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    Alomone Labs anti kv1 3
    PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting <t>Kv1.3</t> in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p
    Anti Kv1 3, 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/anti kv1 3/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family"

    Article Title: Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

    Journal: Redox Biology

    doi: 10.1016/j.redox.2020.101705

    PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p
    Figure Legend Snippet: PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Techniques Used: Solubility, In Vitro, Ex Vivo, Proliferation Assay, Blocking Assay, Derivative Assay, Cell Surface Hydrophobicity, Cell Culture, FACS

    PAP-1-MHEG acts on mitochondria, affecting energetic homeostasis. a) Dissipation of the mitochondrial membrane potential as reported by TMR fluorescence. Representative images (n = 3 separate experiments) of B16F10 cells. FCCP was used as positive control. b) Mitochondrial ROS production as reported by the development of MitoSOX fluorescence. Representative images (n = 3 separate experiments). Antimycin A was used as positive control. c - d) as in a and b, B-CLL cells. The plots show averages ± SEM (n ≥ 3 separate experiments). e) Effect of PAP-1-MHEG on the Oxygen Consumption Rate (OCR) of B16F10 cells. Values were normalized with respect to basal respiration recorded before compound addition. Mean values ± SEM from six experiments are shown. f) ATP content of B16F10 cells after the addition of the indicated compounds. Medium glucose was replaced with 2-deoxyglucose (2DG) to inhibit glycolysis. Oligomycin was used as positive control. Values are reported as percentage of luciferase signal with respect to the untreated sample (“ref”). Data are presented as means ± SEM (n = 5). g) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with 0.1% DMSO, as negative control, or with PAP-1-MHEG as indicated in the figure. The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected using the TOPflash plasmid. The values are reported as the percentage of luciferase signal with respect to the negative control (“ref”). Values are means ± SEM (n = 5). h) Activity of respiratory chain complex I, III, and V was assessed in vitro in isolated mouse liver mitochondria upon addition of PAP-1-MHEG. DMSO was used as negative control. Means ± SEM of 5 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. i) Respiratory chain complex I activity was measured as in h. Where indicated, isolated mitochondria were pre-incubated with the peptide toxin MgTx, to block the Kv1.3 channel before the addition of PAP-1-MHEG. Means ± SEM of 3 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. For all the panels statistical significance (ANOVA or Student's t-test) was determined (* = p
    Figure Legend Snippet: PAP-1-MHEG acts on mitochondria, affecting energetic homeostasis. a) Dissipation of the mitochondrial membrane potential as reported by TMR fluorescence. Representative images (n = 3 separate experiments) of B16F10 cells. FCCP was used as positive control. b) Mitochondrial ROS production as reported by the development of MitoSOX fluorescence. Representative images (n = 3 separate experiments). Antimycin A was used as positive control. c - d) as in a and b, B-CLL cells. The plots show averages ± SEM (n ≥ 3 separate experiments). e) Effect of PAP-1-MHEG on the Oxygen Consumption Rate (OCR) of B16F10 cells. Values were normalized with respect to basal respiration recorded before compound addition. Mean values ± SEM from six experiments are shown. f) ATP content of B16F10 cells after the addition of the indicated compounds. Medium glucose was replaced with 2-deoxyglucose (2DG) to inhibit glycolysis. Oligomycin was used as positive control. Values are reported as percentage of luciferase signal with respect to the untreated sample (“ref”). Data are presented as means ± SEM (n = 5). g) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with 0.1% DMSO, as negative control, or with PAP-1-MHEG as indicated in the figure. The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected using the TOPflash plasmid. The values are reported as the percentage of luciferase signal with respect to the negative control (“ref”). Values are means ± SEM (n = 5). h) Activity of respiratory chain complex I, III, and V was assessed in vitro in isolated mouse liver mitochondria upon addition of PAP-1-MHEG. DMSO was used as negative control. Means ± SEM of 5 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. i) Respiratory chain complex I activity was measured as in h. Where indicated, isolated mitochondria were pre-incubated with the peptide toxin MgTx, to block the Kv1.3 channel before the addition of PAP-1-MHEG. Means ± SEM of 3 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. For all the panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Techniques Used: Fluorescence, Positive Control, Luciferase, Activity Assay, Negative Control, Transfection, Plasmid Preparation, In Vitro, Isolation, Incubation, Blocking Assay

    PAP-1-MHEG induces apoptosis and inhibits respiratory chain complex I. a) Chemical structures of the molecules synthesized and characterized in the present study. b) Whole-cell patch clamp experiments were performed on Jurkat cells. Kv1.3 potassium currents were measured before and after the addition of 1 μM of each indicated compound. Means ± SEM of three independent experiments are shown. c) Viability MTS assays in B16F10 cells. Values are means ± SEM with respect to the untreated sample (n = 4). d) Apoptosis of B16F10 cells assayed using Annexin-V. Cells were treated or not with 20 μM of the indicated compounds for 24 h. 2 μM staurosporine was used as positive control. Shown are the means ± SEM of percentages of Annexin-V-positive cells with respect to control (untreated) (n = 4). e-f) OCR was measured in B16F10 cells. As in e, all compounds were tested at 20 μM. Panel f plots OCR values upon addition of POP-MHEG at different concentrations. Values were normalized with respect to basal respiration recorded before addition of each compound. Mean values ± SEM from 5 experiments are shown . g) Quantitative analysis of OCR values (n = 5). h-j )Activity of respiratory chain complex I ( h ) was measured in vitro in isolated mouse liver mitochondria after the addition of the indicated compounds at 20 μM. DMSO (0.1%) was used as negative control. Mean ± SEM of 3–5 independent experiments are shown and data are expressed as percentage of the control. See also Supplementary Fig. 1a-c . i) Dose-dependent effects of PAP-1 MTEG on complex I activity, measured as in 3h. Values are expressed as means of percentages of controls (0.1% DMSO) ± SEM (n = 7). j) Isolated mitochondria were pre-incubated or not with the peptidic toxin MgTx (500 nM), to block mtKv1.3 before the addition of POP-MHEG. Respiratory chain complex I activity was then measured. Means ± SEM of 3 independent experiments are shown. The data are expressed as percentage of controls (0.1% DMSO).
    Figure Legend Snippet: PAP-1-MHEG induces apoptosis and inhibits respiratory chain complex I. a) Chemical structures of the molecules synthesized and characterized in the present study. b) Whole-cell patch clamp experiments were performed on Jurkat cells. Kv1.3 potassium currents were measured before and after the addition of 1 μM of each indicated compound. Means ± SEM of three independent experiments are shown. c) Viability MTS assays in B16F10 cells. Values are means ± SEM with respect to the untreated sample (n = 4). d) Apoptosis of B16F10 cells assayed using Annexin-V. Cells were treated or not with 20 μM of the indicated compounds for 24 h. 2 μM staurosporine was used as positive control. Shown are the means ± SEM of percentages of Annexin-V-positive cells with respect to control (untreated) (n = 4). e-f) OCR was measured in B16F10 cells. As in e, all compounds were tested at 20 μM. Panel f plots OCR values upon addition of POP-MHEG at different concentrations. Values were normalized with respect to basal respiration recorded before addition of each compound. Mean values ± SEM from 5 experiments are shown . g) Quantitative analysis of OCR values (n = 5). h-j )Activity of respiratory chain complex I ( h ) was measured in vitro in isolated mouse liver mitochondria after the addition of the indicated compounds at 20 μM. DMSO (0.1%) was used as negative control. Mean ± SEM of 3–5 independent experiments are shown and data are expressed as percentage of the control. See also Supplementary Fig. 1a-c . i) Dose-dependent effects of PAP-1 MTEG on complex I activity, measured as in 3h. Values are expressed as means of percentages of controls (0.1% DMSO) ± SEM (n = 7). j) Isolated mitochondria were pre-incubated or not with the peptidic toxin MgTx (500 nM), to block mtKv1.3 before the addition of POP-MHEG. Respiratory chain complex I activity was then measured. Means ± SEM of 3 independent experiments are shown. The data are expressed as percentage of controls (0.1% DMSO).

    Techniques Used: Synthesized, Patch Clamp, Positive Control, Activity Assay, In Vitro, Isolation, Negative Control, Incubation, Blocking Assay

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    Alomone Labs anti kv1 3
    PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting <t>Kv1.3</t> in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p
    Anti Kv1 3, 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/anti kv1 3/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 - by Bioz Stars, 2022-05
    94/100 stars
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    PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Journal: Redox Biology

    Article Title: Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

    doi: 10.1016/j.redox.2020.101705

    Figure Lengend Snippet: PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Kv1.3 (1:200 in TBS, rabbit polyclonal, Alomone Labs APC-101); anti-NDUFA9 (1:1000 in 5% skim milk, mouse monoclonal, Abcam ab14713).

    Techniques: Solubility, In Vitro, Ex Vivo, Proliferation Assay, Blocking Assay, Derivative Assay, Cell Surface Hydrophobicity, Cell Culture, FACS

    PAP-1-MHEG acts on mitochondria, affecting energetic homeostasis. a) Dissipation of the mitochondrial membrane potential as reported by TMR fluorescence. Representative images (n = 3 separate experiments) of B16F10 cells. FCCP was used as positive control. b) Mitochondrial ROS production as reported by the development of MitoSOX fluorescence. Representative images (n = 3 separate experiments). Antimycin A was used as positive control. c - d) as in a and b, B-CLL cells. The plots show averages ± SEM (n ≥ 3 separate experiments). e) Effect of PAP-1-MHEG on the Oxygen Consumption Rate (OCR) of B16F10 cells. Values were normalized with respect to basal respiration recorded before compound addition. Mean values ± SEM from six experiments are shown. f) ATP content of B16F10 cells after the addition of the indicated compounds. Medium glucose was replaced with 2-deoxyglucose (2DG) to inhibit glycolysis. Oligomycin was used as positive control. Values are reported as percentage of luciferase signal with respect to the untreated sample (“ref”). Data are presented as means ± SEM (n = 5). g) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with 0.1% DMSO, as negative control, or with PAP-1-MHEG as indicated in the figure. The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected using the TOPflash plasmid. The values are reported as the percentage of luciferase signal with respect to the negative control (“ref”). Values are means ± SEM (n = 5). h) Activity of respiratory chain complex I, III, and V was assessed in vitro in isolated mouse liver mitochondria upon addition of PAP-1-MHEG. DMSO was used as negative control. Means ± SEM of 5 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. i) Respiratory chain complex I activity was measured as in h. Where indicated, isolated mitochondria were pre-incubated with the peptide toxin MgTx, to block the Kv1.3 channel before the addition of PAP-1-MHEG. Means ± SEM of 3 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. For all the panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Journal: Redox Biology

    Article Title: Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

    doi: 10.1016/j.redox.2020.101705

    Figure Lengend Snippet: PAP-1-MHEG acts on mitochondria, affecting energetic homeostasis. a) Dissipation of the mitochondrial membrane potential as reported by TMR fluorescence. Representative images (n = 3 separate experiments) of B16F10 cells. FCCP was used as positive control. b) Mitochondrial ROS production as reported by the development of MitoSOX fluorescence. Representative images (n = 3 separate experiments). Antimycin A was used as positive control. c - d) as in a and b, B-CLL cells. The plots show averages ± SEM (n ≥ 3 separate experiments). e) Effect of PAP-1-MHEG on the Oxygen Consumption Rate (OCR) of B16F10 cells. Values were normalized with respect to basal respiration recorded before compound addition. Mean values ± SEM from six experiments are shown. f) ATP content of B16F10 cells after the addition of the indicated compounds. Medium glucose was replaced with 2-deoxyglucose (2DG) to inhibit glycolysis. Oligomycin was used as positive control. Values are reported as percentage of luciferase signal with respect to the untreated sample (“ref”). Data are presented as means ± SEM (n = 5). g) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with 0.1% DMSO, as negative control, or with PAP-1-MHEG as indicated in the figure. The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected using the TOPflash plasmid. The values are reported as the percentage of luciferase signal with respect to the negative control (“ref”). Values are means ± SEM (n = 5). h) Activity of respiratory chain complex I, III, and V was assessed in vitro in isolated mouse liver mitochondria upon addition of PAP-1-MHEG. DMSO was used as negative control. Means ± SEM of 5 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. i) Respiratory chain complex I activity was measured as in h. Where indicated, isolated mitochondria were pre-incubated with the peptide toxin MgTx, to block the Kv1.3 channel before the addition of PAP-1-MHEG. Means ± SEM of 3 independent experiments are shown and data are expressed as percentage of the DMSO-treated samples. For all the panels statistical significance (ANOVA or Student's t-test) was determined (* = p

    Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Kv1.3 (1:200 in TBS, rabbit polyclonal, Alomone Labs APC-101); anti-NDUFA9 (1:1000 in 5% skim milk, mouse monoclonal, Abcam ab14713).

    Techniques: Fluorescence, Positive Control, Luciferase, Activity Assay, Negative Control, Transfection, Plasmid Preparation, In Vitro, Isolation, Incubation, Blocking Assay

    PAP-1-MHEG induces apoptosis and inhibits respiratory chain complex I. a) Chemical structures of the molecules synthesized and characterized in the present study. b) Whole-cell patch clamp experiments were performed on Jurkat cells. Kv1.3 potassium currents were measured before and after the addition of 1 μM of each indicated compound. Means ± SEM of three independent experiments are shown. c) Viability MTS assays in B16F10 cells. Values are means ± SEM with respect to the untreated sample (n = 4). d) Apoptosis of B16F10 cells assayed using Annexin-V. Cells were treated or not with 20 μM of the indicated compounds for 24 h. 2 μM staurosporine was used as positive control. Shown are the means ± SEM of percentages of Annexin-V-positive cells with respect to control (untreated) (n = 4). e-f) OCR was measured in B16F10 cells. As in e, all compounds were tested at 20 μM. Panel f plots OCR values upon addition of POP-MHEG at different concentrations. Values were normalized with respect to basal respiration recorded before addition of each compound. Mean values ± SEM from 5 experiments are shown . g) Quantitative analysis of OCR values (n = 5). h-j )Activity of respiratory chain complex I ( h ) was measured in vitro in isolated mouse liver mitochondria after the addition of the indicated compounds at 20 μM. DMSO (0.1%) was used as negative control. Mean ± SEM of 3–5 independent experiments are shown and data are expressed as percentage of the control. See also Supplementary Fig. 1a-c . i) Dose-dependent effects of PAP-1 MTEG on complex I activity, measured as in 3h. Values are expressed as means of percentages of controls (0.1% DMSO) ± SEM (n = 7). j) Isolated mitochondria were pre-incubated or not with the peptidic toxin MgTx (500 nM), to block mtKv1.3 before the addition of POP-MHEG. Respiratory chain complex I activity was then measured. Means ± SEM of 3 independent experiments are shown. The data are expressed as percentage of controls (0.1% DMSO).

    Journal: Redox Biology

    Article Title: Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

    doi: 10.1016/j.redox.2020.101705

    Figure Lengend Snippet: PAP-1-MHEG induces apoptosis and inhibits respiratory chain complex I. a) Chemical structures of the molecules synthesized and characterized in the present study. b) Whole-cell patch clamp experiments were performed on Jurkat cells. Kv1.3 potassium currents were measured before and after the addition of 1 μM of each indicated compound. Means ± SEM of three independent experiments are shown. c) Viability MTS assays in B16F10 cells. Values are means ± SEM with respect to the untreated sample (n = 4). d) Apoptosis of B16F10 cells assayed using Annexin-V. Cells were treated or not with 20 μM of the indicated compounds for 24 h. 2 μM staurosporine was used as positive control. Shown are the means ± SEM of percentages of Annexin-V-positive cells with respect to control (untreated) (n = 4). e-f) OCR was measured in B16F10 cells. As in e, all compounds were tested at 20 μM. Panel f plots OCR values upon addition of POP-MHEG at different concentrations. Values were normalized with respect to basal respiration recorded before addition of each compound. Mean values ± SEM from 5 experiments are shown . g) Quantitative analysis of OCR values (n = 5). h-j )Activity of respiratory chain complex I ( h ) was measured in vitro in isolated mouse liver mitochondria after the addition of the indicated compounds at 20 μM. DMSO (0.1%) was used as negative control. Mean ± SEM of 3–5 independent experiments are shown and data are expressed as percentage of the control. See also Supplementary Fig. 1a-c . i) Dose-dependent effects of PAP-1 MTEG on complex I activity, measured as in 3h. Values are expressed as means of percentages of controls (0.1% DMSO) ± SEM (n = 7). j) Isolated mitochondria were pre-incubated or not with the peptidic toxin MgTx (500 nM), to block mtKv1.3 before the addition of POP-MHEG. Respiratory chain complex I activity was then measured. Means ± SEM of 3 independent experiments are shown. The data are expressed as percentage of controls (0.1% DMSO).

    Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Kv1.3 (1:200 in TBS, rabbit polyclonal, Alomone Labs APC-101); anti-NDUFA9 (1:1000 in 5% skim milk, mouse monoclonal, Abcam ab14713).

    Techniques: Synthesized, Patch Clamp, Positive Control, Activity Assay, In Vitro, Isolation, Negative Control, Incubation, Blocking Assay