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  • 97
    Millipore u73122
    HCV core protein modulates cellular calcium to stimulate inflammasome activation and IL-1β release. (A) Wheat germ agglutinin staining of THP-1 cells treated with HCV. THP-1 cells were exposed to HCV 30-60minutes. Before fixing with paraformaldehyde, cells were labeled with wheat germ agglutinin to mark the plasma membrane. Red stains the plasma membrane (wheat germ agglutinin), green stains HCV core protein within THP-1 cytoplasm and blue (DAPI) stains the nuclei. (B) Calcium flux in THP-1 cells post stimulation with rHCV-Core or ionomycin or rGFP. (C) immunoblot showing the presence and absence of cleaved IL-1β and caspase-1 in HCV treated cells in the presence of u-73343 (inactive phospholipase C inhibitor) or <t>u-73122</t> (active phospholipase C inhibitor, control) or DMSO (vehicle control). (D) ELISA of IL-1β in THP-1 cells post stimulation with HCV polyU/UC and rHCV-core or HCV polyU/UC and Ng in the presence of u-73122 or control u-73343. (E) TNF primed THP-1 cells were treated with rHCV-core or Ng in the presence and absence of calcium inhibitor (u-73122) or inactive analog (u-73433) or vehicle control (DMSO). Lower panel of (E) shows immunoblot of active IL-1β protein post treatment with calcium inhibitor. Experiments were performed in duplicates and are representative of three or more independent experiments. Data are represented as means and +/- SD, for (D) and (E) *P
    U73122, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u73122/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u73122 - by Bioz Stars, 2021-05
    97/100 stars
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    95
    Tocris u73122
    FGFR2-TRPA1 binding event results in FGFR2 activation, but TRPA1 inhibition. a Western blot showing the efficiency of transfection of Strep-tagged FGFR2, FLAG-tagged FGFR2, and TRPA1-GFP in HEK 293T cells. b , c Duolink assay in HEK 293T cells using antibodies targeted against Strep-tagged and FLAG-tagged FGFR2 in the absence (upper panel) or presence (bottom panel) of TRPA1. Scale bar: 50 μm. PLA signals are quantified in ( c ). d Western blot analysis of differentially transfected and treated HEK 293T cells. The blot was probed with the indicated antibodies. e Western blot analysis of HEK 293T cells transfected with FGFR2 in all samples but differentially transfected with TRPA1. The blot was probed with the indicated antibodies. f Western blot analysis of purified FGFR2 dimerization assay (under non-reducing or reducing conditions) in the presence or absence of purified TRPA1. g Western blot analysis of an in vitro receptor kinase assay in the presence or absence of purified TRPA1. h Luminescent kinase assay results of purified full length FGFR2 (F) and intracellular cytoplasmic region of FGFR2 (IntraF) in the presence or absence of full-length TRPA1 (T) or ΔN. Intensity of the luminescence signal is measured in relative luminescence units (RLU). i – k Bar graphs of the averaged calcium imaging results (Δ F340/380) with representative traces of calcium responses. m Example inward currents, from a holding potential of −60 mV, evoked in untransfected (○) and FGFR2-transfected HCC-44 cells (●) by the application of AITC (1:1000 in the perfusate). Currents were recorded at 2 s intervals for a control period, during application of AITC and then during washout. n Representative images of an invasion assay using differentially transfected HEK 293T that were incubated over night (O/N) in serum starvation media. Lower chamber contains 1% FBS-supplemented media. Images were taken at 20× magnification (scale bar: 100 μm). <t>U73122</t> is PLC-γ1 inhibitor and PLC-γ1_KD designates HEK 293T cell line with knocked down PLC-γ1. o Quantification of the results from ( n ), where the number of invaded cells were counted in 6 different microscopic fields/well. p MTT assay results of differentially transfected HEK 293T cells with or without U0126 (MEK inhibitor) treatment. Percentage change in cell viability for each sample with respect to the untransfected control sample (Non) is depicted on the Y-axis. In the above, error bars, s.d. ( n = 3 biological replicates). * P ≤ 0.05 and ** P ≤ 0.01 were determined by two-tailed Student’s t test
    U73122, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u73122/product/Tocris
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u73122 - by Bioz Stars, 2021-05
    95/100 stars
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    86
    U73122 PLC serpina3k
    <t>SERPINA3K</t> inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P
    Serpina3k, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    Image Search Results


    HCV core protein modulates cellular calcium to stimulate inflammasome activation and IL-1β release. (A) Wheat germ agglutinin staining of THP-1 cells treated with HCV. THP-1 cells were exposed to HCV 30-60minutes. Before fixing with paraformaldehyde, cells were labeled with wheat germ agglutinin to mark the plasma membrane. Red stains the plasma membrane (wheat germ agglutinin), green stains HCV core protein within THP-1 cytoplasm and blue (DAPI) stains the nuclei. (B) Calcium flux in THP-1 cells post stimulation with rHCV-Core or ionomycin or rGFP. (C) immunoblot showing the presence and absence of cleaved IL-1β and caspase-1 in HCV treated cells in the presence of u-73343 (inactive phospholipase C inhibitor) or u-73122 (active phospholipase C inhibitor, control) or DMSO (vehicle control). (D) ELISA of IL-1β in THP-1 cells post stimulation with HCV polyU/UC and rHCV-core or HCV polyU/UC and Ng in the presence of u-73122 or control u-73343. (E) TNF primed THP-1 cells were treated with rHCV-core or Ng in the presence and absence of calcium inhibitor (u-73122) or inactive analog (u-73433) or vehicle control (DMSO). Lower panel of (E) shows immunoblot of active IL-1β protein post treatment with calcium inhibitor. Experiments were performed in duplicates and are representative of three or more independent experiments. Data are represented as means and +/- SD, for (D) and (E) *P

    Journal: PLoS Pathogens

    Article Title: Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation

    doi: 10.1371/journal.ppat.1007593

    Figure Lengend Snippet: HCV core protein modulates cellular calcium to stimulate inflammasome activation and IL-1β release. (A) Wheat germ agglutinin staining of THP-1 cells treated with HCV. THP-1 cells were exposed to HCV 30-60minutes. Before fixing with paraformaldehyde, cells were labeled with wheat germ agglutinin to mark the plasma membrane. Red stains the plasma membrane (wheat germ agglutinin), green stains HCV core protein within THP-1 cytoplasm and blue (DAPI) stains the nuclei. (B) Calcium flux in THP-1 cells post stimulation with rHCV-Core or ionomycin or rGFP. (C) immunoblot showing the presence and absence of cleaved IL-1β and caspase-1 in HCV treated cells in the presence of u-73343 (inactive phospholipase C inhibitor) or u-73122 (active phospholipase C inhibitor, control) or DMSO (vehicle control). (D) ELISA of IL-1β in THP-1 cells post stimulation with HCV polyU/UC and rHCV-core or HCV polyU/UC and Ng in the presence of u-73122 or control u-73343. (E) TNF primed THP-1 cells were treated with rHCV-core or Ng in the presence and absence of calcium inhibitor (u-73122) or inactive analog (u-73433) or vehicle control (DMSO). Lower panel of (E) shows immunoblot of active IL-1β protein post treatment with calcium inhibitor. Experiments were performed in duplicates and are representative of three or more independent experiments. Data are represented as means and +/- SD, for (D) and (E) *P

    Article Snippet: Nigericin, ATP, D609, u-73343, u-73122, DMSO, and PMA (Sigma).

    Techniques: Activation Assay, Staining, Labeling, Enzyme-linked Immunosorbent Assay

    FGFR2-TRPA1 binding event results in FGFR2 activation, but TRPA1 inhibition. a Western blot showing the efficiency of transfection of Strep-tagged FGFR2, FLAG-tagged FGFR2, and TRPA1-GFP in HEK 293T cells. b , c Duolink assay in HEK 293T cells using antibodies targeted against Strep-tagged and FLAG-tagged FGFR2 in the absence (upper panel) or presence (bottom panel) of TRPA1. Scale bar: 50 μm. PLA signals are quantified in ( c ). d Western blot analysis of differentially transfected and treated HEK 293T cells. The blot was probed with the indicated antibodies. e Western blot analysis of HEK 293T cells transfected with FGFR2 in all samples but differentially transfected with TRPA1. The blot was probed with the indicated antibodies. f Western blot analysis of purified FGFR2 dimerization assay (under non-reducing or reducing conditions) in the presence or absence of purified TRPA1. g Western blot analysis of an in vitro receptor kinase assay in the presence or absence of purified TRPA1. h Luminescent kinase assay results of purified full length FGFR2 (F) and intracellular cytoplasmic region of FGFR2 (IntraF) in the presence or absence of full-length TRPA1 (T) or ΔN. Intensity of the luminescence signal is measured in relative luminescence units (RLU). i – k Bar graphs of the averaged calcium imaging results (Δ F340/380) with representative traces of calcium responses. m Example inward currents, from a holding potential of −60 mV, evoked in untransfected (○) and FGFR2-transfected HCC-44 cells (●) by the application of AITC (1:1000 in the perfusate). Currents were recorded at 2 s intervals for a control period, during application of AITC and then during washout. n Representative images of an invasion assay using differentially transfected HEK 293T that were incubated over night (O/N) in serum starvation media. Lower chamber contains 1% FBS-supplemented media. Images were taken at 20× magnification (scale bar: 100 μm). U73122 is PLC-γ1 inhibitor and PLC-γ1_KD designates HEK 293T cell line with knocked down PLC-γ1. o Quantification of the results from ( n ), where the number of invaded cells were counted in 6 different microscopic fields/well. p MTT assay results of differentially transfected HEK 293T cells with or without U0126 (MEK inhibitor) treatment. Percentage change in cell viability for each sample with respect to the untransfected control sample (Non) is depicted on the Y-axis. In the above, error bars, s.d. ( n = 3 biological replicates). * P ≤ 0.05 and ** P ≤ 0.01 were determined by two-tailed Student’s t test

    Journal: Nature Communications

    Article Title: TRPA1–FGFR2 binding event is a regulatory oncogenic driver modulated by miRNA-142-3p

    doi: 10.1038/s41467-017-00983-w

    Figure Lengend Snippet: FGFR2-TRPA1 binding event results in FGFR2 activation, but TRPA1 inhibition. a Western blot showing the efficiency of transfection of Strep-tagged FGFR2, FLAG-tagged FGFR2, and TRPA1-GFP in HEK 293T cells. b , c Duolink assay in HEK 293T cells using antibodies targeted against Strep-tagged and FLAG-tagged FGFR2 in the absence (upper panel) or presence (bottom panel) of TRPA1. Scale bar: 50 μm. PLA signals are quantified in ( c ). d Western blot analysis of differentially transfected and treated HEK 293T cells. The blot was probed with the indicated antibodies. e Western blot analysis of HEK 293T cells transfected with FGFR2 in all samples but differentially transfected with TRPA1. The blot was probed with the indicated antibodies. f Western blot analysis of purified FGFR2 dimerization assay (under non-reducing or reducing conditions) in the presence or absence of purified TRPA1. g Western blot analysis of an in vitro receptor kinase assay in the presence or absence of purified TRPA1. h Luminescent kinase assay results of purified full length FGFR2 (F) and intracellular cytoplasmic region of FGFR2 (IntraF) in the presence or absence of full-length TRPA1 (T) or ΔN. Intensity of the luminescence signal is measured in relative luminescence units (RLU). i – k Bar graphs of the averaged calcium imaging results (Δ F340/380) with representative traces of calcium responses. m Example inward currents, from a holding potential of −60 mV, evoked in untransfected (○) and FGFR2-transfected HCC-44 cells (●) by the application of AITC (1:1000 in the perfusate). Currents were recorded at 2 s intervals for a control period, during application of AITC and then during washout. n Representative images of an invasion assay using differentially transfected HEK 293T that were incubated over night (O/N) in serum starvation media. Lower chamber contains 1% FBS-supplemented media. Images were taken at 20× magnification (scale bar: 100 μm). U73122 is PLC-γ1 inhibitor and PLC-γ1_KD designates HEK 293T cell line with knocked down PLC-γ1. o Quantification of the results from ( n ), where the number of invaded cells were counted in 6 different microscopic fields/well. p MTT assay results of differentially transfected HEK 293T cells with or without U0126 (MEK inhibitor) treatment. Percentage change in cell viability for each sample with respect to the untransfected control sample (Non) is depicted on the Y-axis. In the above, error bars, s.d. ( n = 3 biological replicates). * P ≤ 0.05 and ** P ≤ 0.01 were determined by two-tailed Student’s t test

    Article Snippet: U73122 (#1268) was purchased from TOCRIS.

    Techniques: Binding Assay, Activation Assay, Inhibition, Western Blot, Transfection, Proximity Ligation Assay, Purification, In Vitro, Kinase Assay, Imaging, Invasion Assay, Incubation, Planar Chromatography, MTT Assay, Two Tailed Test

    SERPINA3K inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Planar Chromatography, Negative Control, Fluorescence, MTT Assay, Western Blot

    SERPINA3K does not chelate free calcium. Free [Ca 2+ ] was measured by the fluorescence of the Fluo-4 (pentapotassium salt). (A) A Ca 2+ /Fluo-4 binding curve was plotted with different concentrations of CaCl 2 and 10 µM Fluo-4. (B) SERPINA3K (10 µM) was incubated with 10 µM CaCl 2 or a cell lysate in the presence of 10 µM Fluo-4. EGTA (10 µM) and BSA (10 µM) were used as positive and negative controls, respectively. Free [Ca 2+ ] was measured (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K does not chelate free calcium. Free [Ca 2+ ] was measured by the fluorescence of the Fluo-4 (pentapotassium salt). (A) A Ca 2+ /Fluo-4 binding curve was plotted with different concentrations of CaCl 2 and 10 µM Fluo-4. (B) SERPINA3K (10 µM) was incubated with 10 µM CaCl 2 or a cell lysate in the presence of 10 µM Fluo-4. EGTA (10 µM) and BSA (10 µM) were used as positive and negative controls, respectively. Free [Ca 2+ ] was measured (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Fluorescence, Binding Assay, Incubation

    SERPINA3K binds to Müller-derived rMC-1 cells. (A) The rMC-1 cells were incubated with increasing concentrations of FITC-SERPINA3K or FITC-BSA for 1 h. (B) The rMC-1 cells were incubated with 400 nM FITC-SERPINA3K in the presence of different concentrations of unlabeled SERPINA3K or BSA for 1 h. Then the cells were washed three times with PBS. The fluorescence in the cells was measured by a fluorometer using 485/530 nm filter (mean±SEM, n = 8).

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K binds to Müller-derived rMC-1 cells. (A) The rMC-1 cells were incubated with increasing concentrations of FITC-SERPINA3K or FITC-BSA for 1 h. (B) The rMC-1 cells were incubated with 400 nM FITC-SERPINA3K in the presence of different concentrations of unlabeled SERPINA3K or BSA for 1 h. Then the cells were washed three times with PBS. The fluorescence in the cells was measured by a fluorometer using 485/530 nm filter (mean±SEM, n = 8).

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Incubation, Fluorescence

    SERPINA3K blocks intracellular calcium overload induced by H 2 O 2 . (A) Elevated intracellular [Ca 2+ ] in cells exposed to H 2 O 2 . The Müller-derived rMC-1 cells were treated with 400 µM H 2 O 2 for various durations as indicated. TBHP (20 µM) was used as control. Intracellular [Ca 2+ ] was measured by the fluorescence of the probe Fluo-4/AM (mean±SEM, n = 8). (B–E) The cells were pre-treated with 1 µM SERPINA3K or BSA for 1 h followed by the H 2 O 2 exposure. BSA was added to bring the total protein concentration to the same in each well. Representative fluorescence images were captured under a fluorescent microscope from untreated cells (B), cells pre-treated with 1 µM BSA (C) and with 1 µM SERPINA3K (D) followed by a 3-h exposure to H 2 O 2 . (E) [Ca 2+ ] in cells pre-treated with various concentrations of SERPINA3K prior to the exposure to H 2 O 2 (mean±SEM, n = 6). (F) The protective effects of 1 µM SERPINA3K and 10 µM BAPTA/AM (a calcium chelator) were quantified using the MTT assay (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K blocks intracellular calcium overload induced by H 2 O 2 . (A) Elevated intracellular [Ca 2+ ] in cells exposed to H 2 O 2 . The Müller-derived rMC-1 cells were treated with 400 µM H 2 O 2 for various durations as indicated. TBHP (20 µM) was used as control. Intracellular [Ca 2+ ] was measured by the fluorescence of the probe Fluo-4/AM (mean±SEM, n = 8). (B–E) The cells were pre-treated with 1 µM SERPINA3K or BSA for 1 h followed by the H 2 O 2 exposure. BSA was added to bring the total protein concentration to the same in each well. Representative fluorescence images were captured under a fluorescent microscope from untreated cells (B), cells pre-treated with 1 µM BSA (C) and with 1 µM SERPINA3K (D) followed by a 3-h exposure to H 2 O 2 . (E) [Ca 2+ ] in cells pre-treated with various concentrations of SERPINA3K prior to the exposure to H 2 O 2 (mean±SEM, n = 6). (F) The protective effects of 1 µM SERPINA3K and 10 µM BAPTA/AM (a calcium chelator) were quantified using the MTT assay (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Fluorescence, Protein Concentration, Microscopy, MTT Assay

    Protective effects of SERPINA3K on rMC-1 cells against H 2 O 2 -induced necrosis. Müller-derived rMC-1 cells were pre-treated with SERPINA3K or BSA for 1 h and then exposed to 400 µM H 2 O 2 for 8 h (A–C, G) and 4 h (D–F, H, I). The protein concentration in the media was brought to 1 µM in each well by addition of BSA. (A–C) Representative phase contrast images showing cell morphology in untreated control (A), cells treated with 1 µM BSA and H 2 O 2 (B), and cells treated with 1 µM SERPINA3K and H 2 O 2 (C). Scale bar, 50 µm. (G) Viable cells were quantified using the MTT assay (mean±SEM, n = 3). (D–F, H) The viable and necrotic cells were analyzed by flow cytometry following staining with PI and Annexin-V; (D) Untreated control; (E) treated with 1 µM BSA and H 2 O 2 ; (F) treated with 1 µM SERPINA3K and H 2 O 2 ; (H) quantification of necrotic cells as percentages of total cells (mean±SEM, n = 3). (I) DNA laddering analysis showed no apparent DNA fragmentation in the H 2 O 2 -treated cells. Lane 1, control cells; 2, cells treated with H 2 O 2 for 4 h; 3, cells treated with H 2 O 2 and SERPINA3K, and 4, over-grown cells as positive control. # P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Protective effects of SERPINA3K on rMC-1 cells against H 2 O 2 -induced necrosis. Müller-derived rMC-1 cells were pre-treated with SERPINA3K or BSA for 1 h and then exposed to 400 µM H 2 O 2 for 8 h (A–C, G) and 4 h (D–F, H, I). The protein concentration in the media was brought to 1 µM in each well by addition of BSA. (A–C) Representative phase contrast images showing cell morphology in untreated control (A), cells treated with 1 µM BSA and H 2 O 2 (B), and cells treated with 1 µM SERPINA3K and H 2 O 2 (C). Scale bar, 50 µm. (G) Viable cells were quantified using the MTT assay (mean±SEM, n = 3). (D–F, H) The viable and necrotic cells were analyzed by flow cytometry following staining with PI and Annexin-V; (D) Untreated control; (E) treated with 1 µM BSA and H 2 O 2 ; (F) treated with 1 µM SERPINA3K and H 2 O 2 ; (H) quantification of necrotic cells as percentages of total cells (mean±SEM, n = 3). (I) DNA laddering analysis showed no apparent DNA fragmentation in the H 2 O 2 -treated cells. Lane 1, control cells; 2, cells treated with H 2 O 2 for 4 h; 3, cells treated with H 2 O 2 and SERPINA3K, and 4, over-grown cells as positive control. # P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Protein Concentration, MTT Assay, Flow Cytometry, Cytometry, Staining, DNA Laddering, Positive Control

    Lack of effect of SERPINA3K on the intracellular ROS generation in cells exposed to H 2 O 2 . (A, B) The rMC-1 cells were treated with 400 µM H 2 O 2 (A) or 20 µM TBHP (B) for 1, 2, 4 and 8 h in the absence or presence of 1 µM BSA or SERPINA3K. Viable cells were quantified using MTT assay (mean±SEM, n = 3). (C) The rMC-1 cells were exposed to 400 µM H 2 O 2 or 20 µM TBHP for various durations as indicated, to define the time point for the ROS increase. Note that ROS levels were elevated by H 2 O 2 as early as 15 min, while the ROS generation induced by TBHP occurred at 4 h of the treatment. (D E) rMC-1 cells were pre-treated with various concentrations of SERPINA3K for 1 h. The medium was supplemented with BSA to the same total protein concentration in each well. The cells were then exposed to 400 µM H 2 O 2 for 15 min (D) or to 20 µM TBHP for 4 h (E). The intracellular ROS generation was measured using CM-H2DCFDA as a probe. Values are arbitrary fluorescence units (mean±SEM, n = 8). SERPINA3K blocked the ROS increase induced by TBHP but not that by H 2 O 2 . * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Lack of effect of SERPINA3K on the intracellular ROS generation in cells exposed to H 2 O 2 . (A, B) The rMC-1 cells were treated with 400 µM H 2 O 2 (A) or 20 µM TBHP (B) for 1, 2, 4 and 8 h in the absence or presence of 1 µM BSA or SERPINA3K. Viable cells were quantified using MTT assay (mean±SEM, n = 3). (C) The rMC-1 cells were exposed to 400 µM H 2 O 2 or 20 µM TBHP for various durations as indicated, to define the time point for the ROS increase. Note that ROS levels were elevated by H 2 O 2 as early as 15 min, while the ROS generation induced by TBHP occurred at 4 h of the treatment. (D E) rMC-1 cells were pre-treated with various concentrations of SERPINA3K for 1 h. The medium was supplemented with BSA to the same total protein concentration in each well. The cells were then exposed to 400 µM H 2 O 2 for 15 min (D) or to 20 µM TBHP for 4 h (E). The intracellular ROS generation was measured using CM-H2DCFDA as a probe. Values are arbitrary fluorescence units (mean±SEM, n = 8). SERPINA3K blocked the ROS increase induced by TBHP but not that by H 2 O 2 . * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: MTT Assay, Protein Concentration, Fluorescence

    Protective effects of SERPINA3K in retinal cells. Y79 (A), R28 (B), RPE (C) and rMC-1 cells (D) were exposed to 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K or BSA for 8 h, or incubated with 1 µM SERPINA3K alone for 8 h. Viable cells were quantified by MTT assay (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Protective effects of SERPINA3K in retinal cells. Y79 (A), R28 (B), RPE (C) and rMC-1 cells (D) were exposed to 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K or BSA for 8 h, or incubated with 1 µM SERPINA3K alone for 8 h. Viable cells were quantified by MTT assay (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Incubation, MTT Assay