Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Calcium microfluorimetry shows functional TRPA1 and TRPM8 expression in populations of Panc-1 cells. ( A ) Average ΔF/F 0 trace ± SEM (n = 21 cells) during control recordings across three AITC (25 μM, 15 s) challenges. ( B ) Replacement with a Ca 2+ -free extracellular solution before and during the 2nd AITC application abolished the response (average ΔF/F 0 trace ± SEM from n = 65 cells). ( C ) The specific TRPA1 inhibitor A-967079 (10 μM) was applied before and then co-applied with AITC during the 2nd challenge and completely inhibited the response (average ΔF/F 0 trace ± SEM from n = 31 cells). ( D ) Pooled data from the above experiments and statistical analysis. The average ± SEM ΔF/F 0 values are shown for each AITC stimulus in the series (*** p < 0.001, two-sample Student’s t-test; control, n = 45; Ca 2+ -free, n = 65; A-967079, n = 50).

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Functional Assay, Expressing, Control

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Whole-cell current recordings in Panc-1 cells confirm the functional expression of TRPA1 in this cell line. ( A ) AITC (25 μM)-sensitive cells were identified using calcium microfluorimetry (upper green trace from upper left inset). The solution was then replaced with the Ca 2+- free variant, and the responding cells were patch-clamped. Representative example recording showing the time course of the peak outward and inward currents during superfusion with AITC (100 μM). During co-application of A-967079 (10 µM), the currents were almost completely eliminated. The voltage clamp protocol (ramping from − 100 to 100 mV) is shown in the lower left inset. ( B ) Current–voltage plots at the points indicated, with colours and numbers as shown in ( A ). Outward rectification regime is observable at sub-maximal current activation (trace 2), while the current at maximal activation (trace 3) shows an almost linear I-V relationship. ( C ). Pooled data from n = 6 recordings using the above protocols. Average current density (measured from baseline) ± SEM. A-966790 produced a statistically significant inhibitory effect (** p < 0.001, paired-sample Student’s t-test). ( D ) Representative recording performed in Ca 2+ -containing solution showing the time course of the peak outward current and inward current recorded at a constant holding potential of − 80 mV. The currents displayed a transient behaviour in response to AITC (25 μM). A-967079 (10 µM) inhibition is especially noticeable after the second current increase. The calcium microfluorimetry trace used for selecting the cell is displayed in the upper left inset, while the voltage clamp protocol (holding at − 80 mV and ramping from − 80 to 80 mV) is shown in the lower right inset.

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Functional Assay, Expressing, Variant Assay, Activation Assay, Produced, Inhibition

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Expression of TRPA1 protein in PDAC cells A. Immunofluorescence of fixed Panc-1 and HEK-293 T cells either transfected or un-transfected with pcDNA3.1 plasmid encoding human TRPA1. Cells were stained with mouse monoclonal TRPA1 primary antibody (green), whereas DAPI nuclear staining is shown in blue. Scales are included on each image. Bars represent 10 µm.

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Staining

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Effects of TRPA1 activation on tumour cell migration. ( A ) Wound healing assay. Panc-1 cells were grown to confluence in three Petri dishes per condition. The monolayer was wounded and imaged immediately (0 h). Growth medium containing 10 μM AITC, A-967079 (10 µM) or a mixture of both were added. Wound closure was recorded at 12 h and 24 h after scratch. The area of the wound was determined by carefully selecting the cell-free regions as displayed with white contours using ImageJ software. Average ± SEM values were calculated from 20 measurements per time point, and the wound healing percent was calculated at each time point (see also Method section). The experiment was repeated at least three times, and images from a representative experiment are shown. ( B ) Average ± SEM values from the three experiments presented in ( A ). ns, not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001 as calculated with Student’s t-test. ( C ) Cell viability after 24 h treatment with the indicated compounds. Bars represent 3 experiments, which were each performed in triplicate.

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Activation Assay, Migration, Wound Healing Assay, Software

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Effects of siRNA-mediated TRPA1 knockdown on the pharmacological sensitivity of Panc-1 cells. ( A ) Wound healing assay illustrating changes in the migration rates of Panc-1 cells 72 h after transfection with siRNA with and without AITC treatment. ( B ) Average ± SEM values from the three experiments performed in triplicate presented in ( A ). ns, not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Inset: Western blot showing silencing of TRPA1 in lysates collected at the end of the experiment.

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Knockdown, Wound Healing Assay, Migration, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Influence of TRPA1 activation on cell cycle progression of Panc-1 cells. ( A ) Cell cycle distribution of Panc-1 cells under different conditions. Cells were cultured in complete medium and treated for 24 h with AITC (50 µM) or a mixture of AITC and A-967079 (10 µM), as indicated. ( B ) Summary of experiments presented in ( A ), showing the averages ± SEM of n = 3 experiments. Broken Y axes indicate numerically small, but significant differences of sub-G1 phase. The gating strategy of the control sample is presented in the inset.

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Activation Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

doi: 10.1038/s41598-021-81250-3

Figure Lengend Snippet: Calcium microfluorimetry shows functional TRPA1 and TRPM8 expression in populations of Panc-1 cells. ( A ) False colour fluorescence images extracted from a calcium microfluorimetry sequence at baseline during AITC stimulation (100 μM) and during treatment with ionomycin (2 μM) as a control at the end of the time course show that intracellular Ca 2+ increased in a population of Panc-1 cells during stimulation with AITC. The white scale bar indicates 50 μm. Lower left panel: representative individual fluorescence traces from the same experiment during exposure to WS-12 (5 μM), AITC and ionomycin. The arrowheads and numbers indicate the acquisition times of the three images shown above. Lower right panel: Venn diagram showing the relation between AITC-responding cells (grey, 27%,) and WS-12-responding cells (green, 7.6%), which are surrounded by a large proportion of cells insensitive to both agonists (white, 67.5%, n = 329 cells examined). ( B ) Scatter plot displaying the relationship between response amplitudes to AITC and WS-12. Same cells as above (82 cells sensitive to AITC-only, 15 to WS-12 and 10 cells to both agonists). The threshold values for identifying a response were set at 10% ΔF/F 0 . ( C ) The average ± SEM of the response amplitudes to AITC and WS-12 (for 92 AITC-sensitive cells and 25 WS-12-sensitive cells; same cells as in ( A , B )).

Article Snippet: The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C.

Techniques: Functional Assay, Expressing, Fluorescence, Sequencing, Control