Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Sequences of primers used for real-time RT-PCR assays

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Quantitative RT-PCR

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Relative expression levels of TRPM8 mRNA. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. The OVX + E2 group showed a trend for decreased expression of TRPM8 channel mRNA in lumbar skin in comparison with the OVX group, although the difference between the two groups did not reach statistical significance ( P =0.0873>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Expressing, Comparison

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: TRPM8 protein levels. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. There was no statistical difference between mean TRPM8 protein levels in lumbar skin of the OVX and OVX + E2 groups ( P =0.3095>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques:

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Immunohistochemistry. A. NON-OPE: non-operated rats. B. OVX: ovariectomized rats. C. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. TRPM8 channels and PGP9.5-positive nerve fibers in lumbar skin of NON-OPE, OVX, and OVX + E2 rats were visualized by immunohistochemistry. TRPM8 channels were stained red, and PGP9.5-positive nerve fibers were stained green. Cell nuclei are shown in blue. PGP9.5-positive nerve fibers were scattered in a line at the external side of the epidermal tissues (arrows). Scale bar, 10 µ m. Expression of TRPM8 in the OVX rat was greater than in the NON-OPE rat (A, B), whereas expression of TRPM8 in the OVX + E2 rat was lesser than in the OVX rat (B, C).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 1. Menthol-induced intracellular Ca2 release. A, time course of TRPM8 current activation in Ca2-free extracellular solution upon application of 1 mM menthol at 33 °C in HEK293 cells overexpressing TRPM8. The upper panel shows the in and outward current at 90 and 90 mV, whereas the lower panel displays the increase in [Ca2]i. B, current-voltage relations obtained at the time points indicated in panel A.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 2. Influence of temperature on the menthol-induced [Ca2]i release. A, typical time course of [Ca2]i for non-transfected HEK293 cells at 33 °C when applying 1 mM menthol in the absence of extracellular Ca2. A rise in [Ca2]i was only observed during the first menthol application. The inset shows the chemical structure of menthol. B, the left panel shows the averaged [Ca2]i increase upon application of 1 mM menthol for non-transfected cells compared with TRPM8-transfected cells at 33 °C. The right panel compares the time needed to reach half-maximal rise upon menthol application. C, a typical experiment showing that TRPM8- transfected cells in Ca2-containing solution only display a rise in [Ca2]i upon exposure to low menthol concentrations at low temperatures. The upper panel shows the temperature course, the lower panel displays the [Ca2]i increase upon application of 10 M menthol. D, the same as in C, but for untransfected cells exposed to 1 mM of menthol. A rise in [Ca2]i is favored by high temperatures. E, dose-response curves at 23 and 33 °C, respectively, for the [Ca2]i increase upon application of different concentrations of menthol on TRPM8-trans- fected cells. F, same as in E but for non-transfected cells.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Transfection

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 4. Lack of endogenous TRPM8 expression in non-transfected HEK293 cells. Agarose gel electro- phoresis of mRNA products obtained after amplification of 3 different base pair sequences specific for TRPM8 (see Table 1) with reverse transcriptase-PCR. Evaluation of the constituvely expressed actin gene was included as a quality control for the cDNA.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Expressing, Transfection, Agarose Gel Electrophoresis, Amplification, Reverse Transcription, Control

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 6. Comparison of intracellular Ca2 release induced by different TRPM8 agonists. A, typical time course of [Ca2]i for non-transfected HEK293 cells at 33 °C when applying 3 mM eucalyptol in the absence of extracellularCa2.Theinsetshowsthechemicalstructureofeucalyptol.B,sameasinAbutwith20Micilin.The inset shows the chemical structure of icilin. C, same as in A but with 3 mM linalool. D, same as in A but with 3 mM geraniol. E, dose-response curves at 23 and 33 °C for the [Ca2]i increase upon application of different concen- trations of linalool in non-transfected HEK293 cells. The inset shows the chemical structure of linalool. F, same as in E but for geraniol. The inset shows the chemical structure of geraniol.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Comparison, Transfection

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A TRPM8 gene expression in melanoma tissues (red, T; n = 461 ) vs. normal skin (green, N; n = 558 ) from The Genomic Data Commons (GDC) Cancer Genome Atlas (TCGA) database. Data are presented as log2 TPM; p < 0.001 by unpaired t -test. B Representative westen blot showing TRPM8 protein expression in the indicated cell lines. Tubulin was used as a loading control. Immunofluorescence analysis of TRPM8 localization in WM266-4 ( C ) and AMM16 cells ( D ). Cells were stained with anti-TRPM8 antibody (blu), Endoplasmic reticulum (ER)-tracker (green) and Plasma Membrane-tracker (red). Merged images indicate TRPM8 distribution in both plasma membrane and ER compartments. Scale bar: 10 µm. Viability of AMM16 ( E ) and WM266-4 ( F ) cells untreated or treated with compounds 3, 5, 4, 6 and 9 at the concentrations indicated in the legends above. Absorbance values from WST-1 assays after 24, 48 and 72 h are reported. Data are expressed as mean ± standard deviations (SDs) from n independent experiments. * p < 0.05 for the indicated time points vs. the corresponding untreated control.

Article Snippet: The following antibodies were used in WB and Co-IP analyzes: the mouse monoclonal: anti-IκBα (L35A5) (#4814, Cell Signaling); anti-p53 (DO-1) (sc-126, Santa Cruz); anti-IKKα (3G12) (#11930, Cell Signaling); anti-α-tubulin (#E-AB-20036, Elabsciences); anti-GAPDH (#E-AB-20079, Elabsciences); the rabbit polyclonal: anti-phospho-ATM (Ser1981) (D25E5) (#13050, Cell Signaling); anti-phospho-p53 (Ser15) (#AF1043, R&D Systems); anti-phospho-AKT (Ser473) (#9271, Cell Signaling); anti-AKT (#9272, Cell Signaling); anti-phospho-GSK-3α/β (Ser21/9) (#9331, Cell Signaling); anti-caspase-3 (#9662, Cell Signaling); anti-PARP cleavage site (214/215) (#AB3565, Millipore); anti-PARP (#06-557, Upstate); anti-TRPM8 (#NBP1-97311, Novus Biologicals); anti-PMAIP1 (NOXA) (#A9801, Abclonal); the recombinant rabbit monoclonal anti-PUMA (#A3752, Abclonal); anti-ULBP1 (#A21161, Abclonal); anti-IKKβ (D30C6) (#8943, Cell Signaling); anti-phospho-IKKα/β (Ser176/180) (#2697, Cell Signaling); anti-phospho-NF-κB p65 (Ser536) (93H1) (#3033, Cell Signaling); anti-PI3K p85 (#06-195; Millipore, Burlington, Massachusetts, USA); anti-PI3K p110 δ (#AB1678; Abcam, Cambridge, UK).

Techniques: Gene Expression, Expressing, Control, Immunofluorescence, Staining, Clinical Proteomics, Membrane

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A Western blot analysis of WM266-4 cell lysates collected after 60 or 120 min of treatment with TRPM8 modulators, using the indicated antibodies. B WM266-4 cells were untreated (-) or treated for 120 min with TRPM8 antagonists (4 and 9, used at 10 µM). Lysate proteins were immune-precipitated using the anti-TRPM8 (anti-TRPM8) or control (ctrl IgG) antibodies. WB analysis using antibodies against the indicated proteins was done to reveal co-immunoprecipitated proteins. Western blot analysis of cleaved caspase-3 and cleaved PARP in AMM16 ( C ) and WM266-4 ( D ) cells treated with TRPM8 modulators at the indicated concentrations and hours. The α-tubulin was used as loading control. Graphs in the lower part of the figure represent the densitometric analysis of the cleaved PARP/tubulin ratio obtained in three different experiments ( n = 3 ).

Article Snippet: The following antibodies were used in WB and Co-IP analyzes: the mouse monoclonal: anti-IκBα (L35A5) (#4814, Cell Signaling); anti-p53 (DO-1) (sc-126, Santa Cruz); anti-IKKα (3G12) (#11930, Cell Signaling); anti-α-tubulin (#E-AB-20036, Elabsciences); anti-GAPDH (#E-AB-20079, Elabsciences); the rabbit polyclonal: anti-phospho-ATM (Ser1981) (D25E5) (#13050, Cell Signaling); anti-phospho-p53 (Ser15) (#AF1043, R&D Systems); anti-phospho-AKT (Ser473) (#9271, Cell Signaling); anti-AKT (#9272, Cell Signaling); anti-phospho-GSK-3α/β (Ser21/9) (#9331, Cell Signaling); anti-caspase-3 (#9662, Cell Signaling); anti-PARP cleavage site (214/215) (#AB3565, Millipore); anti-PARP (#06-557, Upstate); anti-TRPM8 (#NBP1-97311, Novus Biologicals); anti-PMAIP1 (NOXA) (#A9801, Abclonal); the recombinant rabbit monoclonal anti-PUMA (#A3752, Abclonal); anti-ULBP1 (#A21161, Abclonal); anti-IKKβ (D30C6) (#8943, Cell Signaling); anti-phospho-IKKα/β (Ser176/180) (#2697, Cell Signaling); anti-phospho-NF-κB p65 (Ser536) (93H1) (#3033, Cell Signaling); anti-PI3K p85 (#06-195; Millipore, Burlington, Massachusetts, USA); anti-PI3K p110 δ (#AB1678; Abcam, Cambridge, UK).

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A ) Core body temperature under cold conditions (4 °C). ( B ) The mRNA expression levels of TRPM8, TRPA1, NFκB and TNFα. ( C,D ) The protein expression levels of TRPM8, TRPA1, NFκB and TNFα. Data are shown as the mean ± S.D. from 12 mice in each group. ## v.s . the control (zero hour), P < 0.01; # P < 0.05.

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A ) Intracellular Ca 2+ in the cells under cold conditions (4 °C). The Ca 2+ concentration in the cytoplasm at 4 °C is higher than that at 37 °C. ( B ) The mRNA expression levels of TRPM8, TRPA1, NFκB and TNFα. ( C,D ) The protein expression levels of TRPM8, TRPA1, NFκB and TNFα. Data are shown as the mean ± S.D. from three experiments. # P < 0.05; ## P < 0.01, v.s. the control (zero time).

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Concentration Assay, Expressing, Control

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A ) Construction of a Trpm8 (Dylight 649) knockdown stable cell line. WT represents wild type cells. KD signifies the Trpm8 knockdown cells. ( B,C ) Protein expression levels of TRPM8, TRPA1, NFκB and TNFα. NS: no significance. Data are shown as the mean ± S.D. from three experiments. # P < 0.05; ## P < 0.01, v.s. the control (zero time).

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Knockdown, Stable Transfection, Expressing, Control

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A ) The expression of TRPM8, NFκB and TNFα in wild type cells and Trpm8 knockdown cells at 37 °C and 4 °C. KD signifies Trpm8 knockdown cells. ( B ) In wild type cells, TRPM8 was upregulated and NFκB and TNFα were downregulated under cold conditions. ( C ) In KD cells, TRPM8 showed weak expression and NFκB and TNFα expression levels were increased. ( D ) Co-localization of TRPM8 and TNFα in the cytoplasm (cold condition and 500 nM menthol). Data are shown as the mean ± S.D. from three experiments. * P < 0.05; ** P < 0.01.

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Expressing, Knockdown

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A ) Immunofluorescence assay image of TRPM8 and NFκB in the cytoplasm. WT represents wild type cells. KD represents Trpm8 knockdown cells. ( B ) The results of co-immunoprecipitation (CoIP) of endogenous TRPM8 and NFκB using NFκB antibodies. Western blot analysis was carried out to detect TRPM8 and NFκB. ( C ) Reverse CoIP confirmed interaction between NFκB and TRPM8. CoIPs were also performed with TRPM8 antibodies. Western blot analysis was carried out by TRPM8 and NFκB. Data are shown as the mean ± S.D. from three experiments. * P < 0.05; ** P < 0.01.

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Immunofluorescence, Knockdown, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: TRPM8 in the negative regulation of TNFα expression during cold stress

doi: 10.1038/srep45155

Figure Lengend Snippet: ( A,B ) NFκB in the cytoplasm and in the nucleus. ( A ) WT represents wild type cells. ( B ) KD represents Trpm8 knockdown cells. ( C,D ) Kinetic expression levels of NFκB in both WT and KD cells. C-P65 represents NFκBp65 in the cytoplasm. N-P65 represents NFκBp65 in the nuclei. ( E–H ) The mRNA expression levels of NFκB and TNFα after JSH-23 (8 μM), the inhibitor of NFκB, was applied. ( E , F ) WT cells. ( G , H ) KD cells. Data are shown as the mean ± S.D. from three experiments. ** P < 0.01, v.s. the control (zero time).

Article Snippet: For Western blot analysis, a primary antibody against TRPM8 (NBP1-97311, 127 kDa) was purchased from Novus Biologicals (Littleton, USA), TRPA1 (ab68847, 127 kDa), TNFα (ab199013, 17 kDa) antibodies were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China) and NFκB (SC-8008, 65 kDa) and β-actin (SC-47778, 43 kDa) antibodies were purchased from Santa Cruz Biotechnology Inc. (Texas, USA).

Techniques: Knockdown, Expressing, Control

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: ( A ) Simulation of the temperature dependence of ΔG during TRP channel activation with different ΔCp values. A positive ΔCp generates a convex curve, while a negative ΔCp produces a concave curve. The heat-sensing and cold-sensing regimes of the curves are indicated in red and blue, respectively. The ΔG-temperature profiles were simulated using the equation ΔG = ΔH 0 + ΔCp(T - T 0 ) - ΔS 0 T - TΔCp ln(T/T 0 ), where the values of positive ΔCp, negative ΔCp, T 0 , ΔH 0 and ΔS 0 were set to be 3.00 kcal/(mol . K), −3.00 kcal/(mol . K), 298.15 K (25 °C), −3.00 kcal/mol and –0.01 kcal/(mol·K), respectively. ( B ) The workflow for TRPM8 oxidation site determination. ( C ) Oxidation experiments were performed at least three times with biological replicates. Bar graph for the FCs showing significant changes in oxidation levels of TRPM8 peptides. FC >1.2 or FC <0.83 under 4 and 30 °C. FC fold change. Solid line, 1.0; Dash line, 1.2 and 0.83. All detected TRPM8 residues with FCs are shown. For residues detected more than three times in the oxidation experiments, data were presented as mean ± s.e.m. .

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Activation Assay

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: ( A ) Residues contributing to ΔCp were mutated to be either polar or hydrophobic. The magnitude of ΔCp influences the degree of curvature, with larger absolute values producing greater curvature. The van’t Hoff equation was used to determine ΔH and ΔS from a linear fit of lnK versus 1/T. As the absolute value of ΔCp increases, the slope of the linear fit becomes steeper, resulting in higher absolute values of ΔH and ΔS (gray region). ( B , C ) The workflow for TRPM8 spectral library generation and oxidation site determination and quantification. DIA data-independent acquisition. ( D ) Peptide fragmentation spectra from the spectral library. ( E ) Extracted ion chromatogram (XIC) groups (left: MS1, right: MS2) from the quantification results of an example peptide AFSTNEQDKDNWNGQLK.

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Data-independent acquisition

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: (Related to Fig. ). ( A , B ) Representative whole-cell current recordings of the TRPM8 channel activated by menthol before and after Fenton oxidation. ( C , D ) Representative emission spectra of ANAP incorporated at residue W436 and M1059, respectively. Emission spectra in gray and blue were measured at either 30 or 4 °C, respectively. ( E ) Comparison of shifts in emission spectra peak of ANAP incorporated at residue W436 and M1059 with their FC values measured from HRF-MS. (The y-axis represents the emission spectra peak of ANAP; n = 3 biological replicates; data were presented as mean ± s.e.m.).

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Residue, Comparison

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: ( A ) Left: representative temperature-driven activation of wild-type TRPM8 and its W137L mutant. Right: Measured ΔH (black bars, left axis) and ΔS values (light gray bars, right axis) of TRPM8 and the channel mutants ( n = 3–7 biological replicates; statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test for histograms. Data were presented as mean ± s.e.m.). ( B ) Correlation between SCH and ΔH values. For TRPM8, the W137L residue with more exposed sidechains in cold activation. The hydrophobicity scale of SCH was determined by Hessa et al. ( C ) Left: representative temperature-driven activation of wild-type TRPM8 and its W651L mutant. Middle: Van’t Hoff plots for the cold-activated TRPM8 currents shown in left. Right: Measured ΔH (black bars, left axis) and ΔS values (light gray bars, right axis) of TRPM8 and the channel mutants ( n = 3–7 biological replicates; statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test for histograms. Data are presented as mean ± s.e.m.). ( D ) Correlation between SCH and ΔH values. For TRPM8 W651L residue with more buried sidechains in cold activation. The hydrophobicity scale of SCH was determined by Hessa et al. ( E ) Correlation between SCH and ΔH values for TRPM8 residues with changes in FC values during cold activation. The hydrophobicity scale of SCH was determined by Hessa et al. The slope factors greater than 10 were plotted against the FC values of the corresponding sites. The first and third quadrants, where the sites followed the predictions from the water–protein interaction hypothesis of cold sensing, were shaded in blue. The second and fourth quadrants, where the sites did not follow the predictions from the water–protein interaction hypothesis of cold sensing, were shaded in gray. ( F ) The proportion consistent with the temperature-sensing hypothesis increased as the slope factor increased. The slope factor was calculated by fitting SCH and ΔH values to a linear function for each site with buried/exposed changes. The hydrophobicity scale of SCH was determined by Hessa et al (represented in blue) and Moon et al (represented in green), respectively. The x-axis represents the proportion that aligns with the temperature sensitivity hypothesis. The sites located in the first and third quadrants from panel ( E ) were mapped onto the cryo-EM structure of TRPM8 (PDB ID: 7WRA) with their sidechains shown in blue. ( G ) Representative whole-cell current recordings of TRPM8 mutants that regulate the cold-activated properties of TRPM8 in response to cold. Cold- and menthol-induced currents were measured at +80 mV to record TRPM8 channel activation. To estimate the open probability (Po), current amplitudes under saturating menthol stimulation (1 mM) were normalized, assuming maximal channel opening under these conditions. .

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Activation Assay, Mutagenesis, Residue, Cryo-EM Sample Prep

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: (Related to Fig. ). ( A – C ) Correlation between SCH and ΔH values for TRPM8 residues with changes in FC values during cold activation. The hydrophobicity values shown on the x-axis in ( A ) were based on the SCH hydrophobicity scale reported by Hessa et al, whereas those in Panels ( B ) and ( C ) were based on the scale reported by Moon et al. ( D ) The F912 mutation was excluded due to substantial differences in the hydrophobicity scales reported by Hessa et al and Moon et al.

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Activation Assay, Mutagenesis

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: (Related to Fig. ). ( A ) The proportion that aligns with the temperature sensitivity hypothesis increased as the slope factor increased. The slope factor was calculated by fitting SCH and ΔH values to a linear function for each site with buried/exposed changes. The hydrophobicity scale of SCH was determined by Moon et al. The data were then grouped into five classes, with absolute slope factor values greater than 0, 1, 2, and 4, respectively. The x-axis represents the proportion that aligns with the temperature sensitivity hypothesis. ( B ) Correlation between SCH and ΔH values for TRPM8 residues with changes in FC values during cold activation. The hydrophobicity scale of SCH was determined by Moon et al. The slope factor was plotted against the FC value of the corresponding site. The first and third quadrant, where the sites followed the predictions from the water–protein interaction hypothesis of cold sensing, were shaded in green. The second and fourth quadrant, where the sites did not follow the predictions from the water–protein interaction hypothesis of cold sensing, were shaded in gray. The sites located in the first and third quadrant were mapped onto the cryo-EM structure of TRPM8 with their sidechains shown in green. ( C ) Representative whole-cell current recordings of TRPM8 mutants that deviated from the hypothesis exhibited cold-activated properties.

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Activation Assay, Cryo-EM Sample Prep

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: (Related to Fig. ). ( A ) The location of MHR1-3 domains (colored in blue) in TRPM8. ( B ) Size-exclusion chromatography of the protein of MHR1-3 domains on Superose 6 (GE Healthcare) and SDS-PAGE. ( C ) The location of ARD domains (colored in pink) in TRPV2. ( D ) Size-exclusion chromatography of the protein of ARD on Superose 6 (GE Healthcare) and SDS-PAGE. ( E ) Representative CD spectra of ARD in TRPV2. ( F ) The temperature dependence of the CD spectra. ( G , H ) Representative intrinsic tryptophan emission spectra of ARD in TRPV2 and temperature dependence of intrinsic tryptophan emission peaks, respectively.

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Size-exclusion Chromatography, SDS Page, Circular Dichroism

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: ( A ) Representative CD spectra of the MHR1-3 domains in TRPM8 measured at different temperature levels. ( B ) Comparison of cold activation of TRPM8 current normalized to menthol activation (dots in blue) and changes in mean residue ellipticity (dots in black). ( C ) Representative intrinsic tryptophan emission spectra of the MHR1-3 domains measured at different temperature levels. The inlet showed the cooling-induced shifts in emission peak. ( D ) Temperature dependence of intrinsic tryptophan emission peaks values. ( E ) RMSD (relative to the TRPM8 structure in the apo state) plots of MD simulation trajectories at either 4 °C or 30 °C. All three RMSD curves stabilize within 100 ns. ( F ) The RMSF changes of TRPM8 were observed at 4 and 30 °C, respectively. ( G ) The fractional changes in RMSF (RMSF at 4 minus 30 °C) normalized to RMSF at 4 °C. Dashed lines indicated the 20% significance in fluctuation level, below which any fluctuation was due to nonspecific thermal effects (Wen and Zheng, ). ( H ) The temperature-dependent changes (4 °C minus 30 °C) in the radius of gyration (Rg) for each residue in TRPM8. ( I ) Representative snapshots of MD trajectories showing the buried/exposed state of W137 (residue in blue) and the water molecules nearby (oxygen and hydrogen atoms in water colored in red and white, respectively) at 4 and 30 °C. .

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Circular Dichroism, Comparison, Activation Assay, Residue

Journal: EMBO Reports

Article Title: Rational tuning of temperature sensitivity of the TRPM8 channel

doi: 10.1038/s44319-025-00630-2

Figure Lengend Snippet: ( A ) Representative whole-cell current recordings of cold activation for W137 and its mutants, along with the correlation between SCH and ΔH values for the W137 residue. The hydrophobicity scale of SCH was determined by Hessa et al. ( B ) Transcriptome-wide comparison of mRNA expression between wild-type and W137H mice. ( C ) Representative inside-out patch recordings of DRG neurons in wild-type (left panel) and W137H (right panel) mutant mice activated by cold and 1 mM menthol. ( n = 3 biological replicates; statistical significance was determined using a two-sided Student’s t -test. Data were presented as mean ± s.e.m.). ( D ) Normalized currents of DRG neurons in wild-type and W137H mutant mice. ( n = 3 biological replicates; statistical significance was determined using a two-sided Student’s t -test. Data were presented as mean ± s.e.m.). ( E ) Mice were allowed to move freely in a two-temperature choice test with a control plate (30 °C) and a test plate (ranging from 5 to 30 °C). The percentage of time spent at the control plate was measured every 3 min ( n = 10 biological replicates; statistical significance was determined using a two-sided Student’s t -test. Data were presented as mean ± s.e.m.). ( F ) Representative heat maps (left panel) and traces (right panel) of WT and W137H mice in two-temperature choice assays. ( G ) Workflow of the hypothesis-driven study on temperature-induced mechanisms in the TRPM8 channel. The workflow of this hypothesis-driven study, where the hypothesis testing was composed of two major steps: the detection of temperature-induced buried/exposed changes and the functional validation. Upon cold activation of TRPM8, the intracellular MHR regions were the first to sense the temperature change, initiating conformational shifts in the channel that result in its opening. .

Article Snippet: W137H- trpm8 mice in the C57BL/6J background were commissioned by Cyagen Biosciences, Inc. (Hangzhou, China).

Techniques: Activation Assay, Residue, Comparison, Expressing, Mutagenesis, Control, Functional Assay, Biomarker Discovery

Journal: Indian Journal of Biochemistry and Biophysics

Article Title: Downregulation of TRPM8 channels induce cell death in human DBTRG glioblastoma cells

doi: 10.56042/ijbb.v60i5.950

Figure Lengend Snippet: Fig. 1 — Determination of TRPM8 channel expression levels in siRNA-transfected groups. Bright field images (200x) have shown that relative cellular density decreased in transfected group and normal in control group after 48 h transfection period. Relative intensity of western blotting bands of control and TRPM8 siRNA induced groups has shown in bar graph. a: P< 0.001 vs. the control group

Article Snippet: The TRPM8 siRNA (sc-95009) and Control siRNA (sc-36869) were purchased from Santa Cruz Biotechnology, USA.

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Indian Journal of Biochemistry and Biophysics

Article Title: Downregulation of TRPM8 channels induce cell death in human DBTRG glioblastoma cells

doi: 10.56042/ijbb.v60i5.950

Figure Lengend Snippet: Fig. 4 — The (A) caspase 3; (B) caspase 8; and (C) caspase 9 enzyme activity findings of control and TRPM8 downregulated groups. a: p <0.001 vs. the Control group (mean ± SD; n=6)

Article Snippet: The TRPM8 siRNA (sc-95009) and Control siRNA (sc-36869) were purchased from Santa Cruz Biotechnology, USA.

Techniques: Activity Assay, Control

Journal: Indian Journal of Biochemistry and Biophysics

Article Title: Downregulation of TRPM8 channels induce cell death in human DBTRG glioblastoma cells

doi: 10.56042/ijbb.v60i5.950

Figure Lengend Snippet: Fig. 6 — Determination of apoptosis and cell viability of DBTRG cells. (A) Apoptosis findings of control and TRPM8 downregulated groups. a: P< 0.001 vs. the Control group (mean ± SD; n=6); (B) The 96-well plate image of AMTB hydrochloride incubated DBTRG cells; and (C) Effects of different doses of AMTB hydrochloride incubation for 24 h on cell viability parameter in DBTRG cells. n.s.: no significance with Control group. There is also not significance between 10 µM and 25 µM group. a: P< 0.001 vs. the Control group, b: P< 0.005 vs. the 5 µM group, c: P< 0.001 vs. the 5 µM group (mean ± SD; n=8)

Article Snippet: The TRPM8 siRNA (sc-95009) and Control siRNA (sc-36869) were purchased from Santa Cruz Biotechnology, USA.

Techniques: Control, Incubation