcium signal activation on myoglobin expression in L6 myotubes. (a) L6 myotubes were stimulated with 5 mM caffeine, 0.25 mM 8‐CPT‐cAMP, or 0.5 mM 4‐CmC for 72 h, and then, myoglobin protein level was measured by western blotting ( n = 4 per group). Representative immunoblots and quantification of protein expression levels of myoglobin are shown. (b) L6 myotubes were stimulated with 8‐CPT‐cAMP for 72 h, and then, myoglobin mRNA level was measured by real‐time PCR ( n = 6 per group). (c) L6 myoblasts were incubated with 0, 5, 50, or 500 µM 8‐CPT‐cAMP for 6 h, and CRE‐luciferase activity was then measured by luciferase assay ( n = 3 per group). (d) L6 myotubes were stimulated with 10 µM forskolin for 72 h, and then, myoglobin protein level was measured by western blotting ( n = 3 per group). Representative immunoblots and quantification of protein expression levels of myoglobin are shown. (e) L6 myotubes were stimulated with 10 µM dantrolene for 72 h, and the myoglobin mRNA level was then measured by real‐time PCR ( n = 6 per group). (f) After being treated for 72 h with 5 mM caffeine, 0.25 mM 8‐CPT‐cAMP, or 0.5 mM 4‐CmC, the myotubes were stained with trypan blue. Methanol treatment for 5 min, which induces considerable cell death, was used for the positive control. Distributions of values are depicted as violin plots; dot plots represent individual data points; cross signs depict the median in each group; error bars show 95% confidence interval. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Control. Statistical significance was assessed by (a) parametric or (c) nonparametric multiple comparison tests with the Benjamini–Hochberg method or (b, d, and e) Welch's t test.

Article Snippet: After 5 days of differentiation, myotubes were stimulated in differentiation medium containing 5 mM caffeine (Nacalai Tesque), 5–500 μM 8‐(Bennett et al., ) adenosine 3′,5′‐cyclic monophosphate sodium salt (8‐CPT‐cAMP; Enzo Life Sciences), 0.5 mM 4‐chloro‐ m ‐cresol (4‐CmC; Sigma‐Aldrich), 10 μM forskolin (LKT Laboratories), 10 μM dantrolene (Sigma‐Aldrich), and/or 1 μM KT5720 (Calbiochem).

Techniques: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Luciferase, Activity Assay, Staining, Positive Control, Control, Comparison

Journal: Slas Discovery

Article Title: High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

doi: 10.1177/2472555217716056

Figure Lengend Snippet: mIMCD3 sh Pkd1 cyst growth can be enhanced with forskolin, and this increase can be prevented with positive control compounds rapamycin, sorafenib, roscovitine, and NVP-BEZ-235. ( A ) High-resolution images obtained from an unexposed (0.2% DMSO, top panel) and a forskolin-treated (bottom panel) cyst. ( B ) Using Ominer analysis tools, cyst size could be expressed as a function of forskolin concentration. Data points represent means ± SD of eight replicate wells (technical replicates). ( C ) Representative images of control compounds after coexposure with forskolin, including unstimulated (0.2% DMSO) control. ( D ) Quantification of average cyst size from C. Whiskers represent min to max. Rapamycin, roscovitine, sorafenib, and NVP-BEZ-235 tested in quadruplicate (technical replicates), unstimulated ( n = 16 replicate wells) and stimulated ( n = 8 replicate wells). These experiments have been performed independently more than three times ( Suppl. Figs. S1B,C and S5A,C also show the performance of control molecules in biological replicates).

Article Snippet: Therefore, in addition to potentially providing therapeutically interesting molecules, the selected hits could also be used to relate compound efficacy to the cellular signaling pathways that may be involved. mIMCD3 sh Pkd1 cells were cultured in 3D hydrogels in 384-well plates, in which they formed cysts over a period of 72 h. Subsequently, we coexposed cysts with 2.5 μM forskolin and kinase inhibitors from the SelleckChem compound library in quadruplicate wells at 0.1 and 1 μM for 72 h, as illustrated in .

Techniques: Positive Control, Concentration Assay, Control

Journal: Slas Discovery

Article Title: High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

doi: 10.1177/2472555217716056

Figure Lengend Snippet: SelleckChem kinase inhibitor library screen. Kinase inhibitors were screened in quadruplicate (technical replicates) at 1 and 0.1 µM, in the presence of 2.5 µM forskolin, to stimulate cyst growth (procedure shown in Fig. 1 ). Data were z score normalized to the plate median and subsequently to the unstimulated control median (unstimulated control, black striped line). Cyst growth induction by forskolin is presented by a red striped line. Compound effects are represented by means of quadruplicate wells of two tested concentrations. Top 15 hit molecules are presented in the right cutout. The replicate-adjusted Z′ factor for this primary screen reached −0.93 between stimulated and unstimulated control conditions.

Article Snippet: Therefore, in addition to potentially providing therapeutically interesting molecules, the selected hits could also be used to relate compound efficacy to the cellular signaling pathways that may be involved. mIMCD3 sh Pkd1 cells were cultured in 3D hydrogels in 384-well plates, in which they formed cysts over a period of 72 h. Subsequently, we coexposed cysts with 2.5 μM forskolin and kinase inhibitors from the SelleckChem compound library in quadruplicate wells at 0.1 and 1 μM for 72 h, as illustrated in .

Techniques: Control

Journal: Slas Discovery

Article Title: High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

doi: 10.1177/2472555217716056

Figure Lengend Snippet: Phenotypic analysis discriminates potentially undesirable compound effects. ( A ) Validation of compounds from each target identified in Figure 3 . Mean percent inhibition (technical replicates representing triplicate wells) of cyst growth is depicted by a color scale from yellow (no inhibition of forskolin-induced cyst growth) to blue (complete inhibition of forskolin-induced cyst growth). Standard deviations are not included in this plot; for reference purposes, several dose curves are included in Supplemental Figure S6 . A mean Z′ factor of +0.36 between stimulated and unstimulated control conditions was calculated over six plates. ( B,C ) Multiparametric (PCA) analysis (PCA plot summarizes 84% of variation in the entire dataset) identifies different compound clusters, as shown by the contour plots. Forskolin-stimulated controls (large cysts) are represented as empty circles; unstimulated controls (small cysts) are represented as black dots. Data points represent single wells. ( C ) PCA plot summarizing 84% of variation in the entire dataset. Trajectories of different compound types are indicated by arrows (mTOR inhibitors, blue; CDK inhibitors, green; DNA-PK inhibitors, red; aurora kinase inhibitors, orange). Data points represent single wells; point size correlates with molecule concentration (legend omitted for presentation purposes). ( D ) Representative images from conditions shown as in panels A–C. Novel phenotypes identified in panels B and C are illustrated by images of 100 nM dinaciclib and 1 µM PIK-75.

Article Snippet: Therefore, in addition to potentially providing therapeutically interesting molecules, the selected hits could also be used to relate compound efficacy to the cellular signaling pathways that may be involved. mIMCD3 sh Pkd1 cells were cultured in 3D hydrogels in 384-well plates, in which they formed cysts over a period of 72 h. Subsequently, we coexposed cysts with 2.5 μM forskolin and kinase inhibitors from the SelleckChem compound library in quadruplicate wells at 0.1 and 1 μM for 72 h, as illustrated in .

Techniques: Biomarker Discovery, Inhibition, Control, Concentration Assay

Journal: Slas Discovery

Article Title: High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

doi: 10.1177/2472555217716056

Figure Lengend Snippet: mTOR inhibitors, but not PI3K inhibitors, prevent forskolin-induced cyst swelling. ( A ) PCA plot comprising 84% of variation in the entire dataset. mTOR inhibitors (red circles) cluster together with unstimulated control (black dots), whereas PI3K inhibitors (blue circles) cluster with the stimulated condition (empty circles). Data points represent single wells; inhibitor point size correlates with molecule concentration (legend omitted for presentation purposes). ( B ) Validation of mTOR inhibitors at concentrations of 6–100 nM. Cyst growth inhibition displayed by a color gradient ranging from yellow (no inhibition) to blue (inhibition). The color represents the mean value of triplicate wells (technical replicates; mean Z′ factor over eight plates of 0.28 between stimulated and unstimulated control conditions; also for panels C and D). ( C ) Validation of PI3K inhibitors; color scale as in panel B. ( D ) Validation of dual PI3K/mTOR inhibitors; color scale as in panel B. ( E ) Combination of mTOR inhibitor torin 1 with PI3K inhibitor buparlisib (NVP-BKM120) to assess synergy in mIMRFNPKD 5E4 cells. Values represent mean ± SD of quadruplicate wells (technical replicates; Z′ factor of +0.64 between stimulated and unstimulated control conditions).

Article Snippet: Therefore, in addition to potentially providing therapeutically interesting molecules, the selected hits could also be used to relate compound efficacy to the cellular signaling pathways that may be involved. mIMCD3 sh Pkd1 cells were cultured in 3D hydrogels in 384-well plates, in which they formed cysts over a period of 72 h. Subsequently, we coexposed cysts with 2.5 μM forskolin and kinase inhibitors from the SelleckChem compound library in quadruplicate wells at 0.1 and 1 μM for 72 h, as illustrated in .

Techniques: Control, Concentration Assay, Biomarker Discovery, Inhibition

Journal: Scientific Reports

Article Title: A Chinese herbal decoction, reformulated from Kai-Xin-San, relieves the depression-like symptoms in stressed rats and induces neurogenesis in cultured neurons

doi: 10.1038/srep30014

Figure Lengend Snippet: ( A ) KXS 2012 (0.3 to 3 μg/mL) was applied onto cultured rat cortical neurons (DIV 5) for 96 h. The protein levels of PSD-95 (~95 kDa) and synaptotagmin (~60 kDa) were measured by Western blot assay (upper panel). α-Tubulin (~55 kDa) served as a loading control. Forskolin (Fsk, 50 nM) was used as a positive control. The amount of protein level was quantified (lower panel). (B ) KXS 2012 (3 μg/mL) and BDNF (15 ng/mL) were applied onto cultured hippocampal neurons (DIV 15) for 96 h before pEGFP-N1 transfection. The treatment significantly increased the spine density of control neurons (9–16 dendrites from 6–10 neurons per 10 μm were measured for each condition). The quantification plot was shown in lower right. (C) Representative images show that KXS 2012 (3 μg/mL) and BDNF (15 ng/mL) increased the spine density compared to control. The counted spine density was indicated by asterisk. Scale bars = 10 μm. Values are expressed the fold of change as compared to control (x Basal) or number, Mean ± SEM, n = 4–10. * p < 0.05 and ** p < 0.01 compared to the control.

Article Snippet: The time of herbal treatment was 96 h. Forskolin (Sigma, 50 nM) and BDNF (Alomone lab, Israel, 15 ng/mL) were used as positive controls, respectively .

Techniques: Cell Culture, Western Blot, Control, Positive Control, Transfection

Journal: Scientific Reports

Article Title: A Chinese herbal decoction, reformulated from Kai-Xin-San, relieves the depression-like symptoms in stressed rats and induces neurogenesis in cultured neurons

doi: 10.1038/srep30014

Figure Lengend Snippet: ( A ) The mRNA and protein levels of neurotrophic factors in cultured astrocytes were analyzed by real-time quantitative PCR and ELISA kits (calibration curves of protein amounts were in ). The cultured astrocytes were treated with KXS 2012 (1.5 to15 μg/mL) for 48 h. Forskolin (Fsk, 50 nM) was used as a positive control. ( B ) Cultured astrocytes were serum starved for 5 h. Then KXS 2012 (15 μg/mL) was applied onto the cultures. Fsk (50 nM) or TPA (50 nM) served as a positive control, respectively. Total Erk1/2, phosphorylated Erk1/2 (both at ~42/44 kDa), total CREB and phosphorylated CREB (both at ~40 kDa) were revealed by using specific antibodies at different time. ( C ) Quantification plot of the phosphorylation level at 10 min was shown. ( D ) U0126 (20 μM) and H89 (5 μM) were applied onto astrocytes 3 h before the drug treatment. Quantification plot of mRNA and protein levels of neurotrophic factors was shown. Fsk (50 nM) or TPA (50 nM) served as a positive control, respectively. Values are expressed as the fold of change as compared to control (x Basal), where control value is set as 1, Mean ± SEM, n = 4, * p < 0.05 and ** p < 0.01 compared to the control.

Article Snippet: The time of herbal treatment was 96 h. Forskolin (Sigma, 50 nM) and BDNF (Alomone lab, Israel, 15 ng/mL) were used as positive controls, respectively .

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Positive Control, Phospho-proteomics, Control

Journal: BMC Neuroscience

Article Title: Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

doi: 10.1186/1471-2202-12-86

Figure Lengend Snippet: Odorants induce MAPK and CREB phosphorylation in the olfactory epithelium . (A) Cryosections (12 μm) of mice expressing GFP in every mature OSN (OMP-GFP) were subjected to immunohistochemistry using an antibody specific for phosphorylated p42/44 MAPK in the olfactory epithelium. After 2 hours of odorant exposure a strong anti-phospho-p42/44 MAPK immunoreactivity was observed in the cytoplasm of OSNs. (B) Staining for phospho-CREB (Ser-133). Phospho-CREB was observed in the sustentacular cell layer, in the nuclei of OSNs, and in immature OSNs not expressing OMP, after exposure to a mixture of 100 different odorants for 2 h. Control panel with secondary antibody only or after using a specific blocking peptide shows very low background labeling intensity in the nuclei of the different cell types. (C) WB analysis of control cell lysates (SK-N-MC cells prepared with IBMX and forskolin treatment) and olfactory epithelium samples with CREB and phospho-CREB (Ser-133) specific antibodies showing labeling of the proteins (bands marked by stars), which is abolished when the specific blocking peptide was pre-incubated with the antibody.

Article Snippet: The (positive) control phosphorylated CREB cell extract is total cell extracts from SK-N-MC cells prepared with IBMX and forskolin treatment (Cell Signaling Technology).

Techniques: Phospho-proteomics, Expressing, Immunohistochemistry, Staining, Control, Blocking Assay, Labeling, Incubation