icilin induced rise  (Alomone Labs)

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: RGS2 expression reduces signal transduction after stimulation of TRPM8 channels or Gαq-coupled designer receptors. ( a ) Modular structure of RGS2. The RGS domain is responsible for binding to Gαq, while the N-terminal domain is required for targeting the protein to the plasma membrane. ( b ) HEK293-M8 cells containing the Coll.luc reporter gene were infected with a recombinant lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were serum-starved for 24 h, and then stimulated with icilin (1 μM) in serum-reduced medium for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3, *** p < 0.001). ( c ) HEK293 cells were infected with a lentivirus encoding the Gαq-coupled designer receptor Rαq. Cells were additionally infected with a lentivirus containing the Coll.luc reporter gene. Furthermore, the cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). We incubated the cells in medium containing 0.05% serum for 24 h. Stimulation of the cells was performed with CNO (1 μM) for 24 h in serum-reduced medium. Cells were harvested and analyzed as described in the legend to (n = 5, *** p < 0.001). ( d ) T-REx-TRPM3 cells containing a chromatin-integrated Coll.luc reporter gene were serum-starved for 24 h in the presence of tetracycline (1 μg/mL). The serum-starved cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were stimulated with pregnenolone sulfate (20 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant). ( e ) INS-1 832/13 insulinoma cells were infected with a lentivirus containing the reporter gene Coll.luc. Additionally, we infected the cells with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were incubated in medium containing 0.5% serum and 2 mM glucose for 24 h, and then stimulated with KCl (25 mM) and the FPL64176 (2.5 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant).

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Expressing, Transduction, Binding Assay, Clinical Proteomics, Membrane, Infection, Recombinant, Incubation

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: The transcription factor c-Jun is required to link the stimulation of TRPM8 with the activation of AP-1. ( a ) Modular structure of the transcription factor c-Jun and its dominant-negative c-Jun mutant c-JunΔN. c-JunΔN contains the C-terminal amino acids 188 to 331 of c-Jun, which comprise the bZIP domain. The mutant lacks the transcriptional activation domain. ( b ) Expression of c-JunΔN attenuates icilin-induced activation of AP-1 in HEK293-M8 cells. Cells were infected with a lentivirus containing a Coll.luc reporter gene. Cells were additionally infected with a lentivirus encoding either c-JunΔN or β-galactosidase (mock). Serum-starved cells were stimulated with icilin (1 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; *** p < 0.001).

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Activation Assay, Dominant Negative Mutation, Mutagenesis, Expressing, Infection

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: Pharmacological inhibition of PIP5Kα attenuates intracellular signaling following stimulation of TRPM3 and Ca v 1.2 Ca 2+ channels. ( a ) Modular structure of TRPM3 showing the interaction sites of phosphatidylinositol 4,5-bisphosphate with the channel (preS1 segment, S4-S5 linker, and TRP domain). ( b ) T-REx-TRPM3 cells containing a Coll.luc reporter gene integrated into the chromatin were serum-starved for 24 h in the presence of tetracycline (1 μg/mL) to induce TRPM3 expression. The serum-starved cells were preincubated with ISA-2011B (10 μM) for 3 h, and then stimulated with pregnenolone sulfate (20 μM) for 24 h in the presence of the inhibitor. Cells were harvested and analyzed as described in the legend to (n = 4; *** p < 0.001). ( c ) Modular structure of Ca v 1.2 voltage-gated Ca 2+ channels, consisting of the α1 subunit, which forms the pore, and the auxiliary subunits α2δ, β, and γ. ( d ) INS-1 832/13 insulinoma cells were infected with a recombinant lentivirus containing the Coll.luc reporter gene. Cells were serum-starved in medium containing 0.5% serum and 2 mM glucose for 24 h. Cells were preincubated in the same medium with ISA-2011B (10 μM) for three hours. Stimulation of the cells was performed with KCl (25 mM) and the voltage-gated Ca 2+ channel activator FPL64176 (2.5 μM) in the presence of the inhibitor for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; *** p < 0.001).

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Inhibition, Expressing, Infection, Recombinant

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: RGS2 expression reduces signal transduction after stimulation of TRPM8 channels or Gαq-coupled designer receptors. ( a ) Modular structure of RGS2. The RGS domain is responsible for binding to Gαq, while the N-terminal domain is required for targeting the protein to the plasma membrane. ( b ) HEK293-M8 cells containing the Coll.luc reporter gene were infected with a recombinant lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were serum-starved for 24 h, and then stimulated with icilin (1 μM) in serum-reduced medium for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3, *** p < 0.001). ( c ) HEK293 cells were infected with a lentivirus encoding the Gαq-coupled designer receptor Rαq. Cells were additionally infected with a lentivirus containing the Coll.luc reporter gene. Furthermore, the cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). We incubated the cells in medium containing 0.05% serum for 24 h. Stimulation of the cells was performed with CNO (1 μM) for 24 h in serum-reduced medium. Cells were harvested and analyzed as described in the legend to (n = 5, *** p < 0.001). ( d ) T-REx-TRPM3 cells containing a chromatin-integrated Coll.luc reporter gene were serum-starved for 24 h in the presence of tetracycline (1 μg/mL). The serum-starved cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were stimulated with pregnenolone sulfate (20 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant). ( e ) INS-1 832/13 insulinoma cells were infected with a lentivirus containing the reporter gene Coll.luc. Additionally, we infected the cells with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were incubated in medium containing 0.5% serum and 2 mM glucose for 24 h, and then stimulated with KCl (25 mM) and the FPL64176 (2.5 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant).

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Expressing, Transduction, Binding Assay, Clinical Proteomics, Membrane, Infection, Recombinant, Incubation

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: The transcription factor c-Jun is required to link the stimulation of TRPM8 with the activation of AP-1. ( a ) Modular structure of the transcription factor c-Jun and its dominant-negative c-Jun mutant c-JunΔN. c-JunΔN contains the C-terminal amino acids 188 to 331 of c-Jun, which comprise the bZIP domain. The mutant lacks the transcriptional activation domain. ( b ) Expression of c-JunΔN attenuates icilin-induced activation of AP-1 in HEK293-M8 cells. Cells were infected with a lentivirus containing a Coll.luc reporter gene. Cells were additionally infected with a lentivirus encoding either c-JunΔN or β-galactosidase (mock). Serum-starved cells were stimulated with icilin (1 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; *** p < 0.001).

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Activation Assay, Dominant Negative Mutation, Mutagenesis, Expressing, Infection

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: Signal transduction of TRPM8 channels. The TRPM8 channel is a tetramer embedded into the plasma membrane. The channel interacts with phosphatidylinositol 4,5-bisphosphate, which is essential for its activation. Reducing phosphatidylinositol 4,5-bisphosphate levels by blocking its biosynthesis with a PIP5K inhibitor impairs TRPM8 activation. PIP5K is, therefore, an important regulator of TRPM8 signaling. The stimulation of Gαq-coupled receptors triggers the dissociation of the trimeric G proteins into a GTP-bound α-subunit and the βγ subunits. The Gα subunit interacts with TRPM8 channels and is essential for TRPM8 activation. The α-subunit and the βγ subunits bind to PLCβ and activate the enzyme by increasing k cat and changing the orientation of the enzyme in the membrane to its substrate. This conformational change may remove the blockade of PLCβ to TRPM8 channels. After the activation of TRPM8, Ca 2+ ions flow into the cells through the channel and trigger the activation of ERK1/2, which acts as signal transducer. The kinase translocates into the cell nucleus and activates AP-1, which is composed of the bZIP proteins c-Jun and c-Fos.

Article Snippet: Stimulation was performed with icilin (PubChem CID: 161930; 1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557, dissolved in DMSO), pregnenolone sulfate (PubChem CID: 105074; PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), or clozapine- N -oxide (PubChem CID: 135445691; 1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), respectively, for 24 h in medium containing 0.05% fetal bovine serum.

Techniques: Transduction, Clinical Proteomics, Membrane, Activation Assay, Blocking Assay