Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: ASIC channel activity in A549, WI-38, and HLF cells. (A) Whole-cell configuration of the patch clamp technique and protocol of experiments. Membrane voltage was clamped at –50 mV in all patches. (B) Representative traces showing ASIC1a-like currents activated by a rapid drop of extracellular pH from 7.4 to 5.5, the currents were reversibly inhibited by 10 μM benzamil (derivative of amiloride). (C) Distribution of transient peak amplitudes at pH 5.5 elicited ASIC1a-like currents in A549 cells. (D) The effect of mambalgin-2 on ASIC1a-like currents in A549 cells. (E) ASIC1a-like currents in WI-38 cells. (F) Distribution of transient peak amplitudes in WI-38 cells at pH 5.5 elicited ASIC1a-like currents. (G) The effect of mambalgin-2 on ASIC1a-like currents in WI-38 cells. (H) An absence of acidification-evoked currents in HLF cells.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Activity Assay, Patch Clamp

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Analysis of mambalgin-2 and amiloride action at ASIC1a and ASIC1a/α-ENaC/γ-ENaC channels in Xenopus laevis oocytes. (A) Two-electrode configuration of the patch-clamp technique and protocol of experiments. Representative current traces recorded in X. laevis oocytes expressing the hASIC1a (B) and hASIC1a/α-ENAC/γ-ENAC (C) channels; traces for non-injected oocytes were used as control and are shown in Figure S9 . The pre-incubation with mambalgin-2 was 15 s (shown off time scale), the stimulation phase (pH 5.0) was 7 s, and the recovery phase is not shown. (D) Dose–response curves for mambalgin-2 inhibitory effect. For each mambalgin-2 concentration, the current response was normalized to the control experiment. Each data point represents an average from independent experiments in different oocytes ± SEM (n =11 for both channels). The fitted curves are described by single-component Hill’s equation. (E) Dose–response curves for amiloride inhibitory effect. The data points are shown as normalized average ± SEM (n = 3 for both channels).

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Patch Clamp, Expressing, Injection, Incubation, Concentration Assay

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Influence of knockdown of ASIC1, α-ENaC, and γ-ENaC expression on mambalgin-2 activity in A549 lung cancer cells. Dose–response effects of mambalgin-2 on viability of A549 cells after cell transfection with scramble siRNA or siRNA specific for ASIC1a (A) , α-ENaC (B) , and γ-ENaC. (C) Data are presented as % of the control (untreated cells) ± SEM (n = 4). The gene knockdown confirmation is shown in Figures S1A–E , and the analysis of dose–response curves is given in Table 1 . (D) Representative pictures of wounds for A549 cells incubated at pH 5.5 in the presence of 1 µM of mambalgin-2 upon knockdown of ASIC1a, α-ENaC, and γ-ENaC genes. Scale bar = 100 µm. Analysis of gene knockdown influence on the migration of A549 cells in the absence of mambalgin-2 is given in Figures S1F, G . (E) Wound area occupied by migrating A549 cells. Data are presented as % of the wound surface, occupied by migrating cells ± SEM (n = 8); ### (p < 0.001) and *** (p < 0.001) indicate significant difference between the data groups by one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Expressing, Activity Assay, Transfection, Incubation, Migration

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Parameters describing the effect of ASIC1a, α-ENaC, and γ-ENaC knockdown on the anti-proliferative activity of mambalgin-2 in A549 cells.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Activity Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 1. Expression of ASICs in the rat intervertbral disc. Sagittal sections of the intervertebral disc of embryonic (A and B) and mature rat (C–F) spines. Sections were treated with anti-ASIC3 antibody (B, D, and F) or counterstained with Alcian blue, eosin, and hematoxylin (A, C, and E). Note that nucleus pulposus cells expressed ASIC3 protein (B; arrow; magnification, ×20). ASIC3 expression was also evident in mature rat disc in the inner annulus zone (D; arrows). These cells are surrounded by an Alcian blue–positive matrix and display a round morphology (C, arrows), whereas the outer annulus fibrosus cells are surrounded by eosinophilic collagen rich matrix (C, arrowhead). ASIC3 expression was not detectable in the endplate chondrocytes of mature rat discs (F), which exhibit a round hypertrophic phenotype (E, arrows; magnification, ×20). (G) Expression of ASIC3 and ASIC2b in the intervertebral disc. mRNA was extracted from the nucleus pulposus (NP), the annulus fibrosus (AF), and the brain of adult rats and subjected to RT-PCR analysis. There was expression of ASIC3 and ASIC2b in both the discal tissues. Brain maximally expressed both ASIC isoforms. (H) Western blot analysis of ASIC3 and ASIC2b from adult rat discal tissues. Both nucleus pulposus and annulus fibrosus expressed ASIC3 and ASIC2b. (I) Immunofluorescent detection of ASIC proteins in discal cells. Cells were treated with antibodies to ASIC3 and ASIC2b. Both nucleus pulposus and annulus fibrosus cells expressed ASIC3 and ASIC2b (magnification, ×20). (J) Western blot analysis of ASIC proteins in cell lysates of discal cells. Western blots were performed using antibodies to ASIC3 (two separate antibodies for ASIC3) and ASIC2b. Both ASIC3 antibodies recognized a band at 60 kDa and an additional high molecular weight band at 90 kDa. (K) ASIC3 protein expression in the nuclear, cytosolic, and membrane fractions of disc cells. Fractionated protein extracts of the nucleus pulposus and annulus fibrosus cells were probed by Western blot for the expression of ASIC3. Note the prominent expression of this protein in the all the fractions. (L) Effects of deglycoslylation on ASIC3 protein molecular weight. Cell lysates were deglycosylated using PNGase F for 3 h at 37°C and probed for expression of ASIC3. The molecular weight of ASIC3 peptides were unchanged by treatment with the enzyme. (M) Immunoprecipitation (IP) analysis of ASIC3 and ASIC2b in disc cells. IP analysis was performed on tissue and cell lysates using anti-ASIC3 antibody. Immune complexes were separated by SDS-PAGE and probed with anti-ASIC2b antibody. Note the presence of ASIC2b in the ASIC3 immunoprecipitate of both the nucleus pulposus and annulus fibrosus tissue and nucleus pulposus cells.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, High Molecular Weight, Membrane, Molecular Weight, Immunoprecipitation, SDS Page

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 2. Cloning of rat ASIC3 promoter and its activity in disc cells. (A) Cartoon showing map of successive PCR generated 5 deletion constructs of the rat ASIC3 promoter. The ASIC3 promoter contains three distinct domains: proximal, middle, and a distal activating domain. The ASIC3-D construct consists of a 2925-bp fragment containing 2831 bp of the upstream ASIC3 promoter sequence linked to 94 bp of exon 1 (i.e., −2831 to +94), whereas ASIC3-M and ASIC3-P constructs contain a 1571- (−1477 to +94) and a 1065-bp (−971 to + 94) fragement, respectively. (B) Agarose gel electrophoresis showing PCR amplicons corresponding to different size fragments of the rat ASIC3 promoter. PCR was performed on rat liver genomic DNA using a set of nested forward primers containing a MluI site and a common reverse primer with a XhoI site. The resulting DNA fragments were ligated into MluI and XhoI digested luciferase basic expression vector, pGL3. (C and D) Basal activities of ASIC3 promoter constructs relative to full-length construct ASIC3-D in nucleus pulposus (C) and annulus fibrosus cells (D). Both the cell types showed maximal luciferase activity for the ASIC3-P construct, whereas the longest construct, ASIC3-D, containing all three promoter domains, showed minimal activity. Values shown are mean ± SE of three independent experiments. *p < 0.05.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Cloning, Activity Assay, Generated, Construct, Sequencing, Agarose Gel Electrophoresis, Luciferase, Expressing, Plasmid Preparation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 3. Evaluation of ASIC3 promoter re- sponsiveness to NGF treatment in nucleus pulposus, annulus fibrosus, and PC12 neuro- nal cells. Cells were transfected with full- length ASIC3 plasmid (ASIC3-D) and pRL- TK control vector and luciferase activity was measured 24 h after treatment with NGF (100 ng/ml). Note, in the nucleus pulposus (A) and annulus fibrosus cells (B), ASIC3 promoter activity did not change after NGF treatment, whereas in the PC12 cells (C), there was a 3- to 4-fold induction in promoter activity. NGF-mediated increase in ASIC3 promoter activity was suppressed to its basal level when PC12 cells were treated with anti- TrkA or anti-NGF antibody before addition of NGF. A similar experiment was per- formed using cells transfected with tyrosine hydroxylase (TH) promoter construct, a known NGF responsive gene. In both nucleus pulposus (D) and annulus fibrosus (E) cells, TH promoter activity did not change after NGF treatment. In contrast, in PC12 cells (F), a significant induction in ac- tivity was seen. The increase in TH promoter activity by NGF was suppressed by treatment with anti-TrkA or anti-NGF antibody. Val- ues shown are mean ± SD of three indepen- dent experiments. *p < 0.05.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Construct

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 5. Regulation of ASIC3 basal promoter activity by p75NTR in nucleus pulposus cells. (A) Effect of blocking p75NTR expression on ASIC3 promoter activity. Cells trans- fected with ASIC3-D plasmid were treated with anti-p75NTR an- tibody or isotype antibody (Ctr), and luciferase activity was mea- sured 24 h after the treatment. When p75NTR function was blocked, there was a significant suppression in ASIC3 basal pro- moter activity. (B) Effect of expression of DN-p75NTR on ASIC3 promoter activity. Nucleus pulposus cells were co-transfected with ecdysone-inducible DN-p75NTR and ecdysone receptor pVgRxR plasmids along with ASIC3-D reporter. The cells were treated with 1 g pronesterin-A for 24 h, and reporter activity was mea- sured. Pronesterin-A–induced expression of DN-p75NTR re- sulted in ∼50% suppression of ASIC3 promoter activity. Data shown are mean ± SD of three independent experiments per- formed in triplicate (n 3). *p < 0.05.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Activity Assay, Blocking Assay, Expressing, Plasmid Preparation, Luciferase, Transfection

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 6. Regulation of ASIC3 gene promoter by MEK/ERK signaling. (A) Influence of NGF treatment on ERK activity in nucleus pulposus cells. Cells were treated with NGF (100 ng/ml) for increasing time periods (0–180 min), and cell lysates were analyzed by Western blot. NGF treatment induced pERK1/2 expression within 5 min, and levels remained higher than basal levels for 1 h, although there was a decline after 10 min of exposure. (B) p75NTR-mediated activation of ERK in response to NGF treatment. Cells were co-transfected with ELK1-TAD plasmid (50 ng) or empty backbone vector (Gal4DBD; 50 ng) along with DN-p75NTR, and ELK1 transactivation was measured. NGF treatment significantly increased ELK1-TAD activity; the increase in activity was suppressed by pronesterin A–inducible DN-p75NTR. Backbone vector Gal4DBD showed minimal activity in untreated control cells and cells treated with NGF. (C) Effect of ERK inhibition on ASIC3 promoter activity. Cells were co-transfected with ASIC3-D reporter plasmid along with DN-MEK1 or empty backbone vector (pMCL), and reporter activity was measured. There was ∼30% suppression of basal ASIC3 promoter activity with 50 ng DN-MEK plasmid, which was further suppressed at 100 ng. Further increases in DN-MEK plasmid concentration (up to 400 ng) suppressed basal reporter activity by almost 80%. (C) Effect of overexpression of MEK1 on ASIC3 promoter activity. Cells were co-transfected with CA-MEK1 expression vector or empty vector pMCL along with ASIC3-D reporter, and luciferase activity was measured. A significant induction in ASIC3 promoter activity was observed with 50 ng of MEK1 plasmid. When the dose of MEK1 plasmid was 400 ng, there was a 2.25-fold increase in ASIC3 reporter activity. Values shown are representative of three independent experiments, performed in triplicate; mean ± SD. *p < 0.05.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Activity Assay, Western Blot, Expressing, Activation Assay, Transfection, Plasmid Preparation, Control, Inhibition, Concentration Assay, Over Expression, Luciferase

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Protein expression levels of Cav1.2, Piezo1, CaM, and Src in human LAA tissues. (A) Representative western blots and densitometric analysis of Cav1.2 and Piezo1 proteins in LA tissues of AF patients and those with SR. (B) Representative western blots and densitometric analysis of CaM and Src protein in LA tissues of AF patients and those with SR. GAPDH was the internal control. ** p < 0.01. Values are presented as the mean ± standard error of the mean (SEM).

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effects of hypertension on the incidence of AF, I Ca,L , and Piezo1 expression in Wistar rats and SHRs with and without Val treatment. (A) Representative baseline surface ECG and intra-atrial electrocardiogram (IAEG); (B) The incidence AF in Wistar rats and SHRs treated with and without Val treatment ( n = 8). ** p < 0.01 vs. Wistar rat; ## p < < 0.01 vs. SHRs. (C) Typical surface ECG recordings of rats with AF that spontaneously reverted to SR and typical disorganized amplification of atrial waves (f wave). (D) Representative traces of AP in atrial myocytes from Wistar rats, SHRs, and SHR + Val groups and a histogram of APD in atrial myocytes from each group ( n = 12–15 myocytes from 3–4 rats). * p < 0.05 vs. Wistar rat; # p < 0.05 vs. SHRs. (E) Representative traces of I Ca,L (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in atrial myocytes of each group ( n = 8–14 myocytes from 3–4 rats). (F) Representative examples of immunohistochemical analysis of LA tissues from Wistar rats and SHRs treated with and without Val using Ab against Piezo1. Scale bar, 20 μm. (G) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in LA tissues of Wistar rats and SHRs. GAPDH was the internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Amplification, Activation Assay, Immunohistochemical staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of HHP on the depression of I Ca,L in HL-1 cells. (A) Representative traces of AP in HL-1 cells under various hydrostatic pressures (0, 20, and 40 mmHg) for 24 h. APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 10, and 7 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (B) Representative traces (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L ( n = 8–18 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (C) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in HL-1 cells under various hydrostatic pressure (0, 20, and 40 mmHg) for 24 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: The effects of Piezo1 on perceiving HHP and mediating the decrease of I Ca,L . (A,B) Representative Ca 2+ traces and Δ Ca i 2 + (ΔF/F) are shown. Ca 2+ entry was evoked by 10 μm Yoda1 in HL-1 cells stimulated by HHP in the presence or absence of the Piezo1 inhibitor GsmTx4 ( n = 50) or siRNA specifically knockdown Piezo1 ( n = 53–58). si-C, scrambled (control) siRNA; si-P, siRNA directed against Piezo1. (C) Representative traces of AP in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment (3.0 μM) and APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 7, and 11 at 0, 40, and 40 mmHg + GsmTx4). * p < 0.05, ** p < 0.01 vs. 0 mmHg; # p < 0.05 vs. 40 mmHg. (D) Representative traces (pulse protocol, inset), corresponding current–voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment ( n = 9–15). ** p < 0.01 vs. 0 mmHg; ## p < 0.01 vs. 40 mmHg. (E) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (F) Representative blots and densitometry analysis of Cav1.2 in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of CaM/Src on the decrease of I Ca,L induced by HHP or Yoda1 stimulation. (A) Representative blots and densitometry analysis of CaM and Src in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (B) Representative blots and densitometry analysis of CaM and Src in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. (C) Representative blots and densitometry analysis of Src and p-Src ( n = 4) in HL-1 cells transfected with scrambled (control) siRNA or siRNA directed against Piezo1 for 48 h, then treated with Yoda1 at different dosages (0, 1, and 3 μM) for 15 min. (D) Representative traces of AP and histogram of APD in HL-1 cells ( n = 7–8). * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (E) Current–voltage relationship for I Ca,L ( n = 9–17) in HL-1 cells stimulated by 40 mmHg pressure treated with 15 μM PP1 or W7. * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (F) Representative blots and densitometry analysis of Cav1.2 and Src in HL-1 cells stimulated by 40 mmHg pressure treated with W7 under different concentrations (5, 10, 15, and 20 μM). (G) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure treated with PP1 (15 μM). (H) Current-voltage relationship for I Ca,L ( n = 8–10) in HL-1 cells stimulated by Yoda1(3 μM) treated with PP1. * p < 0.05, ** p < 0.01 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Yoda1. (I) Representative blots and densitometry analysis of Cav1.2 in Yoda1(3μM) -stimulated HL-1 cells treated with PP1 (15 μM). GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Schematic representation of the mechanism for the decrease of I Ca,L induced by HHP. Piezo1 activated by HHP depressed I Ca,L contributing to increased AF susceptibility through the CaM/Src/Pitx2 pathway.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons

doi: 10.1038/srep20924

Figure Lengend Snippet: ( A ) Neurons were infected with syntabulin shRNA lentivirus as indicated at DIV5 and examined at DIV13. Syntabulin knockdown significantly increased total ASIC2 expression but reduced surface ASIC2 levels as compared with the levels in control and scrambled-shRNA control. ( B ) Quantification of normalized ASIC2/GAPDH ratio, total ASIC2/surface ASIC2 ratio and surface ASIC2 levels. One-way ANOVA and Sidak test; ** P = 0.008, and P = 0.009 for Stb sh#1 Total ASIC2/GAPDH and Surface ASIC2/GAPDH compared to control respectively; * P = 0.044 and * P = 0.016 for Stb #1 total ASIC2/GAPDH and Surface/Total ASIC2 ratio compared to scrambled shRNA control respectively; * P = 0.020 for Stb #1 Surface/Total ASIC2 ratio compared to control; “ns” P > 0.05. n = 4 − 6 independent experiments. Error bars represent the SEM. ( C , D ) Hoechst staining of neurons after syntabulin knockdown, in pH 7.4 solution and pH6.0 solution. Knockdown of syntabulin significantly increased cell death as compared with controls in pH6.0. Scale bar = 20 μm. ( E ) Quantification of normalized total Hoechst intensity. One-way ANOVA and Sidak test; * P = 0.035 compared to control; * P = 0.043 compared to scrambled-shRNA control; “ns” P > 0.05; n = 6 independent experimental repeats. Error bars represent the SEM.

Article Snippet: The following antibodies were purchased: rabbit polyclonal ASIC2 antibody, Proteintech (17851-1-AP); mouse myc and β-tubulin antibodies, DSHB (9E10 and E7); mouse GAPDH antibody, Beyotime (GA019); mouse Map2 antibody, Sigma (M9942); and mouse β-actin antibody, Sigma (A5316).

Techniques: Infection, shRNA, Knockdown, Expressing, Control, Staining

Journal: Neuroscience

Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

doi: 10.1016/j.neuroscience.2021.01.036

Figure Lengend Snippet: ASIC1 and ASIC2a are located at the mouse NMJ. A. All of the images shown are maximum projections of 16 images collected at 0.5 μm increments vertically through the field containing the NMJ. α-bungarotoxin (α -BTX) is shown in Red. ASIC2a antibodies are in Green. Arrows point to clusters of ASIC2a staining that are outside the area defining the motor nerve terminal but near perisynaptic Schwann cell nuclei, indicated by the DAPI stain (Blue). Calibration bar, 10 μm. B. All of the images shown are maximum projections of 18 images collected at 0.5 μm increments vertically through the field containing the NMJ. α-bungarotoxin (α -BTX) is shown in Red. ASIC1 antibodies are in Green. Arrows point to clusters of ASIC2a staining that are outside the area defining the motor nerve terminal but near perisynaptic Schwann cell nuclei, indicated by the DAPI stain (Blue). Calibration bar, 10 μm.

Article Snippet: When we applied antibodies to the ASIC2a subunit (ASC-012, Alomone Labs, Jerusalem) we observed clear staining at the NMJ.

Techniques: Staining