Forskolin

Manufactured by Cell Signaling Technology

Sourced in United States

Forskolin is a chemical compound derived from the plant Coleus forskohlii. It is a diterpene that can activate the enzyme adenylate cyclase, leading to an increase in intracellular levels of the secondary messenger cyclic AMP (cAMP). This property makes forskolin a useful research tool for studying cAMP-dependent cellular processes.

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Lab products found in correlation

11 protocols using forskolin

Quantifying BeWo Cell Fusion via Fluorescence

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After seeding cells for 24 hr, BeWo cells were treated with 30 μM forskolin (Cell Signaling Technology, #3828s) for 48 hr to induce cell fusion, with forskolin-containing media changed every 24 hr. After forskolin treatment, cells were stained with Hoechst (Invitrogen, #H3570, 1:2000) and Di-8-ANEPPS (Biotium, #61012, 2 µM) or Wheat Germ Agglutinin Alexa Fluor-488 Conjugate (Invitrogen, #W11261, 1:1000) for 15 min at 37°C and 5% CO₂. For each treatment group, six random fields of view were acquired using Zeiss 780 inverted confocal microscope. Cell fusion is quantified by calculating the FI as described previously (Zhang et al., 2020 (link)). FI is calculated as:

F I = \frac{\sum_{1}^{6} f_{i}}{6} = \frac{\sum_{1}^{6} (\frac{\sum n f}{N})}{6}

where fi represents the FI of each field; nf, the number of fused nuclei in each field; N, the total nuclei number in each field, respectively. To avoid the instances of cell division, cells with two nuclei were not considered as fused cells.

Zhang Y., Liang P., Yang L., Shan K.Z., Feng L., Chen Y., Liedtke W., Coyne C.B, & Yang H. (2022). Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion. eLife, 11, e78840.

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Aporphine Derivatives: Sourcing and Applications

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Aporphine derivatives were obtained from the vendors indicated: R-(−)-apomorphine (Sigma Aldrich, A4393), (−)nuciferine (Cerilliant, PHY83282), D-Glaucine (Santa Cruz Biotechnology, sc-490895), (+)boldine (Sigma Aldrich, 67592), (+)-Bulbocapnine (Santa Cruz Biotechnology, sc-257199). Other chemicals sourced for assays were 3-Isobutyl-1-methylxanthine (Sigma Aldrich, I5879), serotonin (Sigma Aldrich, H9523) and forskolin (Cell Signaling Technology, 3828).

Chan J.D., Acharya S., Day T.A, & Marchant J.S. (2016). Pharmacological profiling an abundantly expressed schistosome serotonergic GPCR identifies nuciferine as a potent antagonist. International Journal for Parasitology: Drugs and Drug Resistance, 6(3), 364-370.

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Cilostazol Modulates Cardiomyocyte Signaling

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HCMs were purchased from Promo Cell (Promo Cell, Cat. No. C-12810, lot. 2120301.2) and were grown in myocyte growth medium (Myocyte Growth Medium Kit: Promo Cell, Cat. No.C-22170). After reaching 80% confluence, HCMs were spread into 12 well plates. Each well had 1.5 × 10⁵ cells per well. After overnight serum starvation, HCMs were incubated with selected concentrations of cilostazol (0, 0.1, 1.0 and 10 μM) for 30 min. Then, HCMs are incubated with/without AngII (0.1 μM) and/or forskolin (Cell Signaling, Cat. No. 9803) (10 μM) and/or H-89 (Cayman chemical, Cat. No. 130964-39-5) (10 μM) and/or cilostazol (1.0 μM) for 24 h. mRNA was collected from each well. All experiments were performed in passages of 6 to 8.

Hada Y., Uchida H.A., Umebayashi R., Yoshida M, & Wada J. (2022). Cilostazol Attenuates AngII-Induced Cardiac Fibrosis in apoE Deficient Mice. International Journal of Molecular Sciences, 23(16), 9065.

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Immunofluorescence Microscopy of Centrosome Proteins

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Anti-CG-NAP and anti-GM130 mouse monoclonal antibodies were purchased from BD Biosciences. Rabbit polyclonal anti-tubulin-γ antibody was from Biolegend. Rabbit polyclonal anti-GM130 was from MBL International. Anti-dynein IC and GAPDH mouse monoclonal antibodies were from Merck Millipore. Anti-PKARIIα monoclonal and polyclonal antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-pericentrin and anti-TGN46 antibodies were procured from Abcam. FITC conjugated anti-α-tubulin, rabbit polyclonal detyrosinated α-tubulin, and anti-human IgG (Fc specific) antibodies, nocodazole, poly-l-lysine (PLL), and DMSO were from Sigma-Aldrich. Phospho-PKA substrate (RRXS*/T*) rabbit monoclonal antibody, rabbit polyclonal anti-acetylated α-tubulin antibody, and forskolin were from Cell Signaling Technology. Secondary antibodies included anti-rabbit and anti-mouse Alexa Fluor 568, Alexa Fluor 488, and Alexa Fluor 633 (Molecular Probes). Rhodamine-phalloidin, Alexa Fluor 488 conjugated anti-α-tubulin, and Hoechst 33342 were from Life Technologies. Brefeldin-A was from Calbiochem. Recombinant human IL-2 and SDF-1α were from Peprotech. Human ICAM-1/CD54 protein was from Sino Biological. Dharmacon pre-designed ON-TARGETplus SMARTpool siRNA against targeting CG-NAP or PKARIIα were from GE Life Sciences.

Ong S.T., Chalasani M.L., Fazil M.H., Prasannan P., Kizhakeyil A., Wright G.D., Kelleher D, & Verma N.K. (2018). Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells. Frontiers in Immunology, 9, 397.

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BeWo cell fusion induced by forskolin

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BeWo cells were incubated with 20 μM forskolin (3828, Cell Signaling Technology) to induce cell fusion. After 48 h, the cultured cells were stained with Hoechst 33342 (H1399, Invitrogen) and 2 μM Di-8-ANEPPS (D3167, Invitrogen) for 30 min, and subsequently imaged by Zeiss LSM 880 Fast Airyscan Confocal system (Carl Zeiss, Jena, Germany).
In fluorescent images of six randomly selected fields from three or more independent experiments, the number of nuclei within non-fused or fused cells was counted. Cells with more than two nuclei were identified as fused cells, and the fusion index in each group was calculated as follows: (the number of nuclei in fused cells) / (total number of nuclei). The fusion index data were expressed relative to the forskolin-untreated WT control.

Lee J.G., Yon J.M., Kim G., Lee S.G., Kim C.Y., Cheong S.A., Kim H.Y., Yu J., Kim K., Sung Y.H., Yoo H.J., Woo D.C., Rho J.K., Ha C.H., Pack C.G., Oh S.H., Lim J.S., Han Y.M., Hong E.J., Seong J.K., Lee H.W., Lee S.W., Lee K.U., Kim C.J., Nam S.Y., Cho Y.S, & Baek I.J. (2024). PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development. Nature Communications, 15, 1487.

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Forskolin-Induced BeWo Cell Syncytialisation

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BeWo cell syncytialisation was induced by forskolin as previously described [9 (link)]. Briefly, BeWo cells were treated with 60 μM forskolin (Cell Signaling Technology, #3828s) for 48 hours to induce cell fusion and the culture medium with 60 μM forskolin was replaced every 24 h. After treatments, BeWo cells were incubated with Hoechst (Invitrogen™, #H3570, 1:1500) and various fluorescent membrane dyes: 2 μM Di-8-ANEPPS (Invitrogen™, # D3167), 5.0 μg/mL Wheat Germ Agglutinin-Alexa Fluor™−488 Conjugate (Invitrogen™, # W11261), 1:200 CellBrite™-Orange Cytoplasmic Membrane Dye (Biotium, #30022), and 50 μg/mL Concanavalin A-Alexa Fluor™−647 Conjugate (Invitrogen, #C21421) in 5% CO₂ at 37 °C for 15–20 minutes. Di-8-ANEPPS stained cells can be directly imaged without extra rinsing steps. The other membrane markers need to be washed twice with the dye-free medium at room temperature.

Zhang Y, & Yang H. (2019). A simple and robust fluorescent labeling method to quantify trophoblast fusion. Placenta, 77, 16-18.

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Mitochondrial Assay with Inhibitors

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Gossypol (G8761-100 mg), H89 dihydrochloride hydrate (B1427), 3-BP and cycloheximide (C4859) were purchased from Sigma-Aldrich. Bortezomib (B-1408) was purchased from LC Laboratories. MitoTracker Red CMXRos (M7512) and MitoTracker Green FM (M7514) were purchased from Thermo Fisher Scientific. Forskolin (3828) was purchased from Cell Signaling Technology.

Li S., Li W., Yuan J., Bullova P., Wu J., Zhang X., Liu Y., Plescher M., Rodriguez J., Bedoya-Reina O.C., Jannig P.R., Valente-Silva P., Yu M., Henriksson M.A., Zubarev R.A., Smed-Sörensen A., Suzuki C.K., Ruas J.L., Holmberg J., Larsson C., Christofer Juhlin C., von Kriegsheim A., Cao Y, & Schlisio S. (2022). Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel–Lindau syndrome. Nature Metabolism, 4(6), 739-758.

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Mitochondrial Dynamics Modulation Assay

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Gossypol (G8761-100 mg), H-89 dihydrochloride hydrate (B1427),
Bromopyruvic acid (3-BP) and Cycloheximide (C4859) were purchased from
Sigma-Aldrich. Bortezomib (B-1408) was purchased from LC Laboratories.
MitoTracker™ Red CMXRos (Cat. M7512) and MitoTracker™ Green FM
(Cat. M7514) were purchased from Thermo Fisher Scientific. Forskolin (#3828) was
purchased from Cell Signaling Technology.

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Renin Induction in Confluent Pericytes

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Confluent pericyte cultures were serum-starved (DMEM/1% PS) for 24 hours. Renin induction was stimulated with cAMP inducers (10-μm forskolin; Cell Signalling, Leiden, Netherlands, and 100 μm IBMX; Sigma-Aldrich) in DMEM/1% PS. Controls consisted of vehicle (medium/ethanol/0.01% dimethyl sulfoxide) and serum-free medium with no additives (DMEM/1% PS). After 24 hours, culture media were collected for renin activity assay. After a PBS wash, cells were harvested for RNA extraction.

Stefanska A., Kenyon C., Christian H.C., Buckley C., Shaw I., Mullins J.J, & Péault B. (2016). Human kidney pericytes produce renin. Kidney International, 90(6), 1251-1261.

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Tanycyte cAMP Signaling Assay

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Primary tanycytes were seeded in 12 wells plates; at 70% confluency, cells were placed into serum-free medium (DMEM/F12 without phenol red, 1% penicillin/streptomycin and 2 mM L-glutamine, all from Thermo Fisher) and 5ug/mL insulin and 100 μM putrescine (last two from Sigma-Aldrich, USA)) for 2 additional days. 1 hr before the experiment, cells were placed into fresh serum-free medium and treated with MCH 1 μM (Bachem, H-1482), 0, 15, 30, 60 min in the presence or absence of Forskolin 10 μM (Cell Signaling, 3828), which was added 15 min before stopping treatment. At the end of the treatment, cells were washed 3 times with ice-cold PBS and 600 μL of 1x lysis buffer was added to each well. Then, cAMP levels were assessed following the instructions of the kit’s datasheet (Cell signaling, 4339).

Jiang H., Gallet S., Klemm P., Scholl P., Folz-Donahue K., Altmüller J., Alber J., Heilinger C., Kukat C., Loyens A., Müller-Fielitz H., Sundaram S., Schwaninger M., Prevot V, & Brüning J.C. (2020). MCH Neurons Regulate Permeability of the Median Eminence Barrier. Neuron, 107(2), 306-319.e9.

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