Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Detection of TRPM8 transcripts and protein in prostate and breast cancer cell lines. A ) Signals for TRPM8 mRNA were found in cell lines derived from prostate cancer-derived (lanes 1–3) and breast cancer-derived (lanes 4–6) cell lines. The normalization with the GAPDH housekeeping control allowed to semi-quantitatively assessing the abundance of the different transcripts present in each sample. B) Signals for TRPM8 protein were detected in all cell lines; the ones for MCF7 and BT-474 were weaker than for the other cell lines (expected mass: 129 kDa). The GAPDH protein signal was used as a loading control. C) Densitometry analysis. The values of TRPM8 expression for mRNA (filled columns) and protein (empty columns) levels were normalized to the values of GAPDH controls.

Article Snippet: The plasmid encoding the full-length human TRPM8 tagged with Myc-DDK was purchased from Origene (#RC220615).

Techniques: Derivative Assay, Control, Expressing

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: The effect of different Ca 2+ signal modulators on menthol-evoked responses. A)- L) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). Each figure represents the results of one representative experiment out of three with similar results. A) ATMB hydrochloride , a TRPM8 blocker administered before menthol had no effect on menthol-evoked Ca 2+ response B ) BCTC, a putative TRPM8 blocker, administered before menthol had no effect on menthol-evoked Ca 2+ responses. C) CapZ, an another putative TRPM8 blocker had no effect on menthol-evoked Ca 2+ responses. D ) Removing the extracellular Ca 2+ ions strongly reduced the menthol-evoked responses E) Cells pre-treated with 50 µM U-73122 did not show oscillations. F) Overexpression of InsP 3 −5-phosphatase hydrolyzing InsP 3 with concomitant BFP expression in transfected cells inhibited menthol-induced oscillations compared to the non-transfected cells. Blue and yellow traces represent average fluorescence recordings from cells with or without InsP 3 −5-phosphatase from the same culture dish, respectively. Asterisks represent significant differences, p < 0.05, student t -test. G-H ) A Du 145 M8KO cell clone responded to menthol ( G ) similar as control Du 145 cells ( H ). I ) Statistical analysis on the integrals of the evoked Ca 2+ responses also showed no significant differences (integrals are from 4 consecutive experiments). J) Icilin did not evoke Ca 2+ oscillations. K ) Suramin, a non-specific purinergic receptor blocker, strongly reduced the menthol-evoked responses. L ) The presence of apyrase in the extracellular milieu also strongly reduced the menthol-evoked responses indicative of a role of extracellular ATP release after menthol treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The plasmid encoding the full-length human TRPM8 tagged with Myc-DDK was purchased from Origene (#RC220615).

Techniques: Fluorescence, Over Expression, Expressing, Transfection, Control

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Menthol-evoked responses in HEK-293 cells. A) Time-lapse image series of Ca 2+ waves. The acquisition rate was set to 3 s. Blue and red colors depict lower and higher fluorescence intensities, respectively. A Ca 2+ wave in HEK-293 cells was evoked by administration of 500 µM menthol. Bar represent 100 µm. B) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). A Ca 2+ response in HEK-293 cells was evoked by administration of 500 µM menthol. C ) Non-transfected HEK-293 cells didn’t respond to 50 µM menthol, but responded, if cells were transfected with a plasmid encoding the human TRPM8 receptor (yellow trace and green traces, respectively). D) Transfected HEK-293 cells showed only very small responses to 50 µM menthol in the absence of extracellular Ca 2+ ions; see magnification of the traces in absence of extracellular Ca 2+ in the boxed red area. This indicates that essentially TRPM8 PM channels are involved in the Ca 2+ responses. C-D ) Traces show average fluorescence values with SD. Asterisks represent significant differences, p < 0.05, Student t -test. E ) EGFP-TRPM8 (green) and mCherry-ER proteins (red) showed partial co-localization (yellow color on the merged image). Bar represent 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The plasmid encoding the full-length human TRPM8 tagged with Myc-DDK was purchased from Origene (#RC220615).

Techniques: Fluorescence, Transfection, IF-cells, Plasmid Preparation

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Effects of cold on TRPM8 channel currents in Du 145 cells with and without TRPM8 (mean ± SD; n = 3 independent experiments). After whole-cell configuration (W.C.), the temperature of the patch chamber was gradually lowered from 27 °C to 15 °C by using a heat-controlled path-chamber and the currents were recorded in the patched cells. A) Recording from a Du 145 M8KO cell with cold stimulation. B) Recording from a Du 145 WT cell with cold stimulation and ACA inhibition. Corresponding I/V- relation (red traces) of currents recorded at the indicated time points 1 and 2. C ) Current densities after cold exposure. Cold-evoked TRPM8 currents were blocked by extracellular ACA (0.025 mM). ( a p < 0.001 vs. the TRPM8 knockout group. b p < 0.001 vs. wild type without ACA treatment). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The plasmid encoding the full-length human TRPM8 tagged with Myc-DDK was purchased from Origene (#RC220615).

Techniques: Inhibition, Knock-Out

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: Activation of TRPM8 in the Du 145 M8 cells by oxidative stress. (mean ± SD). The cells were stained with Fluo-3 calcium dye and mean ± SD of fluorescence in 15 mm 2 of cell as arbitrary unit are presented; n = 10–20 independent experiments. In GSH experiments, the cells were pretreated with GSH (10 mM for 2 hours). The cells were extracellularly stimulated by cumene hyroperoxide (CHPx and 1 mM for 5 min) but they were extracellularly inhibited by ACA (25 μM for 10 min). The samples were analyzed by the laser confocal microscopy fitted with a 40× oil objective. The scale bar was 20 µm. Representative images and fluorescence intensities of the CHPx, ACA and GSH effect on the TRPM8 activation in the laser confocal microscope analyses are shown in ( a , b ) respectively. ( a p ≤ 0.001 versus control. b p ≤ 0.001 versus control + CHPx group. c p ≤ 0.001 versus control + CHPx + ACA group).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Activation Assay, Staining, Fluorescence, Confocal Microscopy, Microscopy

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: There is no activation of TRPM8 in the Du 145 M8 and Du 145 M8KO cells without extracellular Ca 2+ by oxidative stress (CHPx) and menthol. (mean ± SD). (+Ca 2+ , Ca 2+ -containing extracellular buffer; −Ca 2+ , Ca 2+ -free buffer). The cells were stained with Fluo-3 calcium dye and mean ± SD of fluorescence in 15 mm 2 of the cells as arbitrary unit are presented; n = 10–20 independent experiments. The Du 145 M8 and Du 145 M8KO cells with +Ca 2+ and −Ca 2+ buffers in the TRPM8 experiments were stimulated by CHPx (1 mM for 10 min) but they were inhibited by ACA (25 μM for 10 min). The samples were analyzed by the laser confocal microscopy fitted with a 40× oil objective. The scale bar was 20 µm. Representative images and fluorescence intensities of the CHPx, ACA and menthol effects on the TRPM8 activation in the laser confocal microscope analyses are shown in Fig. , respectively. ( a p ≤ 0.001 versus control. b p ≤ 0.001 versus control + CHPx group. c p ≤ 0.001 versus control + CHPx + ACA group. c p ≤ 0.001 versus Du 145 M8 (without Ca 2+ groups).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Activation Assay, Staining, Fluorescence, Confocal Microscopy, Microscopy

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: Effect of oxidative stress (CHPx) and ADPR on the TRPM8 current densities (pA/pF) in the Du 145 M8 and Du 145 M8KO cells. (mean ± SD and n = 3). The TRPM8 currents in the Du 145 M8 and Du 145 M8KO cells were induced either by intracellular ADPR (1 mM in patch-pipette) or extracellular CHPx (10 mM) and 0.1 mM menthol, but they were blocked by extracellular ACA (25 μM) in the patch-chamber. Intracellular GSH (2 mM) was given to the cells in the patch pipette. W.C.: Whole cell. ( a ) Control: Original recordings from control neuron. ( b ) ADPR group. ( c ) Menthol and CHPx group. ( d ) GSH group. ( e ) the Du 145 M8KO cells group. The ( f – h ) were currents densities of ( b – d ) +e patch clamp records, respectively. ( a p ≤ 0.001 versus control. b p ≤ 0.001 versus ADPR and menthol groups. c p ≤ 0.001 versus ADPR + ACA and menthol + ACA groups. d p ≤ 0.001 versus CHPx group).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Transferring, Patch Clamp

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: Activation of TRPM8 in the non-transfected (HEK293) and transfected (HEK293 TM8 ) human HEK293 cells by hydrogen peroxide (H 2 O 2 ). (mean ± SD). The cells were stained with Fluo-3 calcium dye and mean ± SD of fluorescence in 15 mm 2 of the cells as arbitrary unit are presented; n = 10–20 independent experiments. The HEK293 TM8 cells were stimulated by H 2 O 2 (1 mM for 10 min) but they were inhibited by ACA (25 μM for 10 min). The samples were analyzed by the laser confocal microscopy fitted with a 40× oil objective. The scale bar was 5 µm. Representative images ( a ), line ( b ) and column ( c ) of fluorescence intensities of the H 2 O 2 and ACA on the TRPM8 activation in the laser confocal microscope analyses are shown in Figs a–c, respectively. ( a p ≤ 0.001 versus control. b p ≤ 0.001 versus H 2 O 2 group).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Activation Assay, Transfection, Staining, Fluorescence, Confocal Microscopy, Microscopy

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: No activation of TRPM8 in the HEK293 without overexpressing human TRPM2 channel by hydrogen peroxide (H 2 O 2 ). (mean ± SD). The cells were stained with Fluo-3 calcium dye and mean ± SD of fluorescence in 15 mm 2 of the cells as arbitrary unit are presented; n = 10–20 independent experiments. The HEK293 cells were stimulated by H 2 O 2 (1 mM for 10 min) but they were inhibited by ACA (25 μM for 10 min). The samples were analyzed by the laser confocal microscopy fitted with a 40× oil objective. The scale bar was 5 µm. Representative images, line and column of fluorescence intensities of the H 2 O 2 and ACA on the TRPM8 activation in the laser confocal microscope analyses are shown in Figs a–c, respectively.

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Activation Assay, Staining, Fluorescence, Confocal Microscopy, Microscopy

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: Effects of ADPR and oxidative stress (H 2 O 2 ) on the TRPM8 current densities (pA/pF) in the HEK293 and HEK293 TM8 cells. (mean ± SD and n = 6). Fura-2-loaded the HEK293 and HEK293 TM8 cells were stimulated with H 2 O 2 (1 mM) in the presence of normal extracellular calcium (1.2 mM) for 160 seconds. The results were expressed as lines ( a ) and columns ( b ). In patch-clamp experiments, the TRPM8 currents in the HEK293 and HEK293 TM8 cells were induced by intracellular ADPR (1 mM in patch-pipette), but they were blocked by extracellular ACA (25 μM) in the patch-chamber. W.C.: Whole cell. ( c ) Original recordings from HEK293 with ADPR stimulation. ( d ) Original recordings from HEK293 TM8 without ADPR stimulation. ( e ) Original recordings from HEK293 TM8 with ADPR stimulation and ACA inhibition. The f was current densities of e patch clamp records. ( g ) ADPR. Single cell records from HEK293 TM8 cells. ( g ) No ADPR. Control single cell records from HEK293 TM8 cells. ( a p ≤ 0.001 versus HEK293 TM8 group. b p ≤ 0.001 versus HEK293 TM8 + ADPR group).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Patch Clamp, Transferring, Inhibition

Journal: Scientific Reports

Article Title: ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells

doi: 10.1038/s41598-018-37552-0

Figure Lengend Snippet: Effect of CHPx (1 mM) and ACA on the cell viability (MTT) ( a ), apoptosis ( b ), caspase 3 ( c ), caspase 9 ( d ), intracellular ROS production ( e ) and mitochondrial membrane depolarization (JC1) ( f ) levels in the Du 145 wild type (Du145 M8 ) and Du 145-knockout (Du145 M8KO ) cells. (mean ± SD and n = 3). The TRPM8 currents in the Du 145 M8 cells were induced by with CHPx (1 mM for 10 min) but they were blocked by extracellular ACA (25 μM for 10 min). Then, cells in the four groups were further stimulated by CHPx (1 mM). ( a p ≤ 0.001 versus control group. b p ≤ 0.001 versus CHPx group).

Article Snippet: For control experiments, 2 μg of wild type TRPM8 empty vector hTRPM8 (C-terminal FLAG tag) plasmid (OriGene Technologies, Istanbul, Turkey) was used for 24 hours using Lipofectamine 2000 (Invitrogen; Istanbul, Turkey.

Techniques: Knock-Out

Journal: Chemical research in toxicology

Article Title: Electrophilic components of diesel exhaust particles (DEP) activate transient receptor potential ankyrin-1 (TRPA1): a probable mechanism of acute pulmonary toxicity for DEP.

doi: 10.1021/tx200123z

Figure Lengend Snippet: Figure 4. Activation of other human TRP channels by DEP. Over- expressing (TRPA1, M8, V2, V3, and V4), transiently transfected (TRPV1), and untransfected HEK-293 cells were treated with DEP (0.77 mg/mL in-well concentration) or a solution of a prototype TRP channel agonist at a concentration that yielded the maximum response relative to the control HEK-293 cells. Agonists were TRPA1, 150 μM AITC; TRPM8, 20 μM icilin; TRPV1, 20 μM nonivamide; TRPV2, 100 μM Δ9-tetrahydrocannabinol; TRPV3, 300 μM carvacrol; and TRPV4, 12.5 nM GSK 1016790A. Data are the percent maximum response elicited by ionomycin (10 μM) with the vehicle control subtracted. N.D. = no response detected. *, indicates p < 0.05 using ANOVA with Bonferroni correction (n = 3).

Article Snippet: Human TRPM8 cDNA was obtained from Origene (Rockville, MD).

Techniques: Activation Assay, Expressing, Transfection, Concentration Assay, Control

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

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Sequence comparison within the S3 region of selected voltage-gated as well as voltage-independent cation channels, including TRPM8 and TRPM2. The amino acid sequences are shown in single letter code. The highly conserved N-x-x-D motif is highlighted with the outer pair of amino acid residues labeled in red and the inner pair in orange. Further highly conserved amino acid residues upstream of the N-x-x-D-motif are given in bold letters. The glycine residue at position 805 in the sequence of human TRPM8 which is crucial for the icilin sensitivity of the channel is marked in blue. Accession numbers are as follows human TRPM8: Q7Z2W7; human TRPM2: O94759; human TRPA1: O75762; human TRPC3: Q13507, Shaker H4 (KCNAS_DROME): P08510; human sodium channel type 2 alpha subunit (SCN2A), domain 4: Q99250; voltage-gated sodium channel from Bacillus halodurans (NaChBac): Q9KCR8; human L-type calcium channel subunit alpha 1C (CACNA1C) domain 4: Q13936.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Sequencing, Labeling

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Variations of the N-x-x-D motif and corresponding nomenclature of channel variants examined in the study.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques:

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: The variants were wild-type, D802N, N799D, and N799D+D802N. ( A ), Current densities (representing mean ± S.E.M of 26–31 independent experiments) obtained at room temperature during voltage ramps between −150 mV and +150mV applied over 200 ms. Note that only the voltage range from 0 to +150 mV is shown because currents are sizeable exclusively in the outward direction ( B ), Whole-cell patch clamp measurement on HEK-293 cells expressing wild-type TRPM8 during stimulation with menthol (100 µM) or ice-cold bath solution as indicated by the horizontal bars. Between the two stimulations an intermediate wash-step with standard bath solution at room temperature was performed. The holding potential was −60 mV. ( C ), Similar experiment as shown in panel B on cells expressing the TRPM8 variant N799D. Note the different scaling of the ordinates in panels B and C. The inset shows the corresponding current-voltage relation of N799D in comparison to wild-type in the presence of menthol. ( D ), Mean inward current densities of the TRPM8 variants in response to menthol or cold obtained at a holding potential of −60 mV. Note that in double stimulation experiments only the data from the first stimulation were used for the statistical analysis. Each column represents mean ± S.E.M. of 10–16 independent experiments. Values of ***p<0.001 were considered extremely significant.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Patch Clamp, Expressing, Variant Assay

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: ( A ), Maximum increase in the F 340 /F 380 ratio of each variant in response to 300 µM menthol or ice-cold bath solution. In double stimulation experiments only the data from the first stimulation were used for statistical analysis. The miniscule increases in fluorescence observed in mock-transfected controls are subtracted. Each column represents mean ± S.E.M. of 8–17 independent experiments. Significant differences to control are indicated with asterisks. Values of ***p<0.001 were considered extremely significant. ( B ), Western blot of TRPM8 protein on plasma membrane fractions prepared by the differential centrifugation method (“low speed fraction” ref. to ) of HEK-293 cells expressing various TRPM8 variants. Note the dual bands, shown in previous studies to indicate the glycosylated and non-glycosylated channel protein. The glycosylated form is absent in the weakly functional channel variant N799D. As negative control a plasma membrane fraction of mock-transfected HEK-293cells is included.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Variant Assay, Fluorescence, Transfection, Western Blot, Centrifugation, Expressing, Functional Assay, Negative Control

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Whole-cell patch clamp measurements on HEK-293 cells expressing wild-type TRPM2 ( A ) or TRPM2-variant N869D ( B ). Stimulation was performed with ADPR (600 µM in the absence of intracellular Ca 2+ ) infused into the cell through the patch pipette. The holding potential was −60 mV. Insets show the current-voltage relations obtained during voltage ramps within the indicated voltage ranges. Inward currents were repeatedly blocked with NMDG. The TRPM2 variant N869D is analogous to the virtually non-functional TRPM8 variant N799D.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Patch Clamp, Expressing, Variant Assay, Transferring, Functional Assay

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Whole-cell patch clamp measurements on HEK-293 cells expressing variants of TRPM2 and TRPM8 where the inner pair of residues of the N-x-x-D motif is reciprocally exchanged. ( A ), TRPM8 variant V800K+M801L first stimulated with ice-cold bath solution and after an intermediate wash-step with standard bath solution stimulated with 100 µM menthol (as indicated by horizontal bars). ( B ), TRPM2 variant K870V+L871M stimulated by infusion of 600 µM ADPR into the cell through the patch pipette and in the absence of intracellular Ca 2+ . Insets show the corresponding current-voltage relations during stimulation.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Patch Clamp, Expressing, Variant Assay, Transferring