Structured Review

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
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1) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

2) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

3) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

4) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

5) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

6) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

7) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

8) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

9) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

10) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

11) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

12) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

13) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

14) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

15) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

16) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

17) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

18) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

19) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

20) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

21) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

22) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

23) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

24) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

25) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

26) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

27) Product Images from "SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004077

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Incubation, MTT Assay

Related Articles

MTT Assay:

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: .. Correlated with their effects on Ca2+ , MTT assay demonstrated that SERPINA3K, U73122 and BAPTA, but not U0126 and Wortmannin, had protective effects on cell viability under the H2 O2 stress , suggesting that the reduction of intracellular Ca2+ induced by SERPINA3K is not through the ERK or PI3K pathways. .. Consistently, the protective effect of SERPINA3K, but not that of BAPTA, was offset by 10 µM m-3M3FBS, a specific PLC activator, suggesting that SERPINA3K prevents calcium overload through blocking PLC activation.

Incubation:

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: .. The cells treated with SERPINA3K and U73122 showed significantly lower intracellular Ca2+ concentrations than that in the cells treated with U0126, Wortmannin and BSA, after 1.5 to 4 h incubation ( ). .. Correlated with their effects on Ca2+ , MTT assay demonstrated that SERPINA3K, U73122 and BAPTA, but not U0126 and Wortmannin, had protective effects on cell viability under the H2 O2 stress , suggesting that the reduction of intracellular Ca2+ induced by SERPINA3K is not through the ERK or PI3K pathways.

other:

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: At the dose used, SERPINA3K did not induce any detectable decrease in cell viabilities in Müller cells.

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: As SERPINA3K is an extracellular serpin, we have investigated if its protective effect is mediated through a receptor.

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: Cultures were always pretreated for 1 hr with SERPINA3K at concentrations indicated for each experiment, to ensure that the protein bound to the cells before the H2 O2 exposure.

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: Our calcium chelating experiments showed that SERPINA3K did not chelate free calcium ion in a CaCl2 solution or in cell lysates.

Binding Assay:

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
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. .. Cultured rMC-1 cells were incubated with increasing concentrations (16–4096 nM) of FITC-labeled SERPINA3K for 1 h followed by 3 washes with PBS.

Blocking Assay:

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload
Article Snippet: .. Using the CM-H2DCFDA probe, we showed that SERPINA3K did not block the increase of intracellular ROS levels in the cells exposed to H2 O2 , although it reduced cell death. .. Our calcium chelating experiments showed that SERPINA3K did not chelate free calcium ion in a CaCl2 solution or in cell lysates.

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    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
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    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