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    Neuromics human brain mv endothelial cells
    The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular <t>endothelial</t> cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).
    Human Brain Mv Endothelial Cells, supplied by Neuromics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus"

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2022.836634

    The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).
    Figure Legend Snippet: The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).

    Techniques Used: Expressing, Cell Culture, Concentration Assay, Incubation, Real-time Polymerase Chain Reaction, Comparison

    PKCε activation increases PKCε, MnSOD, and VEGF protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were treated with 500 μM TBHP for 1 h and incubated in new culture medium with or without the PKCε activators bryostatin (bry, 25 nM) and DCPLA-ME (DCP, 100 nM) for 3 days. (A,B,E,F,I,J) Immunohistochemistry imaged with confocal microscopy and (C,D,G,H,K,L) western blot analysis of (A–D) PKCε, (E–H) MnSOD, and (I–L) VEGF. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t-test (n = 3 cultures/western blot group).
    Figure Legend Snippet: PKCε activation increases PKCε, MnSOD, and VEGF protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were treated with 500 μM TBHP for 1 h and incubated in new culture medium with or without the PKCε activators bryostatin (bry, 25 nM) and DCPLA-ME (DCP, 100 nM) for 3 days. (A,B,E,F,I,J) Immunohistochemistry imaged with confocal microscopy and (C,D,G,H,K,L) western blot analysis of (A–D) PKCε, (E–H) MnSOD, and (I–L) VEGF. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t-test (n = 3 cultures/western blot group).

    Techniques Used: Activation Assay, Expressing, Cell Culture, Incubation, Immunohistochemistry, Confocal Microscopy, Western Blot, Molecular Weight, Marker, Comparison

    Reactive oxygen species (ROS) affects MnSOD mRNA and protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were incubated with the ROS scavenger N -acetylcysteine (Nac, 5 mM for 15 h) or the cell-permeable SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25 μM for 45 min) and were then treated with TBHP at 500 μM for 1 h and recovered without TBHP, Nac, and MnTMPyP for 3 days. (A,B) Immunohistochemistry and (C,D) western blot analysis were used to study MnSOD protein expression. (E) Quantitative PCR (qPCR) was used to determine MnSOD mRNA expression. Data are reported as mean ± SEM. Asterisks over the bars (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with their according controls, set as 100%. Student’s t -test for mRNA expression and western blot analysis ( n = 4–5 cultures/group) or one-way ANOVA with post hoc Tukey’s multiple comparison test for immunohistochemistry ( n = 42–160 random cells from 3 to 4 cultures per group).
    Figure Legend Snippet: Reactive oxygen species (ROS) affects MnSOD mRNA and protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were incubated with the ROS scavenger N -acetylcysteine (Nac, 5 mM for 15 h) or the cell-permeable SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25 μM for 45 min) and were then treated with TBHP at 500 μM for 1 h and recovered without TBHP, Nac, and MnTMPyP for 3 days. (A,B) Immunohistochemistry and (C,D) western blot analysis were used to study MnSOD protein expression. (E) Quantitative PCR (qPCR) was used to determine MnSOD mRNA expression. Data are reported as mean ± SEM. Asterisks over the bars (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with their according controls, set as 100%. Student’s t -test for mRNA expression and western blot analysis ( n = 4–5 cultures/group) or one-way ANOVA with post hoc Tukey’s multiple comparison test for immunohistochemistry ( n = 42–160 random cells from 3 to 4 cultures per group).

    Techniques Used: Expressing, Cell Culture, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Comparison

    The mRNA-stabilizing protein HuR involved in PKCε-activated MnSOD and VEGF expression in human brain microvascular endothelial cells (HBMEC). HBMEC cells were treated with the HuR inhibitor CMLD-2 (35 μM) or dihydrotanshinone-I (DHTS, 10 μM) for 30 min before and during the 3-day incubation in the presence of the PKCε activator bryostatin (25 nM) or DCPLA-ME (100 nM). (A) Immunohistochemistry of HuR was used to study nuclear export of the HuR protein. (B) Immunohistochemistry and (C,D) western blot analysis of MnSOD protein expression. (E) Quantitative PCR (qPCR) of MnSOD mRNA expression. (F) Immunohistochemistry and (G,H) western blot analysis of VEGF protein expression. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t -test ( n = 3 cultures/western blot group).
    Figure Legend Snippet: The mRNA-stabilizing protein HuR involved in PKCε-activated MnSOD and VEGF expression in human brain microvascular endothelial cells (HBMEC). HBMEC cells were treated with the HuR inhibitor CMLD-2 (35 μM) or dihydrotanshinone-I (DHTS, 10 μM) for 30 min before and during the 3-day incubation in the presence of the PKCε activator bryostatin (25 nM) or DCPLA-ME (100 nM). (A) Immunohistochemistry of HuR was used to study nuclear export of the HuR protein. (B) Immunohistochemistry and (C,D) western blot analysis of MnSOD protein expression. (E) Quantitative PCR (qPCR) of MnSOD mRNA expression. (F) Immunohistochemistry and (G,H) western blot analysis of VEGF protein expression. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t -test ( n = 3 cultures/western blot group).

    Techniques Used: Expressing, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Molecular Weight, Marker, Comparison

    The PKCε activator prevents a decrease in vascular VEGF and MV loss in the CA1 hippocampal stratum radiatum from age-related memory impairment rats. Tissue sections from rats in were used to stained with cytochemistry of the vascular endothelial cell marker IB4. (A) Colocalization of histochemistry of the vascular endothelium marker IB4 and (B) immunohistochemistry VEGF levels. (C) Low magnification of confocal microscope of the vascular endothelium marker IB4 was used to determine (D) . MV density in random hippocampal CA1 areas. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 63–95 random MV cells or 32–119 random areas from 3 to 5 rats/group).
    Figure Legend Snippet: The PKCε activator prevents a decrease in vascular VEGF and MV loss in the CA1 hippocampal stratum radiatum from age-related memory impairment rats. Tissue sections from rats in were used to stained with cytochemistry of the vascular endothelial cell marker IB4. (A) Colocalization of histochemistry of the vascular endothelium marker IB4 and (B) immunohistochemistry VEGF levels. (C) Low magnification of confocal microscope of the vascular endothelium marker IB4 was used to determine (D) . MV density in random hippocampal CA1 areas. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 63–95 random MV cells or 32–119 random areas from 3 to 5 rats/group).

    Techniques Used: Staining, Marker, Immunohistochemistry, Microscopy, Comparison

    Reduction of VEGF protein, but not mRNA, expression and microvascular loss in the CA1 stratum radiatum of autopsy-confirmed AD human hippocampus. (A) Quantitative PCR (qPCR) was used to detect VEGF mRNA at the whole hippocampus level. (B,C) Immunohistochemistry and confocal microscopy were used to determine VEGF protein expression in MV wall cells in hippocampal CA1 area (N, the nucleus of MV wall cell). (D,E) Immunofluorescence of the vascular endothelial cell marker CD31/PECAM was used to determine MV density. AC, age-matched control; AD, autopsy-confirmed Alzheimer’s disease. Although VEGF mRNA was not different among the experiment groups, VEGF and MV density was decreased in AD hippocampi at the early Braak stages II–III, but not AD at the late Braak stages IV–VI, compared to AC group. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.01; two-tailed t -test compared with their according controls ( n = 11 hippocampi per group for qPCR or n = 182–365 random MV cells from 11 hippocampi per group, or 87–105 random CA1 areas from 5 AD Braak II–III, 14 AD Braak IV–VI and 19 AC).
    Figure Legend Snippet: Reduction of VEGF protein, but not mRNA, expression and microvascular loss in the CA1 stratum radiatum of autopsy-confirmed AD human hippocampus. (A) Quantitative PCR (qPCR) was used to detect VEGF mRNA at the whole hippocampus level. (B,C) Immunohistochemistry and confocal microscopy were used to determine VEGF protein expression in MV wall cells in hippocampal CA1 area (N, the nucleus of MV wall cell). (D,E) Immunofluorescence of the vascular endothelial cell marker CD31/PECAM was used to determine MV density. AC, age-matched control; AD, autopsy-confirmed Alzheimer’s disease. Although VEGF mRNA was not different among the experiment groups, VEGF and MV density was decreased in AD hippocampi at the early Braak stages II–III, but not AD at the late Braak stages IV–VI, compared to AC group. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.01; two-tailed t -test compared with their according controls ( n = 11 hippocampi per group for qPCR or n = 182–365 random MV cells from 11 hippocampi per group, or 87–105 random CA1 areas from 5 AD Braak II–III, 14 AD Braak IV–VI and 19 AC).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Confocal Microscopy, Immunofluorescence, Marker, Control, Two Tailed Test

    The PKCε activator protects a reduction of PKCε and MnSOD in the hippocampal CA1 area from Tg2576 transgenic AD mice. Mice at 2 months of age were injected (i.p., twice a week) with normal saline in the presence or absence of bryostatin (30 μg/kg body weight) for a 3-month period. Mice were then studied at the age of 5–6 months old when an increase in soluble amyloid-beta (Aβ) and memory defect were seen in the hippocampus of Tg2576 mice . Bryostatin was withdrawn for 2 weeks to avoid the acute effect of bryostatin. Double immunohistochemistry and confocal microscopy of (A,B) PKCε, (A,C) PKCα, and (D,E) MnSOD in vascular endothelial cells that were marked with PECAM/CD31. Bryostatin (bry) prevented the loss of PKCε and MnSOD and promoted PKCα in Tg2576 (Tg) mice. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells from 3 to 5 mice/group).
    Figure Legend Snippet: The PKCε activator protects a reduction of PKCε and MnSOD in the hippocampal CA1 area from Tg2576 transgenic AD mice. Mice at 2 months of age were injected (i.p., twice a week) with normal saline in the presence or absence of bryostatin (30 μg/kg body weight) for a 3-month period. Mice were then studied at the age of 5–6 months old when an increase in soluble amyloid-beta (Aβ) and memory defect were seen in the hippocampus of Tg2576 mice . Bryostatin was withdrawn for 2 weeks to avoid the acute effect of bryostatin. Double immunohistochemistry and confocal microscopy of (A,B) PKCε, (A,C) PKCα, and (D,E) MnSOD in vascular endothelial cells that were marked with PECAM/CD31. Bryostatin (bry) prevented the loss of PKCε and MnSOD and promoted PKCα in Tg2576 (Tg) mice. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells from 3 to 5 mice/group).

    Techniques Used: Transgenic Assay, Injection, Saline, Immunohistochemistry, Confocal Microscopy, Comparison

    The PKCε activator protects a reduction of VEGF in MV endothelial cells and MV density in the hippocampal CA1 area from Tg2576 transgenic AD mice. Tissue sections from animals in were used. (A) Double immunohistochemistry and confocal microscopy of (B) VEGF in vascular endothelial cells that were marked with PECAM/CD31. (C) Cytochemistry of the vascular endothelial cells marker IB4, imaged with a confocal microscope, was used to determine (D) MV density in random CA1 areas. Bryostatin (bry) prevented the loss of VEGF and MV density in Tg2576 (Tg) mice. Data are represented as mean ± SEM, ** p < 0.01; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells or 19–28 random areas from 3 to 5 mice/group).
    Figure Legend Snippet: The PKCε activator protects a reduction of VEGF in MV endothelial cells and MV density in the hippocampal CA1 area from Tg2576 transgenic AD mice. Tissue sections from animals in were used. (A) Double immunohistochemistry and confocal microscopy of (B) VEGF in vascular endothelial cells that were marked with PECAM/CD31. (C) Cytochemistry of the vascular endothelial cells marker IB4, imaged with a confocal microscope, was used to determine (D) MV density in random CA1 areas. Bryostatin (bry) prevented the loss of VEGF and MV density in Tg2576 (Tg) mice. Data are represented as mean ± SEM, ** p < 0.01; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells or 19–28 random areas from 3 to 5 mice/group).

    Techniques Used: Transgenic Assay, Immunohistochemistry, Confocal Microscopy, Marker, Microscopy, Comparison



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    Neuromics human brain mv endothelial cells
    The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular <t>endothelial</t> cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).
    Human Brain Mv Endothelial Cells, supplied by Neuromics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human brain mv endothelial cells/product/Neuromics
    Average 90 stars, based on 1 article reviews
    human brain mv endothelial cells - by Bioz Stars, 2026-02
    90/100 stars
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    The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Expressing, Cell Culture, Concentration Assay, Incubation, Real-time Polymerase Chain Reaction, Comparison

    PKCε activation increases PKCε, MnSOD, and VEGF protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were treated with 500 μM TBHP for 1 h and incubated in new culture medium with or without the PKCε activators bryostatin (bry, 25 nM) and DCPLA-ME (DCP, 100 nM) for 3 days. (A,B,E,F,I,J) Immunohistochemistry imaged with confocal microscopy and (C,D,G,H,K,L) western blot analysis of (A–D) PKCε, (E–H) MnSOD, and (I–L) VEGF. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t-test (n = 3 cultures/western blot group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: PKCε activation increases PKCε, MnSOD, and VEGF protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were treated with 500 μM TBHP for 1 h and incubated in new culture medium with or without the PKCε activators bryostatin (bry, 25 nM) and DCPLA-ME (DCP, 100 nM) for 3 days. (A,B,E,F,I,J) Immunohistochemistry imaged with confocal microscopy and (C,D,G,H,K,L) western blot analysis of (A–D) PKCε, (E–H) MnSOD, and (I–L) VEGF. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t-test (n = 3 cultures/western blot group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Activation Assay, Expressing, Cell Culture, Incubation, Immunohistochemistry, Confocal Microscopy, Western Blot, Molecular Weight, Marker, Comparison

    Reactive oxygen species (ROS) affects MnSOD mRNA and protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were incubated with the ROS scavenger N -acetylcysteine (Nac, 5 mM for 15 h) or the cell-permeable SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25 μM for 45 min) and were then treated with TBHP at 500 μM for 1 h and recovered without TBHP, Nac, and MnTMPyP for 3 days. (A,B) Immunohistochemistry and (C,D) western blot analysis were used to study MnSOD protein expression. (E) Quantitative PCR (qPCR) was used to determine MnSOD mRNA expression. Data are reported as mean ± SEM. Asterisks over the bars (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with their according controls, set as 100%. Student’s t -test for mRNA expression and western blot analysis ( n = 4–5 cultures/group) or one-way ANOVA with post hoc Tukey’s multiple comparison test for immunohistochemistry ( n = 42–160 random cells from 3 to 4 cultures per group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: Reactive oxygen species (ROS) affects MnSOD mRNA and protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were incubated with the ROS scavenger N -acetylcysteine (Nac, 5 mM for 15 h) or the cell-permeable SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25 μM for 45 min) and were then treated with TBHP at 500 μM for 1 h and recovered without TBHP, Nac, and MnTMPyP for 3 days. (A,B) Immunohistochemistry and (C,D) western blot analysis were used to study MnSOD protein expression. (E) Quantitative PCR (qPCR) was used to determine MnSOD mRNA expression. Data are reported as mean ± SEM. Asterisks over the bars (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with their according controls, set as 100%. Student’s t -test for mRNA expression and western blot analysis ( n = 4–5 cultures/group) or one-way ANOVA with post hoc Tukey’s multiple comparison test for immunohistochemistry ( n = 42–160 random cells from 3 to 4 cultures per group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Expressing, Cell Culture, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Comparison

    The mRNA-stabilizing protein HuR involved in PKCε-activated MnSOD and VEGF expression in human brain microvascular endothelial cells (HBMEC). HBMEC cells were treated with the HuR inhibitor CMLD-2 (35 μM) or dihydrotanshinone-I (DHTS, 10 μM) for 30 min before and during the 3-day incubation in the presence of the PKCε activator bryostatin (25 nM) or DCPLA-ME (100 nM). (A) Immunohistochemistry of HuR was used to study nuclear export of the HuR protein. (B) Immunohistochemistry and (C,D) western blot analysis of MnSOD protein expression. (E) Quantitative PCR (qPCR) of MnSOD mRNA expression. (F) Immunohistochemistry and (G,H) western blot analysis of VEGF protein expression. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t -test ( n = 3 cultures/western blot group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: The mRNA-stabilizing protein HuR involved in PKCε-activated MnSOD and VEGF expression in human brain microvascular endothelial cells (HBMEC). HBMEC cells were treated with the HuR inhibitor CMLD-2 (35 μM) or dihydrotanshinone-I (DHTS, 10 μM) for 30 min before and during the 3-day incubation in the presence of the PKCε activator bryostatin (25 nM) or DCPLA-ME (100 nM). (A) Immunohistochemistry of HuR was used to study nuclear export of the HuR protein. (B) Immunohistochemistry and (C,D) western blot analysis of MnSOD protein expression. (E) Quantitative PCR (qPCR) of MnSOD mRNA expression. (F) Immunohistochemistry and (G,H) western blot analysis of VEGF protein expression. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t -test ( n = 3 cultures/western blot group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Expressing, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Molecular Weight, Marker, Comparison

    The PKCε activator prevents a decrease in vascular VEGF and MV loss in the CA1 hippocampal stratum radiatum from age-related memory impairment rats. Tissue sections from rats in were used to stained with cytochemistry of the vascular endothelial cell marker IB4. (A) Colocalization of histochemistry of the vascular endothelium marker IB4 and (B) immunohistochemistry VEGF levels. (C) Low magnification of confocal microscope of the vascular endothelium marker IB4 was used to determine (D) . MV density in random hippocampal CA1 areas. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 63–95 random MV cells or 32–119 random areas from 3 to 5 rats/group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: The PKCε activator prevents a decrease in vascular VEGF and MV loss in the CA1 hippocampal stratum radiatum from age-related memory impairment rats. Tissue sections from rats in were used to stained with cytochemistry of the vascular endothelial cell marker IB4. (A) Colocalization of histochemistry of the vascular endothelium marker IB4 and (B) immunohistochemistry VEGF levels. (C) Low magnification of confocal microscope of the vascular endothelium marker IB4 was used to determine (D) . MV density in random hippocampal CA1 areas. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 63–95 random MV cells or 32–119 random areas from 3 to 5 rats/group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Staining, Marker, Immunohistochemistry, Microscopy, Comparison

    Reduction of VEGF protein, but not mRNA, expression and microvascular loss in the CA1 stratum radiatum of autopsy-confirmed AD human hippocampus. (A) Quantitative PCR (qPCR) was used to detect VEGF mRNA at the whole hippocampus level. (B,C) Immunohistochemistry and confocal microscopy were used to determine VEGF protein expression in MV wall cells in hippocampal CA1 area (N, the nucleus of MV wall cell). (D,E) Immunofluorescence of the vascular endothelial cell marker CD31/PECAM was used to determine MV density. AC, age-matched control; AD, autopsy-confirmed Alzheimer’s disease. Although VEGF mRNA was not different among the experiment groups, VEGF and MV density was decreased in AD hippocampi at the early Braak stages II–III, but not AD at the late Braak stages IV–VI, compared to AC group. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.01; two-tailed t -test compared with their according controls ( n = 11 hippocampi per group for qPCR or n = 182–365 random MV cells from 11 hippocampi per group, or 87–105 random CA1 areas from 5 AD Braak II–III, 14 AD Braak IV–VI and 19 AC).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: Reduction of VEGF protein, but not mRNA, expression and microvascular loss in the CA1 stratum radiatum of autopsy-confirmed AD human hippocampus. (A) Quantitative PCR (qPCR) was used to detect VEGF mRNA at the whole hippocampus level. (B,C) Immunohistochemistry and confocal microscopy were used to determine VEGF protein expression in MV wall cells in hippocampal CA1 area (N, the nucleus of MV wall cell). (D,E) Immunofluorescence of the vascular endothelial cell marker CD31/PECAM was used to determine MV density. AC, age-matched control; AD, autopsy-confirmed Alzheimer’s disease. Although VEGF mRNA was not different among the experiment groups, VEGF and MV density was decreased in AD hippocampi at the early Braak stages II–III, but not AD at the late Braak stages IV–VI, compared to AC group. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.01; two-tailed t -test compared with their according controls ( n = 11 hippocampi per group for qPCR or n = 182–365 random MV cells from 11 hippocampi per group, or 87–105 random CA1 areas from 5 AD Braak II–III, 14 AD Braak IV–VI and 19 AC).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Confocal Microscopy, Immunofluorescence, Marker, Control, Two Tailed Test

    The PKCε activator protects a reduction of PKCε and MnSOD in the hippocampal CA1 area from Tg2576 transgenic AD mice. Mice at 2 months of age were injected (i.p., twice a week) with normal saline in the presence or absence of bryostatin (30 μg/kg body weight) for a 3-month period. Mice were then studied at the age of 5–6 months old when an increase in soluble amyloid-beta (Aβ) and memory defect were seen in the hippocampus of Tg2576 mice . Bryostatin was withdrawn for 2 weeks to avoid the acute effect of bryostatin. Double immunohistochemistry and confocal microscopy of (A,B) PKCε, (A,C) PKCα, and (D,E) MnSOD in vascular endothelial cells that were marked with PECAM/CD31. Bryostatin (bry) prevented the loss of PKCε and MnSOD and promoted PKCα in Tg2576 (Tg) mice. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells from 3 to 5 mice/group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: The PKCε activator protects a reduction of PKCε and MnSOD in the hippocampal CA1 area from Tg2576 transgenic AD mice. Mice at 2 months of age were injected (i.p., twice a week) with normal saline in the presence or absence of bryostatin (30 μg/kg body weight) for a 3-month period. Mice were then studied at the age of 5–6 months old when an increase in soluble amyloid-beta (Aβ) and memory defect were seen in the hippocampus of Tg2576 mice . Bryostatin was withdrawn for 2 weeks to avoid the acute effect of bryostatin. Double immunohistochemistry and confocal microscopy of (A,B) PKCε, (A,C) PKCα, and (D,E) MnSOD in vascular endothelial cells that were marked with PECAM/CD31. Bryostatin (bry) prevented the loss of PKCε and MnSOD and promoted PKCα in Tg2576 (Tg) mice. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells from 3 to 5 mice/group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Transgenic Assay, Injection, Saline, Immunohistochemistry, Confocal Microscopy, Comparison

    The PKCε activator protects a reduction of VEGF in MV endothelial cells and MV density in the hippocampal CA1 area from Tg2576 transgenic AD mice. Tissue sections from animals in were used. (A) Double immunohistochemistry and confocal microscopy of (B) VEGF in vascular endothelial cells that were marked with PECAM/CD31. (C) Cytochemistry of the vascular endothelial cells marker IB4, imaged with a confocal microscope, was used to determine (D) MV density in random CA1 areas. Bryostatin (bry) prevented the loss of VEGF and MV density in Tg2576 (Tg) mice. Data are represented as mean ± SEM, ** p < 0.01; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells or 19–28 random areas from 3 to 5 mice/group).

    Journal: Frontiers in Aging Neuroscience

    Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

    doi: 10.3389/fnagi.2022.836634

    Figure Lengend Snippet: The PKCε activator protects a reduction of VEGF in MV endothelial cells and MV density in the hippocampal CA1 area from Tg2576 transgenic AD mice. Tissue sections from animals in were used. (A) Double immunohistochemistry and confocal microscopy of (B) VEGF in vascular endothelial cells that were marked with PECAM/CD31. (C) Cytochemistry of the vascular endothelial cells marker IB4, imaged with a confocal microscope, was used to determine (D) MV density in random CA1 areas. Bryostatin (bry) prevented the loss of VEGF and MV density in Tg2576 (Tg) mice. Data are represented as mean ± SEM, ** p < 0.01; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells or 19–28 random areas from 3 to 5 mice/group).

    Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

    Techniques: Transgenic Assay, Immunohistochemistry, Confocal Microscopy, Marker, Microscopy, Comparison