ns1643  (Alomone Labs)


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    Alomone Labs ns1643
    <t>NS1643</t> inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p
    Ns1643, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ns1643/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ns1643 - by Bioz Stars, 2022-01
    94/100 stars

    Images

    1) Product Images from "Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin"

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    Journal: Cancers

    doi: 10.3390/cancers13236025

    NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p
    Figure Legend Snippet: NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p
    Figure Legend Snippet: NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p

    Techniques Used: Migration, Western Blot, Expressing, Wound Healing Assay

    NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p
    Figure Legend Snippet: NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p

    Techniques Used: Activation Assay, Western Blot, Expressing

    NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p
    Figure Legend Snippet: NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p

    Techniques Used: Western Blot, Expressing

    PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .
    Figure Legend Snippet: PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .

    Techniques Used: Expressing, Western Blot, Transfection

    NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p
    Figure Legend Snippet: NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p
    Figure Legend Snippet: NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p

    Techniques Used: Western Blot, Expressing

    2) Product Images from "Preclinical study of a Kv11.1 potassium channel activator as antineoplastic approach for breast cancer"

    Article Title: Preclinical study of a Kv11.1 potassium channel activator as antineoplastic approach for breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22925

    NS1643 treatment increases senescence markers in vivo ( A ) Representative immunoblots and quantification of the effects of NS1643 on senescence markers p16 INK4A and ( B ) p21 waf/cip in tumors extracted from mice receiving DMSO (control) or NS1643. Data is expressed as mean ± SEM; Unpaired t -test. * p
    Figure Legend Snippet: NS1643 treatment increases senescence markers in vivo ( A ) Representative immunoblots and quantification of the effects of NS1643 on senescence markers p16 INK4A and ( B ) p21 waf/cip in tumors extracted from mice receiving DMSO (control) or NS1643. Data is expressed as mean ± SEM; Unpaired t -test. * p

    Techniques Used: In Vivo, Western Blot, Mouse Assay

    Effect of NS1643 on in vivo Cardiac function Transthoracic M-mode echocardiograms echocardiography (Vevo 2100 with MS400 series transducer, Visualsonics Inc.) of ( A ) control ( n = 3) and NS1643-treated mice ( n = 3). Figure represents a motion mode of the left ventricle, which is obtained with a single ultrasound beam transmitted through the heart with the resulting image displayed over time. Light Gray bar=control; Dark gray bar=NS1643. HR=heart rate (beats per min); EF=ejection fraction (%); LVIDs LVIDd=left ventricular internal diameter systole and diastole; LVAWs LVAWd=left ventricular posterior wall systole and diastole (mm). ( B ) Colorimetric assay detecting serum level of creatine phosphokinase and ( C ) serum level of creatine phosphokinase-Myocardial band (CK-MB) assay in blood extracted from DMSO (control) vs NS1643 treated mice. Beat-to-beat detection was achieved using preset detection and analysis settings for mice (typical QRS width 10 ms, R waves > 60 ms apart, Pre-P baseline 10 ms, Maximum PR 50 ms, Maximum RT 40 ms, ST height at 10 ms, averaging 4 beats). Averaged ECG measurements were taken from a minimum of 20 beats per mouse.
    Figure Legend Snippet: Effect of NS1643 on in vivo Cardiac function Transthoracic M-mode echocardiograms echocardiography (Vevo 2100 with MS400 series transducer, Visualsonics Inc.) of ( A ) control ( n = 3) and NS1643-treated mice ( n = 3). Figure represents a motion mode of the left ventricle, which is obtained with a single ultrasound beam transmitted through the heart with the resulting image displayed over time. Light Gray bar=control; Dark gray bar=NS1643. HR=heart rate (beats per min); EF=ejection fraction (%); LVIDs LVIDd=left ventricular internal diameter systole and diastole; LVAWs LVAWd=left ventricular posterior wall systole and diastole (mm). ( B ) Colorimetric assay detecting serum level of creatine phosphokinase and ( C ) serum level of creatine phosphokinase-Myocardial band (CK-MB) assay in blood extracted from DMSO (control) vs NS1643 treated mice. Beat-to-beat detection was achieved using preset detection and analysis settings for mice (typical QRS width 10 ms, R waves > 60 ms apart, Pre-P baseline 10 ms, Maximum PR 50 ms, Maximum RT 40 ms, ST height at 10 ms, averaging 4 beats). Averaged ECG measurements were taken from a minimum of 20 beats per mouse.

    Techniques Used: In Vivo, Mouse Assay, Colorimetric Assay, Mass Spectrometry

    NS1643 generates reactive oxygen species (ROS) and DNA damage in a Ca 2+ -dependent manner ( A ) Effect of NS1643 alone (50 µM), in association with the Kv11.1 blocker E-4031 (10 µM), or in the presence of the Ca 2+ ion chelator EGTA (2.4 mM) on cellular ROS formation in human MDA-MB-231 cells (DCFH-DA to 2′,7′-dichlorofluorescein DCF, Thermo Fisher Sci; Fluorescence was analyzed in a plate reader (PHERAstar FS, BMG LABTECH) with excitation at 485 nm and emission at 520 nm). Data is expressed as mean ± SEM; * p
    Figure Legend Snippet: NS1643 generates reactive oxygen species (ROS) and DNA damage in a Ca 2+ -dependent manner ( A ) Effect of NS1643 alone (50 µM), in association with the Kv11.1 blocker E-4031 (10 µM), or in the presence of the Ca 2+ ion chelator EGTA (2.4 mM) on cellular ROS formation in human MDA-MB-231 cells (DCFH-DA to 2′,7′-dichlorofluorescein DCF, Thermo Fisher Sci; Fluorescence was analyzed in a plate reader (PHERAstar FS, BMG LABTECH) with excitation at 485 nm and emission at 520 nm). Data is expressed as mean ± SEM; * p

    Techniques Used: Multiple Displacement Amplification, Fluorescence

    NS1643 arrests RasV12 tumor growth in Drosophila melanogaster Inhibition of proliferation in Drosophila model by Kv11.1 stimulation (NS1634) in comparison to vehicle alone (control). On the seventh day after egg laying (3 days of drug treatment), larvae were harvested and imaged on an EVOS fluorescent microscope. Data is expressed as Mean ± SEM; * p
    Figure Legend Snippet: NS1643 arrests RasV12 tumor growth in Drosophila melanogaster Inhibition of proliferation in Drosophila model by Kv11.1 stimulation (NS1634) in comparison to vehicle alone (control). On the seventh day after egg laying (3 days of drug treatment), larvae were harvested and imaged on an EVOS fluorescent microscope. Data is expressed as Mean ± SEM; * p

    Techniques Used: Inhibition, Microscopy

    Kv11.1 stimulation inhibits primary tumor growth in xenograft model of breast cancer MDA-MB-231 (ATCC ® ) cells were injected subcutaneously into the flanks of female athymic nude mice. When tumors were palpable the mice were injected intraperitoneally with vehicle alone or Kv11.1 activator NS1643 at 6 mg/kg every two days. ( A ) Representative mice with tumor burdens treated with vehicle (DMSO) alone. ( B ) Representative mice with tumor burdens treated with Kv11.1 activator NS1643. ( C ) Mean tumor volume in mice treated with vehicle alone (control N = 6) and in mice treated with Kv11.1 activator (NS1643 N = 6). Data is expressed as Mean ± SEM; * p
    Figure Legend Snippet: Kv11.1 stimulation inhibits primary tumor growth in xenograft model of breast cancer MDA-MB-231 (ATCC ® ) cells were injected subcutaneously into the flanks of female athymic nude mice. When tumors were palpable the mice were injected intraperitoneally with vehicle alone or Kv11.1 activator NS1643 at 6 mg/kg every two days. ( A ) Representative mice with tumor burdens treated with vehicle (DMSO) alone. ( B ) Representative mice with tumor burdens treated with Kv11.1 activator NS1643. ( C ) Mean tumor volume in mice treated with vehicle alone (control N = 6) and in mice treated with Kv11.1 activator (NS1643 N = 6). Data is expressed as Mean ± SEM; * p

    Techniques Used: Multiple Displacement Amplification, Injection, Mouse Assay

    NS1643 treatment decreases tumor proliferation markers ( A ) Detection of Ki67 (clone Mib-1, Glostrup, Denmark) protein expression in mice treated with DMSO (control) or NS1643 and ( B ) quantification of Ki67-positive cells per mm 2 . Data presented as mean ± S.D. Unpaired t -test. *** p
    Figure Legend Snippet: NS1643 treatment decreases tumor proliferation markers ( A ) Detection of Ki67 (clone Mib-1, Glostrup, Denmark) protein expression in mice treated with DMSO (control) or NS1643 and ( B ) quantification of Ki67-positive cells per mm 2 . Data presented as mean ± S.D. Unpaired t -test. *** p

    Techniques Used: Expressing, Mouse Assay

    NS1643 generates DNA damage in breast cancer ( A ) Single confocal sections of DAPI-stained tumors from control and NS1643-treated mice show fragmentation of nuclei indicative of DNA damage induced by NS1643 treatment. The number of normal (light grey) vs. fragmented (dark grey) nuclei was quantified by manual counting ( n = 4, p
    Figure Legend Snippet: NS1643 generates DNA damage in breast cancer ( A ) Single confocal sections of DAPI-stained tumors from control and NS1643-treated mice show fragmentation of nuclei indicative of DNA damage induced by NS1643 treatment. The number of normal (light grey) vs. fragmented (dark grey) nuclei was quantified by manual counting ( n = 4, p

    Techniques Used: Staining, Mouse Assay

    3) Product Images from "Potassium channel activity controls breast cancer metastasis by affecting β-catenin signaling"

    Article Title: Potassium channel activity controls breast cancer metastasis by affecting β-catenin signaling

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1429-0

    Kv11.1 stimulation regulates the cellular localization of β-catenin. a Representative western blot images are demonstrating subcellular localization of β-catenin. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to cellular fractionation (nuclear and membrane fraction). Quantification of the levels of β-catenin protein was done using NIH ImageJ software. * p
    Figure Legend Snippet: Kv11.1 stimulation regulates the cellular localization of β-catenin. a Representative western blot images are demonstrating subcellular localization of β-catenin. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to cellular fractionation (nuclear and membrane fraction). Quantification of the levels of β-catenin protein was done using NIH ImageJ software. * p

    Techniques Used: Western Blot, Cell Fractionation, Software

    Kv11.1 controls β-catenin nuclear translocation signaling. a Representative western blot images of phospho-AKT T308 , phospho-β-catenin S552 , and total AKT levels in cells or b tumors extracted from mice treated with control or NS1643. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of phospho-AKT and phospho-β-catenin were done using NIH ImageJ software. c Representative western blot images of phospho-GSK3⍰Ser9 and total GSK3⍰ levels in MDA-MB-231. d Representative western blot images of β-catenin levels in cells treated with cycloheximide alone or NS1643 for the indicated time and e quantification of the effects ( n = 3). Quantification of the levels of β-catenin and phospho-β-catenin were done using NIH ImageJ software. f Confocal microscopy images are representing the accumulation of β-catenin at the surface membrane in MDA-MD-231 cells treated with control vehicle or NS1643 for 24 h. g Graph showing the effect of NS1643 on cell–cell contact measured by Dispase assay ( n = 6; mean ± SD; * P
    Figure Legend Snippet: Kv11.1 controls β-catenin nuclear translocation signaling. a Representative western blot images of phospho-AKT T308 , phospho-β-catenin S552 , and total AKT levels in cells or b tumors extracted from mice treated with control or NS1643. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of phospho-AKT and phospho-β-catenin were done using NIH ImageJ software. c Representative western blot images of phospho-GSK3⍰Ser9 and total GSK3⍰ levels in MDA-MB-231. d Representative western blot images of β-catenin levels in cells treated with cycloheximide alone or NS1643 for the indicated time and e quantification of the effects ( n = 3). Quantification of the levels of β-catenin and phospho-β-catenin were done using NIH ImageJ software. f Confocal microscopy images are representing the accumulation of β-catenin at the surface membrane in MDA-MD-231 cells treated with control vehicle or NS1643 for 24 h. g Graph showing the effect of NS1643 on cell–cell contact measured by Dispase assay ( n = 6; mean ± SD; * P

    Techniques Used: Translocation Assay, Western Blot, Mouse Assay, Software, Multiple Displacement Amplification, Confocal Microscopy

    Kv11.1 stimulation inhibits primary tumor growth and metastasis in\ a xenograft model of breast cancer. MDA-MB-231 cells were injected subcutaneously into the dorsal flank of NSG mice. When tumors were palpable, the mice were injected intraperitoneally with vehicle alone or Kv11.1 activator NS1643 at 6 mg/kg every 2 days. a Mean tumor volume in mice treated with either vehicle control ( n = 12) or Kv11.1 activator (NS1643) ( n = 12). b Barchart showing the absolute frequencies of liver macrometastases observed in mice treated with either vehicle control or NS1643. c Representative images of livers demonstrating macrometastases in mice treated with either vehicle control or NS1643. d Representative confocal images of human nuclear antigen (HNA; red) and nuclei (DAPI; blue) expression in liver tissue sections from mice treated with either vehicle control or NS1643. e Representative western blot images of HNA expression in liver and ( f ) lung homogenates from mice treated with either vehicle control or NS1643. Quantification of the levels of HNA protein was done using NIH ImageJ software. Data is expressed as mean ± SEM; *p
    Figure Legend Snippet: Kv11.1 stimulation inhibits primary tumor growth and metastasis in\ a xenograft model of breast cancer. MDA-MB-231 cells were injected subcutaneously into the dorsal flank of NSG mice. When tumors were palpable, the mice were injected intraperitoneally with vehicle alone or Kv11.1 activator NS1643 at 6 mg/kg every 2 days. a Mean tumor volume in mice treated with either vehicle control ( n = 12) or Kv11.1 activator (NS1643) ( n = 12). b Barchart showing the absolute frequencies of liver macrometastases observed in mice treated with either vehicle control or NS1643. c Representative images of livers demonstrating macrometastases in mice treated with either vehicle control or NS1643. d Representative confocal images of human nuclear antigen (HNA; red) and nuclei (DAPI; blue) expression in liver tissue sections from mice treated with either vehicle control or NS1643. e Representative western blot images of HNA expression in liver and ( f ) lung homogenates from mice treated with either vehicle control or NS1643. Quantification of the levels of HNA protein was done using NIH ImageJ software. Data is expressed as mean ± SEM; *p

    Techniques Used: Multiple Displacement Amplification, Injection, Mouse Assay, Expressing, Western Blot, Software

    Kv11.1 stimulation inhibits breast cancer cell motility. a Representative images are showing the effect of Kv11.1 stimulation on breast cancer cell motility in wound healing scratch assays. Cells were treated with indicated concentrations of NS1643 (50 μ m ; n = 5; Dotted lines align with the center of the control border cell cluster). b Single-cell tracking of untreated control cells, vehicle control cells (DMSO) and NS1643-treated cells. Bar graphs show the effect of NS1643 on wound closure. Data are mean values ± S.E; * p
    Figure Legend Snippet: Kv11.1 stimulation inhibits breast cancer cell motility. a Representative images are showing the effect of Kv11.1 stimulation on breast cancer cell motility in wound healing scratch assays. Cells were treated with indicated concentrations of NS1643 (50 μ m ; n = 5; Dotted lines align with the center of the control border cell cluster). b Single-cell tracking of untreated control cells, vehicle control cells (DMSO) and NS1643-treated cells. Bar graphs show the effect of NS1643 on wound closure. Data are mean values ± S.E; * p

    Techniques Used: Single Cell Tracking

    Kv11.1 stimulation suppresses the stemness of breast cancer cells. a Representative images are showing the effect of Kv11.1 stimulation on mammosphere forming efficiency (MFE) in SKBR3 and MCF7. Cells were seeded into Ultra-low attachment 6-well plate under DMSO or 50 μ m of NS1643 for 7 days. b The mRNA levels of Oct4, Nanog, and Sox were represented relative to β-actin transcripts. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to qRT-PCR analysis. Data are expressed as mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Kv11.1 stimulation suppresses the stemness of breast cancer cells. a Representative images are showing the effect of Kv11.1 stimulation on mammosphere forming efficiency (MFE) in SKBR3 and MCF7. Cells were seeded into Ultra-low attachment 6-well plate under DMSO or 50 μ m of NS1643 for 7 days. b The mRNA levels of Oct4, Nanog, and Sox were represented relative to β-actin transcripts. Cells were treated with 50 μ m of NS1643 for 24 h, harvested, and subjected to qRT-PCR analysis. Data are expressed as mean ± SD of three independent experiments. * p

    Techniques Used: Quantitative RT-PCR

    Kv11.1 stimulation inhibits reprograms EMT. a Representative western blot images of EMT markers (N-cadherin, Vimentin, CD44, and E-cadherin) in MDA-MB-231, SKBR3, and MCF7 breast cancer cells treated with either vehicle control or NS1643. Cells were treated with 50 μ m of NS1643 for 24 h, harvested and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of Vimentin, CD44, N-Cadherin, and E-cadherin was done using NIH ImageJ software. Data are expressed as mean ± SEM; *p
    Figure Legend Snippet: Kv11.1 stimulation inhibits reprograms EMT. a Representative western blot images of EMT markers (N-cadherin, Vimentin, CD44, and E-cadherin) in MDA-MB-231, SKBR3, and MCF7 breast cancer cells treated with either vehicle control or NS1643. Cells were treated with 50 μ m of NS1643 for 24 h, harvested and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of Vimentin, CD44, N-Cadherin, and E-cadherin was done using NIH ImageJ software. Data are expressed as mean ± SEM; *p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Software

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    Alomone Labs ns8593
    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with <t>NS8593</t> (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p
    Ns8593, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ns8593 - by Bioz Stars, 2022-01
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    Alomone Labs rjr 2403 oxalate
    Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and <t>RJR-2403</t> Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.
    Rjr 2403 Oxalate, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rjr 2403 oxalate/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    Alomone Labs ns1643
    <t>NS1643</t> inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p
    Ns1643, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ns1643/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ns1643 - by Bioz Stars, 2022-01
    94/100 stars
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    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Incubation, Flow Cytometry

    TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, Mouse Assay, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Plex Assay, Derivative Assay, Mouse Assay, Incubation

    Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.

    Journal: The Journal of Biological Chemistry

    Article Title: Selective coactivation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid–induced synaptic dysfunction

    doi: 10.1016/j.jbc.2021.100402

    Figure Lengend Snippet: Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.

    Article Snippet: The following agonists were used in this study: 1 μM PNU-120596 (Alomone Labs), 2 μM RJR-2403 Oxalate (Alomone labs), 1 μM NS-3861 (Tocris Bioscience), and 1 μM Carbamoylcholine chloride (carbachol) (Tocris Bioscience).

    Techniques: Binding Assay, Activity Assay, Expressing, De-Phosphorylation Assay

    Schematic model. A ) Impact of Aβ oligomers. In the hippocampus, nAChRs are prominently expressed on inhibitory interneurons, thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. CaN dephosphorylates the AMPAR subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of LTP. B) Reversal of Aβ-induced synaptic and neuronal dysfunction by co-stimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, co-stimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal LTP. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, non-specific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective co-stimulation of nAChRs as potential therapeutic approaches.

    Journal: bioRxiv

    Article Title: Selective co-activation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid-induced synaptic dysfunction

    doi: 10.1101/2020.11.05.370080

    Figure Lengend Snippet: Schematic model. A ) Impact of Aβ oligomers. In the hippocampus, nAChRs are prominently expressed on inhibitory interneurons, thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. CaN dephosphorylates the AMPAR subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of LTP. B) Reversal of Aβ-induced synaptic and neuronal dysfunction by co-stimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, co-stimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal LTP. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, non-specific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective co-stimulation of nAChRs as potential therapeutic approaches.

    Article Snippet: The following agonists were used in this study: 1μM PNU-120596 (Alomone labs), 2μM RJR-2403 Oxalate (Alomone labs), 1μM NS-3861 (Tocris Bioscience), and 1μM Carbamoylcholine chloride (carbachol) (Tocris Bioscience).

    Techniques: Binding Assay, Activity Assay, Expressing, De-Phosphorylation Assay

    NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Migration, Western Blot, Expressing, Wound Healing Assay

    NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Activation Assay, Western Blot, Expressing

    NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Western Blot, Expressing

    PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Western Blot, Transfection

    NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Western Blot, Expressing