Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A GST pull-down assays to assess the interaction between purified GST-tagged C-terminus of TRPM8 (GST-M8C) expressing in E. coli BL21 bacteria and different HA-tagged Src family kinases expressing in HEK293T cells. Western blotting (WB) was performed using the indicated antibodies. B – D Co-immunoprecipitation (Co-IP) assays. B HeLa cells were transfected with the indicated constructs along with Flag-tagged full-length TRPM8 (Flag-TRPM8). Immunoprecipitation (IP) was performed with an anti-HA or anti-Flag antibody, and the samples were analyzed by immunoblotting with the indicated antibodies. C Similar Co-IP in ( B ) but with protein extracts from HeLa cells co-transfected with the indicated constructs along with GFP-tagged C-terminus of TRPM8 (GFP-M8C). D Co-IP as in ( B ) and ( C ) but with protein extracts from native PANC-1 cells. E Representative confocal imaging of co-localization of mcherry-LCK and GFP-TRPM8 in HeLa cells. Overlay images show co-localization of green signals (TRPM8) and red signals (LCK), which generated yellow signals in HeLa. Nuclei were stained with DAPI (blue). Scale bars, 10 µm. F Assay of the interaction in vitro between purified His-LCK fusion and GST-tagged C-terminus of TRPM8 (GST-M8C) from E. coli bacteria. G , H HEK293T cells co-expressing HA-LCK constructs with a series of mutant Flag-tagged cytoplasmic domain of TRPM8 were harvested for Co-IP assays. All studies were repeated at least three times. GFP, green fluorescent protein. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Purification, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Imaging, Generated, Staining, In Vitro, Mutagenesis

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A – E Electrophysiological analysis of LCK on whole-cell TRPM8 currents (I TRPM8 ) by patch-clamping experiments. A – C Expression constructs for Flag-TRPM8 and pEGFP-N1 were co-transfected with HA-BLK, HA-LCK, HA-LYN, or control vector into HEK293T cells. EGFP-positive cells were selected for recording I TRPM8 ( n = 5~20 cells per group). The voltage clamp protocol is shown in the inset of figure. A Representative imaging of I TRPM8 . B The relationship of average I TRPM8 densities (I TRPM8 normalized to cell capacitance) and voltage. C Quantification of peak I TRPM8 density on +80 mV as in ( B ). D , E Similar I TRPM8 recordings as in ( A ) but HEK293T cells co-expressing Flag-TRPM8 and pEGFP-N1 with siLCK or negative scramble siRNAs. ( n = 5~20 cells per group). D The relationship of average I TRPM8 densities and voltage. E Quantification of peak I TRPM8 density on +80 mV as in ( D ). F – I Cell‐surface biotinylation assays for detecting TRPM8 PM expression. F Representative WB images of TRPM8 on the PM and total lysates from HEK293T cells co-transfected with or without HA-LCK. G Quantification of PM protein expression levels of TRPM8 in ( F ). H , I Similar experiments in ( F ) and ( G ) but cells co-expressing with siLCK or negative scramble siRNAs. ITGA5 (Intergrin α5) was used as a loading control for PM proteins. *** P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Imaging

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A – D The effect of LCK on the intracellular N-C binding of TRPM8. A , B Expression constructs for Flag-tagged N-terminus of TRPM8 (Flag-M8N) and GFP-M8C were co-transfected with HA-LCK or control vector into HEK293T cells, before harvest for treatment with 10 μM PP2 for 24 h. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the intracellular N-C binding of TRPM8. C , D Similar experiments in ( A ) and ( B ) but cells co-expressing Flag-M8N and GFP-M8C along with siLCK. E – L The effect of LCK on the multimerization of TRPM8. E , F Expression constructs for Flag-TRPM8 were co-transfected with HA-LCK or control vector into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min, a crosslinking agent. The cell lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. G , H Similar experiments in ( E ) and ( F ) but cells co-expressing Flag-TRPM8 along with siLCK. I , J Flag-TRPM8 and GFP-TRPM8 were co-transfected with or without HA-LCK into AsPC-1 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. K , L Similar experiments in ( I ) and ( J ) but cells co-expressing Flag-TRPM8 and GFP-TRPM8 along with siLCK. ** P < 0.01, *** P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Binding Assay, Expressing, Construct, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A MS imaging of phosphotyrosine site of TRPM8 in combination with the NCBI blast (peptide sequences are indicated). B Amino acid sequence alignment showing that tyrosine at position 1022 is highly conserved among multiple species. C , D Expression constructs for Flag-tagged wild-type TRPM8 (Flag-WT-M8) or mutant Y1022F (Flag-M8-Y1022F) were transfected with or without HA-LCK into HEK293T cells, before harvest for treatment with 10 μM saracatinib for 24 h. The lysates were then used for IP with an anti-Flag antibody and then subjected to WB assay with the indicated antibodies to detect the level of TRPM8 phosphotyrosine. E , F Kinase assay in vitro. Purified GST alone, GST tagged wild-type or mutant of C-terminus of TRPM8 fusion proteins expressing in E. coli bacteria were mixed with HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK construct, 1 mM ATP or their combination in kinase assay buffer to determine the level of M8C phosphotyrosine. G Relationship of test potential and averaged densities of I TRPM8 recorded from HEK293T cells co-transfected Flag-WT-M8 or Flag-M8-Y1022F with pEGFP-N1. H Peak current density on +80 mV as in G ( n = 15~20 cells per group). I , J Expression constructs for Flag-WT-M8 or Flag-M8-Y1022F were co-transfected with or without HA-LCK into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min for WB to detect the level of TRPM8 multimerization. K , L Flag-WT-M8 or Flag-M8-Y1022F along with GFP-tagged wild-type TRPM8 (GFP-WT-M8) were co-transfected with or without HA-LCK into AsPC-1 cells to determine the binding of intermolecular TRPM8. *** P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Imaging, Sequencing, Expressing, Construct, Mutagenesis, Transfection, Kinase Assay, In Vitro, Purification, Immunoprecipitation, Binding Assay

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A GST pull-down assay. Purified GST alone or GST-M8C fusion proteins expressed in E. coli BL21 bacteria were incubated with the lysates from HEK293T cells expressing GFP-14-3-3ζ constructs and subjected to WB assay. B Co-IP assay. The lysates of native PANC-1 cells were added to an anti-14-3-3 antibody for IP and then subjected to WB assay with an anti-TRPM8 antibody. C , D Expression constructs for Flag-TRPM8 with or without GFP-14-3-3ζ were transfected into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min. The lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. E , F Similar experiments in ( C ) and ( D ) but cells co-expressing Flag-TRPM8 along with human 14-3-3ζ-specific siRNAs (si14-3-3ζ#1, #2, or #3). G , H AsPC-1 cells were co-transfected Flag-TRPM8 and GFP-TRPM8 with or without si14-3-3ζ, and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. ** P < 0.01, *** P < 0.001. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Pull Down Assay, Purification, Incubation, Expressing, Construct, Co-Immunoprecipitation Assay, Transfection, Binding Assay

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A , B PANC-1 cells were co-transfected Flag-TRPM8 with HA-LCK or control vector and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of TRPM8 and 14-3-3ζ. C , D Similar experiments in ( A ) and ( B ) but cells co-expressing Flag-TRPM8 along with siLCK. E , F Expression constructs for Flag-WT-TRPM8 or Flag-TRPM8-Y1022F were co-transfected with or without HA-LCK into HEK293T cells to determine the binding of TRPM8 and 14-3-3ζ. G , H PANC-1 cells were co-transfected with Flag-TRPM8, HA-LCK, si14-3-3ζ (#1, #2 or #3) or their combination, before harvest for treatment with 1 μM DSS for 30 min, and subjected to WB assay to detect the level of TRPM8 multimerization. I , J AsPC-1 cells were co-transfected with Flag-TRPM8 and GFP-TRPM8, HA-LCK, si14-3-3ζ or their combination, and then harvested for IP with an anti-Flag antibody and subjected to determine the binding of intermolecular TRPM8. *** P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Expressing, Construct

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A , B LCK phosphorylation assay. PANC-1 cells were co-transfected HA-LCK with the control vector, WT-TRPM8 or mutant TRPM8-Y1022 with the indicated antibodies as shown in ( A ), and quantification of the level of LCK phosphorylation shown in ( B) . C – F LCK ubiquitination assay. C , D Expression constructs for HA-LCK and Myc-Ub were co-transfected with control vector, WT-TRPM8 or mutant TRPM8-Y1022 into AsPC-1 cells. The cells were treated with 10 μM MG132 for 6 h before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. E , F Expression constructs for Myc-Ub and WT-TRPM8 or mutant TRPM8-Y1022F were co-transfected with HA-tagged wild-type or mutant LCK into PANC-1 cells. The cells were treated with 10 μM MG132 before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. *** P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Phosphorylation Assay, Transfection, Plasmid Preparation, Mutagenesis, Ubiquitin Assay, Expressing, Construct

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: A – D Cell proliferation assays in vitro. A , B The RFP labeled cell lines of PANC-1 or AsPC-1 cells stably expressing control vector, WT-TRPM8, or mutant TRPM8-Y1022F were constructed and used for EdU incorporation assays (Upper panel) and Ki67 immunofluorescence (Lower panel). Scale bars, 100 µm. C , D Colony formation assays were performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 µm. E – I Cell migration assays. Scale bars, 200 µm. E , F Wound-healing assay was performed in RFP labeled AsPC-1 stably maintained cells. G , H Transwell assay was performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 µm. I – L Animal xenotransplantation engraftment experiments. I Representative confocal microscopy images of 6 days xenotransplantation of zebrafish injecting with RFP labeled PANC-1 stably maintained cells. Scale bars, 200 µm. J Imaging of tumors excised from the mice subcutaneously injecting RFP labeled PANC-1 stably maintained cells by growth for 5 weeks. K Quantification of the expression of TRPM8 mRNA in ( J ). L Weights of the excised tumors in each group in ( J ). M Growth curves showing the changes in the tumor volume in mice in different groups every 5 days from the injection. N Representative H&E staining images and immunohistochemical images of Ki67 in excised tumors tissues. Scale bars, 20 µm. O Quantification of Ki67 expression in ( N ). ** P < 0.01, *** P < 0.001. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: In Vitro, Labeling, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay, Confocal Microscopy, Imaging, Injection, Staining, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

doi: 10.1038/s41419-022-04977-5

Figure Lengend Snippet: Schematic diagram of the biological role of the LCK-14-3-3ζ-TRPM8 axis in the regulation of TRPM8 function and LCK activity.

Article Snippet: Rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503, PTGCN), anti-phosphotyrosine (p-Tyr) (P4110, Sigma), anti-phosphothreonine (#9391, Cell Signaling Technology), anti-phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R& D), anti-TRPM8 (#ACC-049, Alomone, Israel), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

Techniques: Activity Assay

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 1 Rapamycin activates recombinant TRPM8 channels and potentiates cold-evoked responses. (a) Average ± SEM Fura2 ratio time course in HEK293 cells stably expressing rat TRPM8 (CR#1 cells) during application of rapamycin (RAP) at different concentrations. A single dose was applied in individual experiments (n = 38 to 185 cells, eight experiments). (b) Dose– response curve of rapamycin effects. Data shown are means ± SEM obtained from individual peak amplitudes from experiments shown in a. (c) Average ± SEM Fura2 ratio time course in HEK293 cells stably expressing rat TRPM8 during a protocol in which rapamycin responses were monitored in the presence (2.4-mM Ca2+) and in the absence (0 Ca2+) of extracellular calcium (n = 103 cells). (d) Bar histogram summarizing the amplitude ± SEM of rapamycin responses in the presence and absence of extracellular Ca2+ (n = 257 cells, five experiments). Data shown are means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by Bonferroni post- hoc test. (e) Average ± SEM Fura2 ratio of cold-evoked responses in CR#1 HEK293 cells in control and 30-μM rapamycin (n = 98). (f) Bar histogram summarizing the effect of vehicle or 1-, 10-, 30-μM rapamycin on cold-evoked responses. Individual cold responses have been normalized to the amplitude during the first cold ramp (71 to 92 cells per condition). Ratios >20 have been excluded from the analysis. Data shown are individual values with means ± SEM. *P<0.05, significantly different from vehicle; one-way ANOVA followed by Bonferroni post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Recombinant, Stable Transfection, Expressing, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 2 Rapamycin activates mouse and human TRPM8 orthologues and the menthol-insensitive mutant. (a) Average ± SEM Fura2 ratio time course in HEK293 cells expressing mouse TRPM8. Three different behaviours were observed; cells which responded to a first cold ramp in control conditions (blue trace), cells which did not respond to this cold ramp in control conditions but responded to cold in the presence of rapamycin (RAP) or menthol (Ment; green trace), and cells which did not show any response to the applied stimuli (black trace). (b) Bar histogram summarizing the individual and mean responses of the cold-sensitive HEK293 cells (n = 162, five experiments). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA followed by Bonferroni post-test. (c) Average ± SEM Fura2 ratio time course in HEK293 cells expressing human TRPM8. The different traces represented the three behaviours described in a. (d) Bar histogram summarizing the responses of the cold-sensitive HEK293 cells (n = 178, five experiments) transfected with human TRPM8. Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA followed by Bonferroni post-test. (e) Average ± SEM Fura2 ratio time course in HEK293 cells expressing mouse TRPM8-Y745H. The different traces represented the three behaviours described in a. (f) Bar histogram summarizing the responses of the cold-sensitive HEK293 cells (n = 182, five experiments) transfected with mouse TRPM8-Y745H. Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated, n.s., not significant; one-way ANOVA followed by Bonferroni post-test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Mutagenesis, Expressing, Control, Transfection

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 3 Rapamycin (RAP) activates TRPM8 currents. (a) Representative time course of whole-cell current at 100 and +100 mV in a HEK293 cell transiently transfected with mTRPM8 during the sequential application of different agonists. The bottom trace shows the simultaneous recording of the bath temperature. (b) Current–voltage (I-V) relationship obtained by a 400-ms voltage ramp from 100 to +150 mV during the experiment shown in (a). The colour of individual traces matches the colour at each particular time point in (a). (c) Bar histogram of current density values at 100 and +100 mV evoked by the different stimuli shown in (a), with the same colour code (n = 9 cells). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA, followed by Bonferroni's post-hoc test. (d) V1/2 values calculated from fitting individual I-V curves to a linearized Boltzmann equation. Data shown are individual values with means ± SEM. *P<0.05, significantly different from control, &P<0.05, significantly different as indicated; one-way ANOVA, followed by a Bonferroni's post-hoc test. (e) Representative whole-cell current at 100 and +100 mV in a HEK293 cell transiently transfected with mTRPM8 during a protocol in which the effect of AMTB on rapamycin response was explored. Bottom trace corresponds to the simultaneous recording of bath temperature. (f) Current–voltage relationship (I-V) obtained with a 400 ms voltage ramp from 100 to +150 mV of the responses plotted in e. the colour of the I-V curves matches the coloured time points in e. (g) Bar histogram of individual and mean ± SEM current density values at +100 mV evoked by the different stimuli shown in ( e). Data shown are individual values with means ± SEM; n = 5 cells. *P<0.05, significantly different from control, &P<0.05, significantly different as indicated; one-way ANOVA, followed by Bonferroni's post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Transfection, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 4 Rapamycin activates single TRPM8 channels. (a) Single channel recordings at +100 mV in the cell-attached configuration in HEK293 cells transfected with mTRPM8. The same patch in control (i.e., high K+), rapamycin (RAP; 30 μM) and WS-12 (10 μM). The coloured ticks mark the expanded traces shown below. (b) All-point histograms of current amplitudes for the different recording conditions. The histograms have been fitted with the sum of two Gaussians (red line). (c) Single channel amplitudes of individual patches at different membrane potentials. The dotted lines represent linear fits to the data. (d) Open probability of channel current at different potentials. The dotted lines are fits to the Boltzmann function.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Transfection, Control, Membrane

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 6 TRPM8 is the principal determinant of rapamycin responses in mouse DRG neurons. (a) Representative traces of Fura2 ratio fluorescence in a Trpm8EGFPf/+ DRG culture. Consecutive applications of cold, rapamycin (RAP, 30 μM), menthol (100 μM), AITC (100 μM), capsaicin (100 nM) and high K+ (30 mM) were used to phenotype each neuron. The green trace corresponds to a EGFPf(+) neuron, which responds to cold, rapamycin, menthol and capsaicin. The other two traces (blue and red) correspond to EGFPf() neurons. Bottom trace shows the simultaneous recording of bath temperature. (b) Venn diagram summarizing the responses to rapamycin in EGFPf(+) and EGFPf() neurons in Trpm8EGFPf/+ DRG cultures. (c) Bar histograms of the amplitude of the responses to different agonists in EGFPf(+) rapamycin-sensitive neurons (n = 20, five experiments). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by a Bonferroni's post-hoc test. (d) Correlation between the amplitude of the cold-evoked response and the rapamycin response (n = 20, six experiments). The horizontal dotted line marks the threshold level for rapamycin sensitivity. Green circles represent the three EGFPf(+), cold-sensitive, rapamycin-insensitive neurons recorded. (e) Representative traces of Fura2 ratios in cultured neurons from Trpm8 KO mouse. The protocol was the same as in (a). Orange trace corresponds to a EGFPf(+) neuron that was rapamycin- sensitive. Green trace represents a EGFPf(+) neuron which was not sensitive to any of the agonists tested and the red and blue traces are examples of two EGFPf() neurons. Bottom trace corresponds to the simultaneous recording of the bath temperature during the experiment. (f) Venn diagram summarizing the responses to rapamycin in EGFPf(+) and EGFPf() neurons in Trpm8EGFPf/EGFPf DRG cultures (eight experiments).

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Fluorescence, Cell Culture

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 7 Rapamycin (RAP) increases the excitability of cold-sensitive DRG neurons. (a) Representative whole-cell recording in the voltage- clamp configuration (Vhold = 60 mV) of a TRPM8-expressing, cold-sensitive DRG neuron identified by EYFP expression. Bottom trace corresponds to the simultaneous recording of bath temperature. Rapamycin activates a small inward current and potentiates the response to cold. (b) Temperature dependence of the cold-evoked current in the same neuron in control conditions and in the presence of 30 μM rapamycin. (c) Bar histogram of peak inward current density values evoked by cold, rapamycin and cold in the presence of rapamycin (n = 12). Data shown are individual values with means ± SEM. #P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by Bonferroni's post-hoc test. (d) Representative recording of a cold-sensitive neuron in the whole-cell current-clamp configuration. Cold and rapamycin elicited the firing of action potentials. The combined application of cold and rapamycin led to faster firing, followed by a strong depolarization and the blockade of spikes. (e) Bar histogram of responses, measured as average firing frequency, during the different stimuli applied (n = 11). Firing frequency for cold was the average from the first to the last spike during the cooling ramp. Firing frequency in control conditions was calculated during the 60 s previous to rapamycin application. Rapamycin-evoked firing was calculated from the first spike during rapamycin application to the start of the cold ramp. *P<0.05, significantly different from control, #P<0.05, significantly different as indicated; one- way ANOVA for repeated measures followed by Bonferroni's post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Expressing, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 9 Mathematical modelling of the effects of rapamycin (RAP) on cold thermoreceptor activity. (a) Membrane potential (blue trace), temperature (red trace), and action potential firing frequency (blue dots), as a function of time. The green bar indicates the period of application of agonist (i.e. a change of -100 mV in V1/2 in this example). The red dots in the temperature trace indicate where the temperature threshold has been reached. The black dashed lines in the lower panel indicate the value of the average firing frequency, and the time range where it has been calculated (i.e. the duration of the temperature ramps). (b) Firing temperature threshold as a function of the shift in V1/2. Each trace corresponds to a cell with different parameters, which have been selected in order to encompass a wide range of thresholds under control conditions. The larger dot corresponds to the cell and conditions simulated in panel (a). (c) Same as in panel B for the average firing frequency during cold stimulation. (d) TRPM8 current density (blue trace) as a function of time, and changes in temperature (red trace) in voltage-clamp simulations. The green horizontal bar represents the application of agonist, i.e. a shift of -100 mV in V1/2 as in (a). (e) TRPM8 current density as a function of temperature with (green trace) or without agonist (blue trace). (f) Peak cold-evoked current as a function of the shift in V1/2 for the same cells as shown above with the corresponding colours. Values for V1/2 = 0 correspond to the cold-evoked current in the absence of agonist. The magenta line corresponds to a neuron with very small TRPM8 currents that did not fire action potentials and thus is not shown in the current-clamp panels above. Color code is the same for panels b, c, e and f. The set numbers in panel f correspond to the row numbers within the table found in Extended Data Figure 11-1 of Rivera et al. (2021), detailing the simulation parameter values.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Activity Assay, Membrane, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 10 Rapamycin stimulates tearing by a TRPM8-dependent mechanism. Effect of topical solutions of rapamycin (RAP; 1%) or vehicle on tearing in mice. Tearing is represented as the length of staining in the threads (in mm) placed in the eye. Each dot corresponds to the tearing measured in an eye of an individual mouse (15 WT and 15 Trpm8 KO). Data shown are individual values of evoked tearing, with means ± SEM. *P < 0.05, significantly different as indicated; n.s., not significant; Mann– Whitney test).

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Staining, MANN-WHITNEY

Journal: Neurology: Genetics

Article Title: Trigeminal Neuralgia TRPM8 Mutation

doi: 10.1212/NXG.0000000000000550

Figure Lengend Snippet: Average changes in the Fura-2 ratio of human embryonic kidney 293 cells transfected with wild-type TRPM8 (A) and R30Q mutant (B), in the continued presence of menthol (100 μM; arrow indicates time of addition). (C) Average increase in Fura-2 ratio in response to menthol (100 μM) in nontransfected cells (NT), wild-type TRPM8, and R30Q transfected cells (n = 10 experiments, 4 transfections). * p < 0.05, unpaired Student's t test. (D) Basal fura-2 ratio in wild-type TRPM8 and R30Q mutant transfected cells (n = 15 experiments, 4 transfections). * p < 0.05, unpaired Student t test. TRPM8 = transient receptor potential melastatin 8.

Article Snippet: The wild-type TRPM8 plasmid was obtained from GenScript, in which the insert was cloned in-frame with 2A-GFP at the C-terminus of the channel in the vector pcDNA3.1.

Techniques: Transfection, Mutagenesis

Journal: Neurology: Genetics

Article Title: Trigeminal Neuralgia TRPM8 Mutation

doi: 10.1212/NXG.0000000000000550

Figure Lengend Snippet: (A) Representative whole-cell current traces through wild-type TRPM8 and R30Q transfected cells, in response to the indicated voltage step protocol. (B) Steady state current-voltage relationships of the basal whole-cell currents for wild-type TRPM8 and R30Q mutant (n = 12, ** p < 0.01, *** p < 0.001, 2-way analysis of variance with Bonferroni post hoc test). (C) Steady-state activation curves of wild-type TRPM8 and R30Q transfected cells. The normalized conductance (G/Gmax) was plotted against voltage and fitted with a Boltzmann function, giving rise to V 1/2 and slope factor as follows: TRPM8, 153 ± 8 mV and 30 ± 3 mV (n = 12 cells, 3 transfections); R30Q, 115 ± 5 mV and 28 ± 2 mV (n = 12 cells, 3 transfections). ** p < 0.01, unpaired Student's t test. (D) Representative time courses (left) recorded at +80 mV and −80 mV (black and grey curves, respectively) and I-V traces (right) of whole cell currents through wild-type TRPM8 transfected cells, in the presence of 100 μM menthol, at the indicated time intervals. (E) Same as D), except that time courses and I-V traces are recorded from R30Q transfected cells. (F) Pooled data of whole-cell current (at +80 mV and −80 mV) evoked by 100 μM menthol, from wild-type TRPM8 and R30Q transfected cells. Each column represents mean ± SEM of n = 8 cells, 3 independent experiments. * p < 0.05 (unpaired Student's t -test). TRPM8 = transient receptor potential melastatin 8.

Article Snippet: The wild-type TRPM8 plasmid was obtained from GenScript, in which the insert was cloned in-frame with 2A-GFP at the C-terminus of the channel in the vector pcDNA3.1.

Techniques: Transfection, Mutagenesis, Activation Assay