Journal: Technology in Cancer Research & Treatment

Article Title: High ASIC3 Expression Correlates with Poor Prognosis in Lung Cancer Patients and Mediates Hypercapnic Acidosis-Induced EMT in A549 Cells

doi: 10.1177/15330338261434666

Figure Lengend Snippet: Effects of hypercapnic acidosis on the expression of ASIC3 in A549 cells. A. Representative images of ASIC3 expression in different groups of A549 cells (immunofluorescence staining). The green fluorescence shows ASIC3, the blue fluorescence shows the nucleus, and the combined pink shows the expression of ASIC3 in A549 cells. B. ASIC3 fluorescence intensity results were showed in each group. The data are shown as the mean ± sd. ** P < .01, n = 5for each experiment. C. RT-qPCR analysis of the expression levels of ASIC3.The data are shown as the mean ± sd. ** P < .01, n = 3 denotes independent biological replicates from separate cell cultures.

Article Snippet: The rabbit anti-human antibody ASIC3 was purchased from Cell Signaling Technology Inc., and anti-rabbit secondary antibodies were purchased from KangChen Bio-tech.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR

Journal: Technology in Cancer Research & Treatment

Article Title: High ASIC3 Expression Correlates with Poor Prognosis in Lung Cancer Patients and Mediates Hypercapnic Acidosis-Induced EMT in A549 Cells

doi: 10.1177/15330338261434666

Figure Lengend Snippet: HCA induces an epithelial-to-mesenchymal transition of A549 cells. A. A549 cells were incubated with HCA or amiloride (block ASIC3) and stained with the indicated antibodies of E-cadherin. Images were visualized under a microscope. Magnification, 20×. Scale bar, 20 μm. B. Statistical analysis of E-cadherin protein expressions was performed as above. C. A549 cells were stimulated withHCA or amiloride for 5 days, and cell lysates were subjected to western blot analysis with the indicated antibodies of E-cadherin and Vimentin. E-cadherin (D.) and Vimentin (E.) protein relative expressions. The data are shown as the mean ± sd. ** P < .01. n = 3 denotes independent biological replicates from separate cell cultures.

Article Snippet: The rabbit anti-human antibody ASIC3 was purchased from Cell Signaling Technology Inc., and anti-rabbit secondary antibodies were purchased from KangChen Bio-tech.

Techniques: Incubation, Blocking Assay, Staining, Microscopy, Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: High ASIC3 Expression Correlates with Poor Prognosis in Lung Cancer Patients and Mediates Hypercapnic Acidosis-Induced EMT in A549 Cells

doi: 10.1177/15330338261434666

Figure Lengend Snippet: siASIC3 inhibits HCA-induced EMT in A549 cells. A. Successful silencing of ASIC3 and downregulation of ASIC3 mRNA were achieved. B. The downregulation of ASIC3 mRNA was verified by RT-PCR. C. The expression levels of the epithelial marker E-cadherin, and mesenchymal markers N-cadherin, fibronectin, and vimentin were detected. D. Quantitative analysis of the relative protein expression levels was performed. The data are shown as the mean ± sd. ** P < .01. n = 3 denotes independent biological replicates from separate cell cultures.

Article Snippet: The rabbit anti-human antibody ASIC3 was purchased from Cell Signaling Technology Inc., and anti-rabbit secondary antibodies were purchased from KangChen Bio-tech.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: A Example traces of the effect of 30 µM RPRFa (red) on ASIC1a, ASIC2a, and ASIC3 in response to an acid stimulus (pH 6.3 for ASIC1a and ASIC3, pH 4.0 for ASIC2a), tested by manual patch clamp. Dashed line on ASIC3 trace shows the time 3 s after peak (I 3s ), which was used to calculate I 3s /I pk . B Concentration-response curve for RPRFa on ASIC3 tested on MPC. The y-axis, I 3s /I pk , displays the ratio of the current remaining after 3 s to the peak current as a measure of peptide activity. Data collected from six cells. C Example protocol (top) and trace (bottom) from a cell plotted in ( B ). Steps indicate change in pH, red shows application of RPRFa. Effect of RPRFa and C-terminal modified peptides ( D ) N-terminal extension with alanines ( E ) and aromatic amino acids ( F ) tested on the QPatch II. Black dashed lines indicates 0 (no change) and red dashed lines show the average response of RPRFa. Solid black lines depict geometric mean. G Traces showing the effect of 30 µM WRPRFa (red) on ASIC1a, ASIC2a, and ASIC3 during acid stimulation (pH 6.3 for ASIC1a and ASIC3, pH 4.0 for ASIC2a), tested by MPC. H Concentration-response curve of WRPRFa on ASIC3 tested on MPC. Dotted line depicts the RPRFa concentration-response curve shown in ( B ). Data collected from seven cells. I Example protocol (top) and trace (bottom) from a cell plotted in ( H ). Steps indicate change in pH, red shows application of WRPRFa.

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: Patch Clamp, Concentration Assay, Activity Assay, Modification

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: A pH-dependence of activation for ASIC3 without (black) or with (red) 30 µM WRPRFa. Current from vehicle-treated channels is measured as I pk and from peptide-treated channels as I 3s . Data collected from 11 cells for vehicle-treated and 18 cells for WRPRFa-treated. B Representative traces of data depicted in ( A ). Protocol is shown above each trace; steps indicate change in pH. Red trace indicates entire protocol was performed in the presence of 30 µM WRPRFa. C Time-dependence of WRPRFa binding to closed-state channel. Left, a trace showing application of 30 µM WRPRFa applied for increasing durations of time. A final step with 30 s application time to achieve maximum effect was used for normalization. Right, graph showing the rate of WRPRFa interaction with the closed state of ASIC3, fit with a monoexponential decay function. Data collected from 25 cells. D Time-dependence of WRPRFa wash off from ASIC3 using pH 6.3 (blue) or pH 8.0 (black). Data were fit with a monoexponential decay function. Data collected from five cells for pH 8.0 wash off and 5 cells for pH 6.3 wash off. E Traces of data from ( D ). Top shows the current decay over 1 min of pH 6.3 stimulus. Bottom shows wash off using pH 8.0. Every 20 s a 5 second pH 6.3 stimulus was given to measure the fraction of sustained current remaining as indicator of bound WRPRFa. Above each trace, steps in the protocol indicate change in applied pH and red bars show when 30 µM WRPRFa was applied to cells. F Left shows protocol (above) and trace (below) depicting binding of 30 µM WRPRFa to the activated state of ASIC3. V (vehicle), A (activated), and C (closed) represent specific channel states and blue bars where measurements were taken. Right, graph showing the sustained current (I 3s /I pk ) before treatment (V), with co-application of WRPRFa (A), and to the closed state (C). Data collected from five cells. G Desensitized state binding of 30 µM WRPRFa, following same outline as ( F ). D indicates the desensitized state. Data collected from three cells. Significance in ( F ) and ( G ) were calculated with two-tailed ratio paired t-test.

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: Activation Assay, Binding Assay, Two Tailed Test

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: A Alignment of chicken ASIC1a (1a) and ASIC3 (3) showing conserved residues highlighted in gray, the β11-12 linker in yellow, and mutated palm domain glutamates in red and cyan. β-sheets composing the palm domain are indicated by black boxes. B Structure of cASIC1a (PDB: 6AVE) in resting state. The β11-12 linker is colored yellow, the upper glutamates in red, and the lower glutamate pair cyan. C Representative traces of WT ASIC3, E78Q, E378Q, E416Q, and E421Q before (black) and after (red) 30 µM WRPRFa application, in response to a pH 6.3 stimulus. D Graph depicting the fold-increase in peak current (I pk /I pk ) after 30 µM WRPRFa treatment. Data collected from four cells for E78Q, three cells for E378Q, three cells for E416Q, four cells for E421Q, and five cells for WT. Solid black lines depict geometric mean. E Graph depicting the I 3s /I pk for each channel shown in ( C ). Data collected from the same cells as in ( D ). Solid black lines depict geometric mean. F pH-dependence of activation for E78Q before (black) and after (red) treatment with 30 µM WRPRFa. Data collected from 11 cells for vehicle-treated and 10 for WRPRFa-treated. Dashed lines indicate the pH-dependence of activation curves for WT ASIC3 before (black) and after (red) treatment with WRPRFa, replotted from Fig. . G pH-dependence of activation for the E421Q mutant. Data collected from five cells. Dashed line indicates the pH-dependence of activation curve for WT ASIC3, replotted from Fig. .

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: Activation Assay, Mutagenesis

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: Concentration-response curve of WRPRFa on E78Q ( A ), E378Q ( B ), E416Q ( C ), and E421Q ( D ) depicted in red, with the concentration-response curves for WRPRFa shown as a black dashed line, replotted from Fig. . Data collected from six cells for all mutants. E Concentration-response curves of WIPRFa on WT ASIC3 (black) and E78Q (red). Data collected from four cells for WT and six cells for E78Q. F Concentration-response curves of WRP{ADMA}Fa on WT ASIC3 (black) and E78Q (red). Data collected from six cells for WT and five cells for E78Q. G , H display a quadrant box containing EC 50 values for each combination of peptide and channel used to calculate the coupling coefficient Ω. Concentration-response curves for WIPRFa on E378Q ( I ) and E416Q ( J ) on WT ASIC3 (black) and mutant (red). Data collected from five cells for E378Q and E416Q. K A quadrant box containing EC 50 values for each combination of peptide and channel used to calculate the coupling coefficient Ω for the E416Q and WIPRFa mutations.

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: Concentration Assay, Mutagenesis

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: A Diagram showing subunit composition of ASIC3 and the concatemeric heterotrimers ASIC313 and ASIC131. Black circles indicate ASIC3 subunits and red ASIC1a subunits. B pH-dependence of acute desensitization for ASIC1a (red), ASIC3 (black), ASIC313 (blue), and ASIC131 (mustard). Data collected from four cells for ASIC1a, 11 cells for ASIC3, eight cells for ASIC313, and nine cells for ASIC131. Symbols represent geometric mean and error bars geometric standard deviation. C Protocol (above) and traces (below) for cells plotted in ( B ), ( D ), and ( E ). Top is ASIC313 and bottom ASIC131. Steps in the protocol represent change in pH. D pH-dependence of activation of ASIC313, before (black) and after (red) application of 30 µM WRPRFa. Current from vehicle-treated channels is reported as I pk and from peptide-treated channels as I 3s . Data collected from 13 cells for vehicle-treated and 11 cells for WRPRFa-treated. E pH-dependence of activation of ASIC131, before (black) and after (red) application of 30 µM WRPRFa. Current from vehicle-treated channels is reported as I pk and from peptide-treated channels as I 3s . Data collected from 13 cells for vehicle-treated and 11 cells for WRPRFa-treated. F Concentration-response curves of WRPRFa against ASIC313 (blue) and ASIC131 (mustard). Dashed black line is for ASIC3, replotted from Fig. . Data collected from six cells for ASIC313 and eight cells for ASIC131. G Protocol (above) and traces (below) for ASIC313 (top) and ASIC131 (bottom) from cells plotted in ( F ). Red bars indicate application of WRPRFa.

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: Standard Deviation, Activation Assay, Concentration Assay

Journal: Communications Chemistry

Article Title: Identification, characterization, and structure-activity relationship of the ASIC3-selective peptide WRPRFa

doi: 10.1038/s42004-025-01786-7

Figure Lengend Snippet: A Decrease in ASIC3 current resulting from repetitive acid stimulations. pH 4.0 stimulus in circles and pH 5.0 triangles. Vehicle conditions are black and 30 µM WRPRFa-treated in red. Solid lines represent fits with a monoexponential decay function. Data collected from six cells for pH 5.0 with vehicle, five cells for pH 5.0 with WRPRFa, four cells for pH 4.0 with vehicle, and seven cells for pH 4.0 with WRPRFa. B Traces depicting data presented in ( A ). Expanded traces at right shows pulse #1 in dark lines and pulse #10 in light line, indicated with carets. C Current loss during hold at pH 3.0 (squares), pH 4.0 (circles), and pH 5.0 (triangles). Cells treated with 30 µM WRPRFa shown in red. Data collected from 20 cells for pH 4.0, 15 cells for pH 3.0, 10 cells for pH 5.0, and 15 cells for pH 4.0 with WRPRFa. D Representative traces of data shown in ( C ). Above, liquid protocol used in ( C ). Below, traces of ASIC3 current at indicated pH. Protocol in red indicates entire experiment was done in presence of 30 µM WRPRFa.

Article Snippet: CHO cells stably expressing human ASIC3 ( NM_004769.1 ) (CT6173; Charles River Laboratories) were grown in F12K medium (Thermo), 10% FBS, tetracycline free (Sigma), and 1% penicillin-streptomycin (Thermo) with 10 μg/ml Blasticidin (Thermo) and 400 μg/ml Zeocin included as selection antibiotics.

Techniques: