Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Primer sequences used for RT-PCR analysis of selected cytokine genes.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Effects of cold temperature or/and CSE on TRPM8 expression in 16HBE cells relative quantification of transient receptor potential melastatin 8 (TRPM8) mRNA and protein in 16HBE cells exposed to18°C or/and CSE. (A) The levels of the TRPM8 protein were determined by western blot analysis. (B) The levels of TRPM8 mRNA were determined by real-time RT-PCR, and a comparative Ct method (2 −ΔΔCt ) was used for the relative mRNA quantification. (C) Densitometry quantification of the bands in A was performed using Quantity One software, and the results are expressed as the ratio of the expression of TRPM8 to β-actin. The values in B and C are shown as the means ± SD; n = 4. ∗ p < 0.05 vs. control.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Quantitative RT-PCR, Software, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of cold temperature or/and CSE in TRPM8 mediated increase in intracellular Ca 2+ level in 16HBE cells. Intracellular Ca 2+ levels were measured by Fluo3-AM fluorescent probe assay. (A) Cells were exposed to 18°C or/and CSE for 30 min. (B) Representative images of fluorescence-positive cells were exposed to18°C for 6 min, to CSE for 3 min, to both cold temperature and CSE for 6 min and with or without transfection of TRPM8 shRNA or scramble shRNA. (C) Fluorescence intensity for intracellular calcium concentration was analyzed with the quantification tools. Data in each group are mean ± SD; n = 4. ∗ p < 0.05 vs. control, # p < 0.05 vs. 18°C alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Fluorescence, Transfection, shRNA, Concentration Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF- κ B inhibitor (BAY 11-7085; BAY), p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD n = 3. ∗ p < 0.05 vs. control.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, shRNA, Transfection, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Physiology

Article Title: Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

doi: 10.3389/fphys.2019.00285

Figure Lengend Snippet: Roles of MAPKs/NF-κB signaling pathway in the synergistic effect of CSE on the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to both CSE and 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF-κB inhibitor (BAY 11-7085). p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD; n = 3. ∗ p < 0.05 vs. control, # p < 0.05 vs. cold alone, Δ p < 0.05 vs. CSE alone.

Article Snippet: Exon 18-specific TRPM8 shRNA and scramble shRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, shRNA, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Control

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

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemistry using anti-TRPM8 in pancreatic tissues. ( A ) Normal pancreatic tissues. ( B ) Chronic pancreatitis. ( C ) Pancreatic intra-epithelial neoplasm. ( D ) Intraductal papillary mucinous neoplasm.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemical analysis of TRPM8 in malignant pancreatic tumors. ( A , B ) Adenocarcinoma. ( C , D ) Adenosquamous carcinoma. ( E , F ) Solid pseudo-papillary neoplasm ( G , H ) Acinar cell carcinoma. ( I , J ) Neuroendocrine tumor. ( A , C , E , G , I ) H and E, original magnification ×200. ( B , D , F , H , J ) Immunohistochemistry using anti-TRPM8 antibodies, original magnification ×400.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Immunohistochemical staining, Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression of TRPM8 in various types of histopathology in pancreatic tumors with regard to the intensity of immunoreactivity and the percentage of positive cells. The total number of cases examined is 308. The values represent the number of specimens with the corresponding proportions in parentheses.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Expressing, Histopathology

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression levels of TRPM8 in pancreatic adenocarcinoma. Images in ( A , C , E ) (original magnification ×200) represent the H&E sections of the same tumor as the TRPM8 immunohistochemical staining in ( B , D , F ) (original magnification ×400), respectively. Expression levels of TRPM8: ( B ) no-to-low; ( D ) moderate; ( F ) high.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Anti-TRPM8 immunoreactivity in pancreatic adenocarcinoma positively correlates with primary tumor size and stages. The distribution of the IHC score for expression of TRPM8 is displayed against age/gender, histological grade, primary tumor (T), and stage. The total number of specimens of pancreatic adenocarcinoma examined is 280. Polyserial correlation between the primary tumor size (T) or the tumor stages and the log-transformed IHC score is 0.13 and 0.10, respectively.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Expressing, Transformation Assay

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: The percent coverage ± standard error for expression of TRPM8 in pancreatic adenocarcinoma with respect to the intensity and the tumor stage. The numbers in parentheses represent the patient counts. NA, not applicable.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Expressing

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Over-expression of TRPM8 protein in human pancreatic adenocarcinoma cell lines. ( A ) The protein levels of TRPM8 in H6c7, MIA PaCa-2, PANC-1, and BxPC-3 cells were determined using immunoblotting with anti-TRPM8 antibodies. The GAPDH protein levels were analyzed as internal controls. ( B ) The relative protein levels of TRPM8 are expressed as % (mean ± standard error) of that in H6c7.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: Over Expression, Western Blot

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Short hairpin RNA-mediated silencing of TRPM8 impaired invasion of pancreatic adenocarcinoma cells. BxPC-3 and MIA PaCa-2 cells transfected with either anti- TRPM8 shRNA or NC shRNA were analyzed for cell invasion using the trans-well assay. Cell invasion is expressed % (mean ± standard error) of that in the cells transfected with control shRNA. Representative images of the invaded cells stained with crystal violet are shown at 200× magnification.

Article Snippet: Four plasmids containing different shRNA directed against human TRPM8 and a plasmid containing non-targeting shRNA were obtained from Superarray Biosciences/Qiagen (Valencia, CA, USA) and tested for efficiency of gene silencing.

Techniques: shRNA, Transfection, Staining