Journal: The Korean Journal of Pain

Article Title: Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

doi: 10.3344/kjp.2014.27.4.326

Figure Lengend Snippet: Effect of a nefopam pretreatment on the transient receptor potential ankyrin 1 (TRPA1) and melastatin 8 (TRPM8) protein expression levels in the dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). To evaluate the expressions of TRPA1 (A) and TRPM8 (B) proteins located in the L4-L6 DRG, a Western blot analysis was performed 4 weeks after the final drug administration. The specific signals for TRPA1 and TRPM8 were quantified and plotted (lower panel). β-actin was used as an internal loading control. STZ injections did not significantly affect the basal level of the TRPA1 protein in the DRG, which was not altered by the nefopam pretreatment. The TRPM8 protein level in the DRG significantly increased 4 weeks after the STZ injection, which was reduced by the nefopam pretreatment. *** P < 0.001 compared to the control group. ††† P < 0.001 compared to the STZ group.

Article Snippet: After blocking with 5% goat serum, 0.3% Triton X-100, and 1% bovine serum albumin in phosphate-buffered saline (PBS-T), double-label immunofluorescence was conducted by incubating the paraffin sections overnight at 4°C with rabbit polyclonal antibodies against TRPM8 (1:1000, Abcam), with the IgG fraction of the mouse polyclonal antibody against the N52 clone of neurofilament 200 (NF200, 1:200; Sigma-Aldrich), followed by incubation with Alexa Flour 555 goat anti-rabbit IgG (red; 1:500; Invitrogen, Carlsbad, CA, USA) and Alexa Flour 488 goat anti-mouse IgG (green; 1:500; Invitrogen) for 2 hours at 20°C.

Techniques: Expressing, Injection, Western Blot, Control

Journal: The Korean Journal of Pain

Article Title: Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

doi: 10.3344/kjp.2014.27.4.326

Figure Lengend Snippet: Typical double immunofluorescence staining of transient receptor potential melastatin 8 (TRPM8) and neurofilament 200 (NF200) in the rat dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Four weeks after the final drug treatment, L5 DRG samples were immunostained with TRPM8 and neurofilament 200 (NF200), a marker of myelinated neurons. The colocalization of TRPM8 was visualized in a merged image.

Article Snippet: After blocking with 5% goat serum, 0.3% Triton X-100, and 1% bovine serum albumin in phosphate-buffered saline (PBS-T), double-label immunofluorescence was conducted by incubating the paraffin sections overnight at 4°C with rabbit polyclonal antibodies against TRPM8 (1:1000, Abcam), with the IgG fraction of the mouse polyclonal antibody against the N52 clone of neurofilament 200 (NF200, 1:200; Sigma-Aldrich), followed by incubation with Alexa Flour 555 goat anti-rabbit IgG (red; 1:500; Invitrogen, Carlsbad, CA, USA) and Alexa Flour 488 goat anti-mouse IgG (green; 1:500; Invitrogen) for 2 hours at 20°C.

Techniques: Double Immunofluorescence Staining, Injection, Marker

Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 4 TRPM8 level determination and TRPM8-silencing cell function experiments. (A–D) TRPM8 inhibition in PLC/PRF/5 and HUH-7 cells transfected with shTRPM8 lentivirus was verified by Western blot and qRT-PCR. (E–I) Effect of TRPM8 loss-of-function on PLC/RPF/5 and HUH-7 cell proliferation was determined by colony formation assay (E), CCK-8 (F-G) and EdU assay (H, I). (J, K) The effects of TRPM8 inhibition on the cell migration and invasion of PLC/PRF/5 and HUH-7 cells were detected using Transwell assays. Scale bars: 100 μm. *p < 0.05.

Article Snippet: Primary antibodies against TRPM8 (Zen- bioscience, Chengdu, China), RTP3 (Epigentek, USA), STAT3 (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were employed.

Techniques: Cell Function Assay, Inhibition, Transfection, Western Blot, Quantitative RT-PCR, Colony Assay, CCK-8 Assay, EdU Assay, Migration

Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 6 AD80 promoted the expression of TRPM8 and inhibited the proliferation, migration and invasion of HCC cells in vitro. (A, B) Calcium imaging was used to detected the changes of the fluorescence intensity of WS12 (A) and AD80 (B) groups on time in SNU-449 cells. (C, D) Effects of WS12 (C) and AD80 (D) on the expression of TRPM8 in SNU-449 cells were detected by Western blot. (E-G) EdU (E) and CCK-8 assays (F) were used to detect the effect of WS12 on the proliferation of SNU-449. (G) The effects of WS12 on the cell migration and invasion of SSNU-449 cells were detected using Transwell assays. (H–J) EdU (H) and CCK-8 assays (I) were used to detect the effect of WS12 on the proliferation of SSNU-449. (J) The effects of AD80 on the cell migration and invasion of SNU-449 cells were detected using Transwell assays. Scale bars: 100 μm. *p < 0.05.

Article Snippet: Primary antibodies against TRPM8 (Zen- bioscience, Chengdu, China), RTP3 (Epigentek, USA), STAT3 (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were employed.

Techniques: Expressing, Migration, In Vitro, Imaging, Fluorescence, Western Blot, CCK-8 Assay

Journal: Cancer medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

doi: 10.1002/cam4.70109

Figure Lengend Snippet: FIGURE 7 Schematic depicting the possible mechanism of the TRPM8- mediated RTP3/STAT3 pathway in HCC.

Article Snippet: Primary antibodies against TRPM8 (Zen- bioscience, Chengdu, China), RTP3 (Epigentek, USA), STAT3 (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were employed.

Techniques:

Journal: Neuroscience

Article Title: Diminished Cold Avoidance Behaviours after Chronic Cold Exposure - Potential Involvement of TRPM8.

doi: 10.1016/j.neuroscience.2021.06.014

Figure Lengend Snippet: Fig. 1. Schematic summary of experiments. Male C57BL/6 mice were acclimated for four weeks to appropriate thermal conditions in two separate climate chambers by gradually lowering the ambient temperature (Ta) to 6 C (Cold group; indicated by blue), or increasing to thermoneutral temperature 27 C (Thermoneutral group; indicated by red). After acclimation, mice were assigned to three different experiments as indicated: First, thermosensory preference behaviour testing using Two Plate Choice Test and Thermal Gradient Test. Second, injections with either TRPM8 agonist icilin or vehicle prior to Thermal Gradient Test, and third, Evaluation of cold acclimation responses including thermography imaging and molecular analyses of TRPM8 and UCP1 expression in dorsal root ganglion (DRG) and interscapular brown adipose tissue (iBAT) homogenates, respectively.

Article Snippet: Then the membranes were incubated at 4 C over night with a fluorescent antibody against TRPM8 (REF#NBP1-97311C; TRPM8 monoclonal antibody conjugated with Dylight 650 fluorochrome, Novus Biologicals, USA) diluted 1:2000 in Immobilon Block-FL, or with a primary mouse antibody against UCP1 (REF # MAB6158, R&D Systems) diluted 1:1000 in Immobilon Block-FL, which was detected using a 1:5000 dilution of fluorescent Goat anti-Mouse secondary antibody (Alexa Fluor 647, # A32728, Thermo Fisher Scientific, USA).

Techniques: Imaging, Expressing

Journal: Neuroscience

Article Title: Diminished Cold Avoidance Behaviours after Chronic Cold Exposure - Potential Involvement of TRPM8.

doi: 10.1016/j.neuroscience.2021.06.014

Figure Lengend Snippet: Fig. 4. The effect of TRPM8 agonist icilin on thermal preference of cold-acclimated mice. Mice were acclimated at cold or thermoneutral temperature for four weeks, and then injected with icilin or vehicle (DMSO and saline). (A) WDS behaviours were counted for 30 min after the injection. The results are plotted at 5-minute time intervals. There was no significant differ- ence between the Cold + Icilin and Ther- moneutral + Icilin groups in the number of WDSs at any time-point (n = 6 in each group). Icilin injection increased WDSs in both groups, whereas vehicle had no effect (n = 6 in icilin- and n = 5 in vehi- cle-injected groups; ***P < 0.001). (B) In parallel, jumping behaviours were regis- tered. There was no significant difference between the two groups in the number of jumps after icilin injection (F1, 5 = 1.594, P = 0.2625) (n = 6 in each group). The bars represent jumping behaviours 0– 15 min after icilin injection, as they ceased completely after 15 min. (C, D) Thermal place preference behaviours were assessed using the Thermal Gradient Test defined by ten virtual zones (mid- zone temperature is indicated). Mice in the Cold + Vehicle group showed less cold avoidance compared with the mice in the Thermoneutral + Vehicle group (n = 5 in each group; *P < 0.05 and **P < 0.01). Icilin injection enhanced the cold avoid- ance behaviours of Thermoneutral + Icilin and Cold + Icilin group (n = 6 in each group; *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the vehicle- injected groups). (E) The effect of icilin in different test zones is displayed relative to the vehicle alone (baseline or 0%). Ther- moneutral + Icilin group showed a signif- icantly greater change in cold avoidance after icilin injection than the Cold + Icilin group. (*P < 0.05, **P < 0.01 and ***P < 0.001). Data are expressed as mean ± SEM.

Article Snippet: Then the membranes were incubated at 4 C over night with a fluorescent antibody against TRPM8 (REF#NBP1-97311C; TRPM8 monoclonal antibody conjugated with Dylight 650 fluorochrome, Novus Biologicals, USA) diluted 1:2000 in Immobilon Block-FL, or with a primary mouse antibody against UCP1 (REF # MAB6158, R&D Systems) diluted 1:1000 in Immobilon Block-FL, which was detected using a 1:5000 dilution of fluorescent Goat anti-Mouse secondary antibody (Alexa Fluor 647, # A32728, Thermo Fisher Scientific, USA).

Techniques: Injection, Saline

Journal: Neuroscience

Article Title: Diminished Cold Avoidance Behaviours after Chronic Cold Exposure - Potential Involvement of TRPM8.

doi: 10.1016/j.neuroscience.2021.06.014

Figure Lengend Snippet: Fig. 5. The effect of cold acclimation on levels of TRPM8 cold sensor in DRG. (A) western blot shows TRPM8 band (140 kDa) detected in tissue homogenates of dorsal root ganglion (DRG) and TG as a positive control, but not liver as a negative control. Total protein stain was used to control gel loading. (B) Blocking peptide (the amino acid sequence corresponding to the antibody TRPM8 epitope) dose dependently blocked immune detection of TRPM8 in DRG homo- genates. From left to right: unblocked control (1:2000 TRPM8 antibody); + 1:2000 blocking peptide; + 1:400 blocking peptide; and + 1:200 blocking peptide. Each dilution of blocking peptide was run on different gels and blotted separately. Then, one lane from each blot were cut, put together and imaged (C) western blot analyses of L4 and L5 DRG tissue homogenates. TRPM8 levels were significantly higher in the Thermoneutral group compared to the Cold group (F1,

Article Snippet: Then the membranes were incubated at 4 C over night with a fluorescent antibody against TRPM8 (REF#NBP1-97311C; TRPM8 monoclonal antibody conjugated with Dylight 650 fluorochrome, Novus Biologicals, USA) diluted 1:2000 in Immobilon Block-FL, or with a primary mouse antibody against UCP1 (REF # MAB6158, R&D Systems) diluted 1:1000 in Immobilon Block-FL, which was detected using a 1:5000 dilution of fluorescent Goat anti-Mouse secondary antibody (Alexa Fluor 647, # A32728, Thermo Fisher Scientific, USA).

Techniques: Western Blot, Positive Control, Negative Control, Staining, Control, Blocking Assay, Sequencing

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: 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: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

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 Representative Western blot showing TRPM8 protein expression in the indicated cell lines. Tubulin was used as loading control. Viability of human melanocytes ( B ) and human dermal fibroblasts ( C ) untreated or treated with compounds 4 and 9 at the concentrations indicated in the legends on the right. Absorbance values from WST-1 assays at 24, 48, and 72 h are shown. Data are presented as mean ± SD of three independent experiments. n.s . indicates not significant. Representative Live/Dead assay images of human melanocytes ( D ) and human dermal fibroblasts ( E ) treated for 24 h with compounds 4 and 9 (1 or 10 μM). Viable cells are shown in green (acridine orange; total cells), while dead cells are shown in red (propidium iodide; dead cells). Overlay images are shown. Scale bar, 100 μm. Quantification of cell death is displayed to the right of each overlay image. The percentage of dead cells was calculated as: (red-stained dead cells/green-stained total cells) × 100.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Western Blot, Expressing, Control, Live Dead Assay, Staining

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: Representative images from Live/Dead assays in AMM16 ( A ) and WM266-4 ( D ) melanoma cells treated for 24 h with TRPM8 modulators 4 and 9 (1 µM). Total cells are stained in green (acridine orange), while dead cells are labeled in red (propidium iodide). Scale bar: 100 µm. Quantification of cell death in AMM16 ( B , C ) and WM266-4 ( E , F ) cells treated with compounds 4 and 9 at 1 or 10 µM, respectively, for 24 h. The percentage of dead cells was calculated as: (dead red-stained cells/total green-stained cells) × 100. Representative images of AMM16 ( G ) and WM266-4 ( I ) melanoma cells untreated or treated for 6 h with TRPM8 modulators 4 and 9 (1 or 10 μM) and stained with Annexin V–FITC. Contrast phase (CP), Annexin V–FITC, and merged images (overlay) are shown. Scale bar, 100 μm. Quantification of Annexin V–FITC fluorescence in AMM16 ( H ) and WM266-4 ( L ) cells untreated or treated as in G , I , measured using a multiwell fluorescence reader (excitation/emission settings optimized for FITC as reported in Methods). Data represent mean ± SD from n independent experiments. Data in B , C , E , F , H , L are presented as mean ± SD from n = 3 independent experiments. Statistical significance was determined. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Staining, Labeling, Fluorescence

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 – C WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TRPM8-targeting siRNA (siRNA TRPM8). A Western blot analysis was performed on total cell lysates using the indicated antibodies. B Cells were then left untreated or treated with compound 4 or 9 (1 μM, 24 h), and cell death was assessed by PI staining. Total cells were stained in green with acridin orange. C Quantification of PI-positive cells corresponding to ( B ). Representative Western blots showing transient TRPM8 overexpression (TRPM8 OE) in WM266-4 ( D ) and AMM16 ( F ) melanoma cells. E , G Densitometric analysis of TRPM8 and GAPDH protein levels, represented as TRPM8/GAPDH ratios (from three independent experiments). WM266-4 ( H ) and AMM16 ( I ) cells transfected with control plasmid (ctrl plasmid) or TRPM8 plasmid (TRPM8 OE) were left untreated or treated with compounds 4 and 9 (1 or 10 μM) for 6 h. Representative images (contrast phase, PI staining, and overlays) and corresponding quantitative graphs (below panels) are shown. Scale bar, 100 μm. In C , E , G , H , I Data are presented as mean ± SD from n independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. In H , I Red asterisks indicate statistical significance between ctrl and OE groups at the corresponding treatment conditions.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Transfection, Control, Western Blot, Staining, Over Expression, Plasmid Preparation

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 Representative immunofluorescence images showing intracellular ROS levels in AMM16 and WM266-4 cells untreated or treated for 4 h with TRPM8 modulators 4 and 9 (1 or 10 µM). ROS were detected as reported in Methods section. Scale bar: 100 µm. B , C Quantification of intracellular ROS levels in AMM16 and WM266-4 cells treated as indicated, in the absence or presence of the antioxidants α-tocopherol (Toc) or the mitochondrial ROS scavenger MitoTEMPO. Fluorescence intensity was measured using a TECAN plate reader and expressed as fold increase over the basal levels. D Representative images of mitochondrial ROS levels in AMM16 and WM266-4 cells treated for 1.5 h with TRPM8 modulators 4 and 9 (1 or 10 µM). Mitochondrial ROS were detected using a mitochondria-specific ROS probe. Scale bar: 75 µm. E , F Quantification of mitochondrial ROS levels under the same treatment conditions described in D , with or without antioxidant co-treatment. Fluorescence intensity was measured using a TECAN plate reader and expressed as fold increase over the basal levels. G Viability of WM266-4 cells treated for 24 h with compounds 4 and 9 (1 or 10 µM) in the absence or presence of α-tocopherol or MitoTEMPO. Viability is expressed as fold change relative to untreated controls. Data in B , C , E – G are shown as mean ± standard deviation (SD) from n = 3 independent experiments. Statistical analysis was performed. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the corresponding untreated control.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Immunofluorescence, Fluorescence, Standard Deviation, Control

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 , D WM266-4 cells were treated with TRPM8 modulators, as indicated, for 6 h. A Representative phase contrast and fluorescence microscopy images. JC-1 staining reveals mitochondrial depolarization following TRPM8 inhibition, as indicated by a fluorescence shift from red (JC-1 aggregates in polarized mitochondria) to green (JC-1 monomers in depolarized mitochondria). Scale bar, 10 μm. B Quantification of red-to-green fluorescence ratio, calculated as described in the Methods section. Data are presented as mean ± SD from three independent experiments ( n = 3 ). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the corresponding untreated control. C Western blot analysis of cell lysates obtained after 24 h of treatment, using the indicated antibodies. D Representative confocal microscopy images showing cytochrome c (green) localization and mitochondria (violet) in melanoma cells. Fluorescence signals were acquired using identical exposure settings. Scale bar, 10 μm.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Fluorescence, Microscopy, Staining, Inhibition, Control, Western Blot, Confocal Microscopy

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 cell lysates collected after 7 h of treatment with TRPM8 modulators, using the indicated antibodies. B Immunofluorescence analysis of cells left untreated or treated with TRPM8 modulators (10 μM, 7 h), stained for γH2AX (red punctate nuclear foci) and nuclei (blue). Overlayed images are shown. Scale bar, 2.5 μm. C Western blot analysis of cell lysates using the indicated antibodies. D p53 localization was expressed as percentage of cells showing a nuclear localization. Means and SDs are shown; n represents the number of experiments. *** P < 0.001 for the indicated experimental points versus the corresponding untreated control cells. E Representative Immunofluorescence images of WM266-4 cells left untreated or treated with TRPM8 modulators (10 μM, 7 h), stained for p53 (red) and nuclei (blue) and quantified in D . Overlayed images are shown. Scale bar, 10 μm. In B , E fluorescence signals were acquired using identical exposure settings.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Fluorescence

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: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Western Blot, Control, Immunoprecipitation

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 Representative images of crystal violet-stained colonies, derived from WM266-4 cells, after 21-day treatment with TRPM8 modulators. B Western blot analysis showing ULBP1 expression in WM266-4 cells after treatment with TRPM8 modulators. The α-tubulin was used as a loading control. C WM266-4 derived spheroids treated for 21 days as indicated, in absence (upper panel; -NK cells) or presence (lower panel; + NK cells) of NK cells. Images are representative of three different experiments. Bar, 100 µm. D The graph represents the dead cells/total cells. Values of dead (red stained cells) and total cells (green stained cells) were analyzed using NIH Image J. They derive from red fluorescence mean/green fluorescence mean intensity and are expressed as mean ± SD of 3 different experiments ( n = 3); ** p < 0.01; *** p < 0.001. E NK cell cytotoxicity assay. WM266-4 cells pre-treated with TRPM8 modulators were co-cultured with primary NK cells at the indicated effector:target (E:T) ratios. Where indicated, neutralizing antibodies against ULBP1 or NKG2D were added 1 h before co-culture to melanoma cells or NK, respectively. Data are presented as percentage of lysis. * p < 0.05; ** p < 0.01. F WM266-4 cells were transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53) at two different concentrations (300 pmol and 400 pmol, respectively). After 4 days, cells were collected, lysed, and Western blot analysis was performed on cell lysates using the indicated antibodies. α-Tubulin was used as a loading control. G WM2664 cells transfected with control siRNA (siRNA ctrl) or TP53-targeting siRNA (siRNA p53; 300 pmol) were unstimulated or stimulated with compounds 4 and 9 (at 1 μM) for 4 days and then collected and lysed. Western blot analysis was performed using the indicated antibodies. α-Tubulin was used as a loading control. H Phosphorylated AKT (Ser473) levels in melanoma cells treated with the PI3K agonist, 740 Y-P, in absence or presence of TRPM8 modulators (used at 1 μM) for 4 days, analyzed by Western blot. GAPDH was used as a loading control.

Article Snippet: They were co-cultured with melanoma cells pre-treated with TRPM8 modulators for 21 days, at effector-to-target (E:T) ratios of 25:1 and 50:1, for 8 h. Where indicated, blocking antibodies against NKG2D (sc-53501) or ULBP1 (#A21161; Abclonal) were added 60 min prior to co-culture.

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Control, Fluorescence, Cytotoxicity Assay, Cell Culture, Co-Culture Assay, Lysis, Transfection

Journal: Cancer Medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3 / STAT3 pathway

doi: 10.1002/cam4.70109

Figure Lengend Snippet: TRPM8 regulated the RTP3/STAT3 pathway in HCC. (A‐B) Heat map showed 50 genes positively correlated gene (A) and negatively correlated gene with TRPM8 (B). (C) The co‐expression network of TRPM8 in hepatocellular carcinoma. (D) The co‐expression network of TRPM8 in adjacent liver tissues. (E–H) TRPM8‐related genes were screened by qRT‐PCR and RTP3 was found. (I–L) RTP3 protein expression in TRPM8 overexpressing and silencing cells. (M) The effects of TRPM8 overexpressing HCC cells on the levels of RTP3 and STAT3 were detected by Western blot. (N) The effects of RTP3 overexpressing HCC cells on the levels of STAT3 and TRPM8 were detected by Western blot. (O)Western blot analysis of RTP3/STAT3 axis in TRPM8‐overexpressing HCC cells, and RTP3 overexpression reversed the effect ofTRPM8 on the STAT3.

Article Snippet: Primary antibodies against TRPM8 (Zen‐bioscience, Chengdu, China), RTP3 (Epigentek, USA), STAT3 (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were employed.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Cancer Medicine

Article Title: TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3 / STAT3 pathway

doi: 10.1002/cam4.70109

Figure Lengend Snippet: Schematic depicting the possible mechanism of the TRPM8‐mediated RTP3/STAT3 pathway in HCC.

Article Snippet: Primary antibodies against TRPM8 (Zen‐bioscience, Chengdu, China), RTP3 (Epigentek, USA), STAT3 (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) were employed.

Techniques:

Journal: International Journal of Oncology

Article Title: Androgen regulated TRPM8 expression: A potential mRNA marker for metastatic prostate cancer detection in body fluids

doi: 10.3892/ijo_00000518

Figure Lengend Snippet: Figure 3. Expression of TRPM8 in prostate cancer cell lines: total RNA or whole cell extracts prepared from exponentially growing LNCaP, BPH-1, DU145, and PC-3 cells were subjected to RT-PCR (A) or Western blot analysis (B), respectively to determine TRPM8, AR, and PSA expression as described in Materials and methods.

Article Snippet: Individual membranes were probed with monoclonal antibodies against TRPM8 (Abcam, Cambridge, MA), AR, PSA or ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Oncology

Article Title: Androgen regulated TRPM8 expression: A potential mRNA marker for metastatic prostate cancer detection in body fluids

doi: 10.3892/ijo_00000518

Figure Lengend Snippet: Figure 5. Expression of TRPM8, AR and PSA in synchronized LNCaP cells progressing from G0/G1 to S phase. Exponentially growing LNCaP cells synchronized by isoleucine-deprivation were released into complete media. At 6-h intervals after releasing from isoleucine-block, cells were pulse-labeled with [3H]-thymidine (3H-TdR) and the incorporation of 3H-TdR into DNA was measured to monitor the progression of cells from G1 to S phase (A). Total RNA and whole cell extracts prepared at the same intervals were subjected to RT-PCR (B) and Western blot analysis (C), respectively, to determine TRPM8, AR, and PSA levels as described in Materials and methods. E, exponentially growing cells.

Article Snippet: Individual membranes were probed with monoclonal antibodies against TRPM8 (Abcam, Cambridge, MA), AR, PSA or ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Blocking Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Grancalcin Accelerates Wound Healing by Improving Angiogenesis in Diabetes.

doi: 10.1002/advs.202305856

Figure Lengend Snippet: Figure 5. TRPM8 is a receptor of GCA in endothelial cells. A) TRPM8 protein binding to GCA was identified by LC-MS/MS analysis. B) TRPM8 expression in HUVECs was determined by Western blotting after incubation with normal or high glucose. Immunoprecipitation (IP) analysis of Flag-TRPM8 C) and Myc-GCA D) binding. GCA and siRNA-Trpm8 treatments induced functional changes in HUVECs (n = 3), including migration, tube formation, and expression of various angiogenic factors. Live cell images E) were captured 12 h after the wound-healing assay (scale bars, 100 μm), and the wound-

Article Snippet: [58] The membranes were probed with specific antibodies against GCA (PA577127, 1:1000, Invitrogen), TRPM8 (DF7966, 1:2000, Affinity), ERK (4695T, 1:2000, Cell Signaling Technology), p-ERK (4370S, 1:2000, Cell Signaling Technology), HIF-1α (3281T, 1:2000, Cell Signaling Technology), and VEGF (50661S, 1:2000, Cell Signaling Technology), and GAPDH (5174, 1:2000, Cell Signaling Technology), and subsequently reprobed with the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Incubation, Immunoprecipitation, Binding Assay, Functional Assay, Migration, Wound Healing Assay