human asic3  (Charles River Laboratories)

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: ASIC3 is expressed in almost all CSCs and can be activated by GMQ. ( a ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for ASIC3 (red) and for hoechst (blue). Arrowhead points to an ASIC3 negative cell. ( b ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for CD133/Prominin1 (green) and for hoechst (blue). ( c ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for Ki67 (green) and for hoechst (blue). ( d ) Histogram showing the percentage of positive cells for ASIC3, CD133 and Ki67 n ≥ 3 independent experiments. The graph was made with GraphPad Prism v9 ( e ) High magnification image of ASIC3 (left), hoechst (middle) and double staining (right), arrowheads point to a membrane enriched ASIC3 staining. ( f ) Representative traces of a GMQ-induced current in GBM-MES 0315 cells in the presence or absence of APETx2 (black and red traces respectively). ( g ) Box plot of membrane current density upon stimulation with GMQ alone (red) or in the presence of APETx2 (black). Plot displays the mean (+), median (internal horizontal line), first and third quartiles (upper and lower box edges) and standard deviation (whiskers) of data distribution (n = 11). Dots represent individual cells. Significant difference was assessed by Mann–Whitney non-parametric test (****p < 0.0001). Scale Bar = 20 µm for ( a–c ) and 10 µm for ( e ). All data were analysed by using the Prism 8 (GraphPad) software and traces were generated by Origin 2018 (Origin Lab.).

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Double Staining, Membrane, Standard Deviation, MANN-WHITNEY, Software, Generated

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: The ASIC3 isoform is enriched in glioblastoma CSCs. ( a ) Western blot analysis of ASIC3 and ASIC1a expression in human primary GBM-CSC lines from the three GBM subtypes ( PN proneural, MES mesenchymal, CL classical). ASIC3 (n ≥ 3) ( b ) and ASIC1a (n = 3) ( b ’) protein expression quantification normalized on actin levels in the different GBM-CSC lines. ( c ) Western Blot analysis of CD133/prominin1 and ASIC3 expression in a healthy human brain lysate, in the GBM-MES CSCs 0315 line and in a biopsy from the same patient used to generate the CSC line 0315. ( c ’) ASIC3 and CD133/prominin1 protein expression quantification normalized for actin levels, in healthy human brain, GBM MES 0315 line and MES 0315 patient’s biopsy (n = 4). Statistic was performed by a two-way Anova with Bonferroni’s post-test:(****) p < 0,0001. ( d ) Data from publically available Repository for Molecular BRAIN Neoplasia Data (TCGA datasets Affymetrix HT HG U133A) were used to determine the relative expression of ASIC3 mRNA (ACCN3) in four human glioma subtypes (Classical, Mesenchymal, Neuronal, and Proneuronal), as well as in healthy patients. All 454 glioma patients with available mRNA were examined. ( e ) Western Blot analysis of ASIC3 expression in GBM-CSCs MES 0315 growth in 3D and 2D culture conditions after 24hin culture. Original blots were horizontally cropped at the appropriate MW region in order to perform multiple staining on the same blot. In the figure, portions of the same blot are separated by white lines. Black lines are used to separate independent blots presented in the same figure. Original blots are shown in supplementary figure S1 and S4. Dots represent independent experiments. Measurements were taken from distinct samples. Graphs for ( b,b’,c’ ) were analyzed with GraphPad Prism 9, graph for ( d ) was obtained from https://www.betastasis.com/glioma/tcga_gbm/ .

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Western Blot, Expressing, Staining

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: Generation of a virtual 3D model of the human ASIC3 channel. ( a ) AlphaFold model of human ASIC3 represented as cartoons colored by model quality (from low quality pLDDT = 50 in blue, to high quality pLDDT = 100 in red); inset: close up view of the extracellular domain of ASIC3, highlighting with spheres amino acids Glu78 and Glu421, known to be involved in GMQ binding . ( b ) Superposition of the two structural models (depicted as cartoon) selected after MD simulation using cluster analysis: 682 (68.2 ns) as blue and 812 (81.2 ns) as cyan and extent of the region explored during in silico docking analysis. ( c,d) Close-up views of the interaction between charged (yellow carbon atoms) and non-charged (orange carbons) GMQ with protein residues [sticks with carbon atoms colored in cyan (812) or blue (682)].

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Binding Assay, In Silico

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: In silico docking analysis .

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: In Silico

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: ASIC3 downregulation and its pharmacological blocking reduces the effect of GMQ. ( a ) Blot analysis of ASIC3 expression in GBM CSCs MES 0315 cells after infection with lentiviruses carrying specific anti-ASIC3 shRNAs (shRNA1 and shRNA2). Original blots were horizontally cropped at the appropriate MW region in order to perform multiple staining on the same blot. Portions of the same blot are separated by white lines. ( a ’) ASIC3/Actin protein expression ratio of MES 0315 before and after infection with Lentiviruses carrying shRNA1 or shRNA2 (n = 7) and normalized on control samples. CTRL vs shRNA1: P = 0,0007; CTRL vs shRNA2 P = : 0,0007. Original blots are shown in supplementary figure S9a and S9b. ( b ) Effect of ASIC3 downregulation on GMQ-induced GBM CSCs growth. Plot represents the number of MES 0315 cells after 96 h of GMQ treatments (3 and 10 μM), normalized to untreated samples: control (n = 4); shRNA1 (n = 3); shRNA2 (n = 3). (****) P < 0,0001. ( c ) Plot represents the % of GBM CSCs MES 0315 after 96 h of 3 μM GMQ treatment, in the presence or absence of 1 μM APETx2 toxin, normalized to the untreated sample, (n = 3); Unt vs GMQ p = 0.0214; Untr vs APETX2 + GMQ p = 0.9058; APETX2 vs GMQ p = 0.0189; GMQ vs APETX2 + GMQ p = 0.0029. Unpaired T-test for ( a ’), two-way Anova with Bonferroni’s post-test for ( b ) and One-way Anova with Bonferroni’s post-test for ( c ). Dots represent independent experiments. All graphs were analysed with GraphPad Prism v9.

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Blocking Assay, Expressing, Infection, Staining, Control

Journal: Marine Drugs

Article Title: Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds

doi: 10.3390/md16120500

Figure Lengend Snippet: Effects of ligands in an acetic acid-induced writhing test. Pretreatment of mice with APETx2, sevanol, and Ugr9-1 (2 h before testing) attenuated the response to the intraperitoneal administration of acetic acid. ( A ) Efficacy of ASIC3 antagonists at a dose of 1 mg/kg. ( B – D ) Dose-dependent chart of ligands’ effects: APETx2 ( B ), sevanol ( C ), and Ugr9-1 ( D ). Results are presented as mean ± SEM ( n = 8). ** p < 0.01, *** p < 0.001 versus saline group (ANOVA followed by a Tukey’s test).

Article Snippet: The cRNA transcripts were synthesised from a NaeI-linearized ASIC3 cDNA template (pcDNA3.1+humanASIC3 subcloned from clone EX-Q0260- B02 (GeneCopoeia, Inc., Rockville, MD USA) using a HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol for capped transcripts.

Techniques:

Journal: Marine Drugs

Article Title: Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds

doi: 10.3390/md16120500

Figure Lengend Snippet: Effect of ligands on the CFA-induced thermal hyperalgesia test. Test was performed 2 h after intramuscular administration of ASIC3 antagonists. ( A ) Comparison between ligands at the dose of 1 mg/kg; ( B – D ) Dose-dependent chart of ligands’ effect. APETx2 ( B ), sevanol ( C ), and Ugr9-1 ( D ) reversed CFA-induced thermal hyperalgesia and prolonging withdrawal latency of the inflamed hind paw on a hot plate. Results are presented as mean ± SEM ( n = 7–8). * p < 0.05, ** p < 0.01, *** p < 0.001 versus saline group (ANOVA followed by a Tukey’s test).

Article Snippet: The cRNA transcripts were synthesised from a NaeI-linearized ASIC3 cDNA template (pcDNA3.1+humanASIC3 subcloned from clone EX-Q0260- B02 (GeneCopoeia, Inc., Rockville, MD USA) using a HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol for capped transcripts.

Techniques:

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: