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: TRPM8 expression in different datasets. (A) The mRNA expression level of TRPM8 was lower in HCC cells and Primary liver tissues than that in normal tissues in the MERAV. (B) The expression of TRPM8 in GEO database dataset GSE25097. (C) The expression of TRPM8 in GEO database dataset GSE54236. (D) The expression of TRPM8 in GEO database dataset GSE76427.(E) The expression of TRPM8 in different HCC datasets. (F) Radar map of TRPM8 overall expression among different types of tissues.

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

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: Relationship between TRPM8 expression and clinical value in hepatocellular carcinoma. (A) Western blot results showed that TRPM8 protein level in HCC tissues was lower than that in paired normal tissues. (B) TRPM8 expression in HCC adjacent tissues and tumour tissues tested by IHC. (C)The expression of TRPM8 was negatively correlated with tumour grade. (D)The expression of TRPM8 was negatively correlated with nodal metastasis status. (E) The low expression of TRPM8 in Kaplan–Meier Plotter is associated with poor overall survival. (F) The low expression of TRPM8 in Kaplan–Meier Plotter is associated with disease free survival. Scale bars: 250 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, Western Blot

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: Effect of TRPM8 in inhibiting HCC cell proliferation in vitro and in vivo. (A, B) The protein and mRNA level of TRPM8 in HCC cells as determined using western blot and qRT‐PCR analyses, respectively. (C, D) TRPM8 overexpression was confirmed by western blot and qRT‐PCR after transfection with TRPM8‐overexpressing plasmids. (E–G) The effect of TRPM8 overexpression on HCC cell proliferation was determined by colony formation assays (E) and CCK‐8 (F, G). (H, I) SNU‐449 and HLE cells were seeded onto coverslips and DNA synthesis was assessed via EdU immunofluorescence staining. The graph on the left depicts the percentage of EdU‐positive nuclei. (J, K) The effects of TRPM8 overexpression on the cell migration and invasion of SNU‐449 and HLE cells were detected using Transwell assays. (L–N) Effect of TRPM8 gain‐of‐function in SNU‐449 cells on subcutaneous tumour growth (L). Tumour volumes (M). The mice were sacrificed 28 d after cell injection and the tumours were removed and weighed (N). 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: In Vitro, In Vivo, Western Blot, Quantitative RT-PCR, Over Expression, Transfection, CCK-8 Assay, DNA Synthesis, Immunofluorescence, Staining, Migration, Injection

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 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: 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: 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: 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: 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: PLOS ONE

Article Title: Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

doi: 10.1371/journal.pone.0289395

Figure Lengend Snippet: A . Agarose gel electrophoresis of PCR products from MEG-01 cells, using two sets of TRPM8 primers. Arrows indicate the size of the expected amplicons: 379 bp for the 1410F/1788R primer set, and 607 bp for the 1410F/2017R set. P1 and P2 indicate unique MEG-01 passages. The preparation was tested for contamination by using mock samples including the primers and the PCR reaction kit without cDNA. B . Anti-TRPM8 western blot of MEG-01 cell lysate using the primary anti-TRPM8 antibody (#NBP1-97311, raised against the peptide corresponding to the intracellular epitope) and the secondary goat anti-rabbit HRP-conjugated antibody. C . Confocal images of MEG-01 immunostaining with anti-TRPM8 (yellow; primary antibody raised against the peptide corresponding to the extracellular epitope + goat anti-rabbit secondary), Hoechst nuclear staining (blue), integrin IIb/IIIa (CD41, magenta), DIC (gray) and TRPM8 + nucleus overlay. The scale bar is 10 μm. (D-G) Calcium imaging of Fluo4-AM loaded MEG-01 cells. D . TRPM8-dependent calcium influx in MEG-01 cells. Average responses to TRPM8 agonists over time from responsive MEG-01 cells. Vehicle EtOH 0.1% (gray), menthol 500 μM (blue), WS-12 4 μM (magenta), icilin 100 μM (orange). Fluo4-AM fluorescence (F), baseline (F 0 ) corrected and normalized to F max obtained after ionophore 23187 application. Thick lines indicate average and dotted lines indicate SEM. The shaded area indicates the duration of agonist application. The delay in the WS-12 response might be due to the delay in perfusion combined with the low working concentration. E. Average response over time to ADP 50 μM. F . Percent MEG-01 cells showing positive (dark gray) and negative (light gray) responses to agonists. G . Change in calcium levels from individual “positive” cells in response to agonists: vehicle EtOH 0.1% (dark gray), menthol 500μM (blue), WS-12 4 μM (magenta), icilin 100 μM (orange), and ADP 50 μM (light gray). Error bars indicate mean ± SEM. Symbols above scatter graphs indicate p-values from unpaired Student’s t-test comparison with vehicle (ns–p > 0.05, *–p < 0.05, **–p < 0.01, ***–p < 0.005).

Article Snippet: Where specified, prior to staining, the primary anti-TRPM8 antibody was incubated with the corresponding blocking peptide (Cat # BLP-CC049, Alomone Labs, Israel) at a 1:1 ratio at room temperature for 1hr.

Techniques: Agarose Gel Electrophoresis, Western Blot, Immunostaining, Staining, Imaging, Fluorescence, Concentration Assay, Comparison

Journal: PLOS ONE

Article Title: Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

doi: 10.1371/journal.pone.0289395

Figure Lengend Snippet: (A-F) Flow cytometry analysis of human platelets in plasma. Cell count vs fluorescence intensity histograms of unstained (A) , stained with only secondary antibody (B) and anti-TRPM8 primary with secondary FITC-conjugated antibody staining (C) . TRPM8 (+) platelets defined based on secondary control samples, brackets indicate the TRPM8-positive platelets. 100,000 events were measured for each sample. D . Percent of TRPM8 positive platelets from several different healthy donors (n = 7). E . Representative images of random CD41 and TRPM8-positive platelets from one healthy donor (out of five) by imaging flow cytometry. 20,000 events were measured for each sample. The scale bar is 7 μm. F . Percentage of spheroid and discoid cells within TRPM8-positive (+) and -negative (-) platelet populations. Platelets were assigned spheroid or discoid shapes as described in (Özpolat et al., 2023). (G-H) Confocal microscopy of anti-TRPM8 immunostaining of washed platelets on poly-lysine coated glass coverslips. G . Representative images of platelets from two healthy donors stained with primary anti-TRPM8 and secondary antibodies (left) or secondary-only control (right). The white outline indicates the footprint of the platelet as identified by the R18 membrane stain. The scale bar is 1 μm. H . Background subtracted average pixel intensity (AU) of platelets from two healthy donors (stained with primary anti-TRPM8 and secondary antibodies (268 cells) or secondary only control (142 cells). Error bars indicate Mean ± SEM. Asterisks indicate the p-value <0.001 from the unpaired t-test with Welch’s correction for unequal distribution comparing background subtracted ROI intensities.

Article Snippet: Where specified, prior to staining, the primary anti-TRPM8 antibody was incubated with the corresponding blocking peptide (Cat # BLP-CC049, Alomone Labs, Israel) at a 1:1 ratio at room temperature for 1hr.

Techniques: Flow Cytometry, Cell Counting, Fluorescence, Staining, Control, Imaging, Confocal Microscopy, Immunostaining, Membrane

Journal: PLOS ONE

Article Title: Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

doi: 10.1371/journal.pone.0289395

Figure Lengend Snippet: A. Expression of the active form of integrin αIIbβ3 on platelets from blood collected at 22˚C (blue symbols), 37˚C (pink), and exposed to 4˚C (green). Samples were either treated with vehicle (DMSO, filled symbols) or TRPM8 inhibitor PF 05105679 (2 μM, abbreviated as PF, open symbols). Samples were isolated from the blood of healthy volunteers (n = 3 to 5) who denied taking any platelet function-modifying medications for 14 days prior to the experiment. Analyses were performed using flow cytometry on LSR II. Platelets in PRP were distinguished by expression of CD61 using anti-CD61 PerCP-conjugated antibody. For each sample, 10,000 CD61 positive events were acquired. CD61 positive events were identified by gating approximately 1% of the appropriate isotype antibody in the positive gate. B . Percent of platelet microaggregates in PRP samples analyzed using imaging flow cytometry, n = 4. ( C and D ) representative images of two groups of platelet shape ( C— spheroid cells, D —discoid cells). Samples were stained against integrin αIIbβ3 (PAC-1, green), p-Selectin (CD62P, yellow), and CD61 (red) fluorescent antibodies, and analyzed by imaging flow cytometry. The scale bar is 7 μm. ( E and F ). Percent spheroid ( E ) and discoid ( F ) shaped platelets in the PRP samples from blood collected at 22˚C (blue symbols), 37˚C (pink), and exposed to 4˚C (green), n = 4. Samples were analyzed using imaging flow cytometry. Platelets were assigned spheroid or discoid shapes as described in (Özpolat et al., 2023). Lines connecting data points indicate the same donor. Statistical analysis was performed using paired Student t-test, where asterisks indicated a p-value lower than 0.05 for *, 0.01 for **, and 0.005 for *** respectively, and “ns” indicates a p-value >0.05. Symbols above bars indicate paired comparison between treatment groups, and without bars indicate comparison to vehicle.

Article Snippet: Where specified, prior to staining, the primary anti-TRPM8 antibody was incubated with the corresponding blocking peptide (Cat # BLP-CC049, Alomone Labs, Israel) at a 1:1 ratio at room temperature for 1hr.

Techniques: Expressing, Isolation, Medications, Flow Cytometry, Imaging, Staining, Comparison

Journal: PLOS ONE

Article Title: Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

doi: 10.1371/journal.pone.0289395

Figure Lengend Snippet: PRP isolated from blood collected by venipuncture at 37˚C (pink background) or 22˚C (white background) was subjected to light transmission aggregometry measured with stirring at 37˚C. A. ADP-induced aggregation normalized to ADP-only maximum (dotted line indicates 1). PRP from healthy donors was pre-treated with either vehicle DMSO (filled symbols) or PF 05105679 (2 μM, open symbols) for 5 minutes, then treated with either vehicle ETOH, menthol (500 μM, blue), WS-12 (2 μM, purple) or icilin (100 μM, orange) in presence of 1mM Ca 2+ for 5 more minutes. Subthreshold ADP concentration (previously identified for each donor (0.8 to 4 μM) as less than 60% maximal aggregation) was added and maximal aggregation was measured (for 37˚C n = 6, for 22˚C n = 10). Arrow indicates a hyper-responsive subject represented in B. B. Aggregation over time traces for the donor with especially strong WS-12 response. Subthreshold ADP was added at the time indicated by arrows. C. Effect of TRPM8 inhibitors on ADP-induced aggregation. PRP was pre-treated with either vehicle DMSO (filled squares), PF 05105679 (2 μM, open circles, n = 10) or AMTB (10 μM, open diamonds, n = 5). D. Effect of TRPM8 inhibitors on collagen-induced aggregation (n = 5). Subthreshold collagen concentration (previously identified for each donor (0.25 to 1 μg/ml) as less than 60% maximal aggregation). Values were normalized to the maximum aggregation achieved by collagen alone (dotted line). E. Effect of TRPM8 inhibitors on the convulxin-induced aggregation (n = 5). Subthreshold convulxin concentration (previously identified for each donor (1 to 10 ng/ml) as less than 60% maximal aggregation). Values were normalized to the maximum aggregation achieved by convulxin alone (dotted line). F. Effect of TRPM8 inhibitors on the SOCE-induced aggregation (n = 5). PRP was pre-treated with thapsigargin (5 μM) for 10 minutes prior to the addition of 1 mM Ca 2+ (no other aggregation agonists were used). Raw percent aggregation values are reported. Lines connecting data points indicate the same donor. Statistical analysis was performed using paired Student t-test, where asterisks indicated a p-value lower than 0.05 for *, and “ns” indicates s p-value >0.05. Symbols above bars indicate paired comparison between treatment groups, and without bars indicate comparison to vehicle.

Article Snippet: Where specified, prior to staining, the primary anti-TRPM8 antibody was incubated with the corresponding blocking peptide (Cat # BLP-CC049, Alomone Labs, Israel) at a 1:1 ratio at room temperature for 1hr.

Techniques: Isolation, Transmission Assay, Concentration Assay, Comparison