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℃ 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℃.

Techniques: Expressing, Injection, Western Blot

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℃ 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℃.

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