Journal: bioRxiv

Article Title: Small-Molecule Modulators of Lipid Raft Stability and Protein-Raft Partitioning

doi: 10.1101/2024.10.28.620521

Figure Lengend Snippet: A) The average current traces of TRPM8 menthol response before and after exposure to the compounds in stably expressing full-length human TRPM8 HEK293T cells. Each dot is when the pulse program is applied. No perfusion was done in the first 5 minutes of the experiment to allow the automated patch clamp plate to equilibrate to 30 °C. Menthol was applied at 100 µM for 75 seconds 4 times to allow the menthol response to saturate before applying either 0.03% DMSO (control), 10 µM VU0619195, (See Supp. Fig. 8A for VU0607402, PD and MBCD). 0.03% DMSO concentration was kept consistent throughout the experiment. Menthol was then applied at 100 µM with the corresponding compounds twice for 75 seconds. Each n refers to the single sum of 20 cells from an amplifier on the automated patch clamp ensemble plate. B) The average ratio of menthol response from each compound in TRPM8 HEK293T cells. The ratio of menthol response uses the data from panel A and (Supp. Fig 8A), where the last two menthol response before compound application were averaged and compared to the two menthol response after compound application. Each n refers to the single sum of 20 cells from an amplifier on the automated patch clamp ensemble plate and are jittered. P-values were determined by ANOVA followed by Dunnett’s tests. Bars are means ± SD. C) Results of Western blot analysis of phospho EGFR (left) and ERK (right) from HeLa cells treated with compounds for 15 min then stimulated with EGF for 1 min. n =3, bars are means ± SD. All comparisons are not significant.

Article Snippet: Cells were then labeled with either of two TRPM8 primary antibodies targeted to an extracellular epitope (Alomone, Cat. No. ACC-049 Abcepta, Cat. No. AP8181D).

Techniques: Stable Transfection, Expressing, Automated, Patch Clamp, Control, Concentration Assay, Western Blot, Analysis

Journal: PLoS ONE

Article Title: TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

doi: 10.1371/journal.pone.0143171

Figure Lengend Snippet: (A) TRPM8 mRNA expression by semi-quantitative PCR in superior mesenteric artery with endothelium removed (n = 3). C, control; P1, 1 μL prostate RT reaction; P2, 2 μL prostate RT reaction, Me, 2 μL mesenteric artery RT reaction. (B) Western blot analysis of TRPM8 protein expression in rat vascular tissue. Molecular weight of TRPM8 was 125 KDa. Bottom panel: β actin used to demonstrate protein loading (at 42kDa).

Article Snippet: The membrane was blocked with 5% (wt/vol) milk in TBS containing 0.1% Tween 20 (TBST) for 1 h at room temperature, followed by overnight incubation at 4°C with the specific primary rabbit polyclonal antibody against TRPM8 (1:1000 dilution; Abcam, Cambridge, Massachusetts, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Molecular Weight

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: 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