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

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

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

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

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

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

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

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

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11 induces pancreatic stellate cell activation and invasion. ( a ) t-distributed stochastic neighbour embedding (tSNE) plots showing scRNA-seq expression of Il11ra1 , Il6ra and Il-6st ( gp130 ) in mouse pancreatic tissue. Cell clusters were identified using the Tabula Muris web tool ( https://tabula-muris.ds.czbiohub.org/ (accessed on 21 February 2022)). . Black arrows indicate pancreatic stellate cells (PSCs). ( b ) Representative immunostaining images of gp130, IL11RA, IL6RA in PSCs. Cells were counterstained with DAPI. Scale bars: 50 µm. ( c ) Western blot analysis of phosphorylated and total ERK and STAT3 and αSMA in lysates from PSC treated with IL11 (10 ng/mL) or IL 6 (10 ng/mL) across the indicated time-points (0 to 24 h). GAPDH serves as a loading control. ( d ) ELISA-based quantification of secreted MMP2 levels in PSC supernatants. ( e , f ) Representative immunofluorescence images and quantification of αSMA +ve cells, Collagen I intensity/area and EdU +ve cells at baseline and after 24 h treatment with either recombinant human TGFβ1 (5 ng/mL), IL11 (5 ng/mL), bFGF (10 ng/mL), CTGF (50 ng/mL), PDGF (200 ng/mL) or EDN1 (250 ng/mL). Cells were counterstained with DAPI. Scale bar: 200 µm. ( g ) Matrigel invasion capacity of PSCs was determined at baseline and after 24 h treatment with PDGF (20 ng/mL) or with increasing concentrations of IL11 (5–20 ng/mL). Scale bars: 150 µm. AU: Arbitrary Unit. Data are represented as mean ± SD in panel ( d ) and median and whiskers extending from minimum to maximum values in panels ( f , g ). p values were determined by one-way ANOVA with Dunnet’s correction. BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Activation Assay, Expressing, Immunostaining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: Autocrine IL11 signalling is important downstream of several pancreatitis factors. ( a ) ELISA of secreted IL11 from PSCs after 24 h treatment with recombinant human TGFβ1 (5 ng/mL), bFGF (10 ng/mL), CTGF (10 ng/mL), PDGF (200 ng/mL), EDN1 (250 ng/mL). ( b , c ) Representative immunofluorescence images and quantification of αSMA +ve cells and collagen I immunostaining of PSCs treated with IgG or the neutralizing IL11RA antibody (X209, 24 h) and profibrotic cytokines listed in panel ( a ). Cells were counterstained with DAPI. Scale bars: 200 µm. ( d ) ELISA of secreted MMP2 and ( e ) Sirius Red quantification of secreted collagen in the culture supernatant of PSCs treated as depicted in panel ( c ). ( f ) Western blot analysis of phosphorylated and total ERK and STAT3, and αSMA in lysates of PSCs treated with various profibrotic stimuli with either IgG or X209 (2 µg/mL, 24 h). ( g ) Matrigel invasion capacity of PSCs treated with IgG or X209 (2 µg/mL) and PDGF (20 ng/mL). Scale bars: 150 µm. AU: arbitrary unit. Data are represented as mean ± SD in ( a , d , e , g ) or as median and whiskers extending from minimum to maximum values in panel ( b ). p values were determined by one-way ANOVA with Dunnett’s correction in panel a , two-way ANOVA (Sidak’s correction) in panels ( b , d , e ) and by Student’s t test in ( g ). BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Immunofluorescence, Immunostaining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11 drives pancreatic stellate cell activation via ERK signalling and post-transcriptional effects. ( a ) RNA expression of IL11 , ACTA2 , COL1A1 and TIMP1 in PSCs treated with recombinant human TGFβ1 or IL11 (5 ng/mL; 24 h). ( b ) Representative immunofluorescence images and quantification of αSMA +ve cells, Collagen I intensity/area and EdU +ve cells at baseline and after 24 h treatment with IL11 (5 ng/mL) and ERK inhibitor U0126 (10 µM). Data are represented as mean ± SD in panel ( a ) and as median and whiskers extending from minimum to maximum values in panel ( b ). Scale bars: 100 µm. p values were determined by one way ANOVA (Dunnet’s correction) in ( a ) and one way ANOVA (Tukey’s correction) in ( b ). BL: baseline.

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Activation Assay, RNA Expression, Recombinant, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11RA antibody treatment reduces pancreatic fibrosis in a mouse model of pancreatitis. ( a ) Schematic of the induction of pancreatitis in wildtype C57BL/6 mice by pancreatic duct ligation (PDL) and Western blot analysis of IL11 and fibronectin (FN1) expression in pancreatic lysates post-sham or 14 days post-PDL surgery (n = 3/group). ( b ) Schematic of the administration timepoints of neutralizing IL11RA antibody (X209) or IgG control antibody treatment in the PDL model. ( c ) Gross pancreas anatomy and the tissue weights of the ligated splenic lobe in X209 or IgG treated mice. ( d ) Hematoxylin and eosin staining of pancreatic sections from the healthy or ligated splenic lobes of X209 or IgG treated mice. Scale bars: 100 µm. ( e ) Masson’s trichrome staining images and collagen quantification of fibrosis in the ligated splenic lobes of X209 or IgG treated mice (n = 3–4). Scale bars: 1000 µm. AU: arbitrary unit. ( f ) Collagen I immunostaining of the ligated splenic lobes of X209 or IgG treated mice. Scale bars: 50 µm. Data shown as median and whiskers extending from minimum to maximum values. p values were determined by Student’s t -test in ( c ) and one way ANOVA (Tukey’s correction) in ( e , f ).

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Ligation, Western Blot, Expressing, Control, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

doi: 10.3390/ijms23073549

Figure Lengend Snippet: IL11RA antibody treatment attenuates pancreatic inflammation and pathological signalling in a mouse model of pancreatitis. ( a ) Western blot analysis of IL11, IL6, IL1β, TNF, FN1, αSMA, cleaved (Clv.) and total caspase-3 and ( b ) phosphorylated and total protein levels of ERK, STAT3 and NF-kB in lysates from the ligated splenic lobes of X209 or IgG treated mice (n = 4/group). GAPDH served as loading control in ( a ). p values were determined by one way ANOVA (Tukey’s correction).

Article Snippet: Primary human pancreatic stellate cells (PSCs; ScienCell, Carlsbad, CA, USA; Cat #3830, Lot #14289) were isolated from human pancreas healthy samples and were maintained in Stellate cell medium (SteCM, Cat #5301, ScienCell, Carlsbad, CA, USA) supplemented with stellate cell growth supplement(ScienCell, Carlsbad, CA, USA Cat #5352), 2% fetal bovine serum (ScienCell, Carlsbad, CA, USA; Cat #0010) and 1% Penicillin-streptomycin (ScienCell, Carlsbad, CA, USA Cat #0503), in an incubator at 37 °C and with an atmosphere of 5% CO 2 .

Techniques: Western Blot, Control

Journal: Biomedicines

Article Title: The Effects of Antipsychotics in Experimental Models of Krabbe Disease

doi: 10.3390/biomedicines11051313

Figure Lengend Snippet: Antipsychotics prevent psychosine toxicity of human astrocytes. ( a ) The experimental schematic diagram for treatment timelines prior to analysis. ( b ) Light microscopy images of astrocytes. ( c ) MTT assays of psychosine-induced cell toxicity in human astrocytes. Positive control of 20% DMSO. Kruskal-Wallis, Dunn’s multiple comparison tests ( n = 5–10). ( d ) LDH assays of psychosine-induced cell toxicity in human astrocytes. One-way ANOVA, Tukey’s multiple comparison tests ( n = 5). MTT assays showed that typical ( e ) 1 µM or ( f ) 10 µM and atypical ( h ) 1 µM or ( i ) 10 µM antipsychotics did not alter cell viability compared to control, while DMSO (20%) and psychosine (10 µM) significantly reduced cell viability compared to control ( p < 0.0001 #### ). All antipsychotics (1 µM or 10 µM) (except aripiprazole (Ari) at 10 µM) significantly prevented psychosine (10 µM) induced reductions in cell viability ( p < 0.05 *, p < 0.01 **, p < 0.001 *** and p < 0.0001 ****). Kruskal-Wallis, Dunn’s multiple comparison tests ( n = 5–10). LDH assays showed that ( g ) typical and ( j ) atypical antipsychotics at 1 µM were not toxic to cells compared to control, whereas DMSO (20%) and psychosine (10 µM) significantly increased cell toxicity compared to control. All antipsychotics (1 µM) prevented psychosine (10 µM) toxicity. One-way ANOVA, Tukey’s multiple comparison test ( n = 5). ( k ) Typical antipsychotics and ( l ) atypical antipsychotics (1 µM) had no effect alone on astrocyte morphology compared to control, while DMSO (20%) and psychosine (10 µM) reduced the number of astrocyte extensions. ( k ) Typical antipsychotics and ( l ) atypical antipsychotics (1 µM) attenuated morphological changes induced by 10 µM psychosine. Representative confocal cell fluorescent images labelled for GFAP (green), Vimentin (red) and Hoescht (blue). The number of astrocyte projections of 20–30 cells per treatment group was analysed. Images were analysed using ImageJ software. One-way ANOVA, Tukey’s multiple comparison tests ( n = 6). Typical antipsychotics included: Haloperidol (Hal), chlorpromazine (Chl) and sulpiride (Sul). Atypical antipsychotics included: clozapine (Clo), olanzapine (Ola), amisulpride (Ami), quetiapine (Que), risperidone (Ris) and aripiprazole (Ari).

Article Snippet: Human astrocytes (ScienCell Research Laboratory, Carlsbad, CA, USA; Cat No. #1800, Lot No. 9063) were cultured exactly as we have described previously [ , , , , , , , , , ].

Techniques: Light Microscopy, Positive Control, Comparison, Control, Software

Journal: Biomedicines

Article Title: The Effects of Antipsychotics in Experimental Models of Krabbe Disease

doi: 10.3390/biomedicines11051313

Figure Lengend Snippet: Selective D 2 and 5HT 2A antagonists prevent psychosine-induced toxicity. ( a ) Pharmacology Heat Map. Receptor binding profiles of antipsychotics are shown. Inhibitory constant (Ki): 100 nM < Ki < 10,000 nM (weak, light grey), 10 nM < Ki < 100 (moderate, mid grey), 1 > Ki < 10 (strong, dark grey). All drugs are antagonists or inverse agonists at D2 and 5HT2A receptors apart from aripiprazole which is a partial agonist at these receptors. Haloperidol (Hal), chlorpromazine (Chl), sulpiride (Sul), clozapine (Clo), olanzapine (Ola), amisulpride (Ami), quetiapine (Que), risperidone (Ris) and aripiprazole (Ari) ( b ) MTT assays showed that 1 µM Eticlopride (ETI) (selective D 2 antagonist) and 1 µM Volinanserin (VOL) (selective 5HT 2A antagonist) did not alter cell viability compared to control, while DMSO (20%) and psychosine (10 µM) significantly reduced cell viability compared to control. Both 1 µM Eticlopride (ETI) and 1 µM Volinanserin (VOL) prevented significant reductions in cell viability induced by 10 µM psychosine Kruskal-Wallis, Dunn’s multiple comparison tests ( n = 5). ( c ) LDH assays showed that 1 µM Eticlopride (ETI) and 1 µM Volinanserin (VOL) did not induce cell toxicity compared to the control. In contrast, DMSO (20%) and psychosine (10 µM) significantly increased cell toxicity compared to the control (####). Both drugs prevented significant cell toxicity induced by 10 µM psychosine. One-way ANOVA, Tukey’s multiple comparison tests ( n = 5). Representative confocal cell fluorescent images labelled for ( d ) GFAP (green) and ( e ) Vimentin (red), with Hoescht (blue) staining. Eticlopride (ETI) and Volinanserin (VOL) at 1 µM had no effect alone on astrocyte morphology compared to control, while DMSO (20%) and psychosine (10 µM) reduced the number of astrocyte extensions. Both selective antagonists attenuated morphological changes induced by 10 µM psychosine. The number of ( f ) GFAP (green) and ( g ) Vimentin (red) positive astrocyte projections of 20–30 cells per treatment group were analysed. Images were analysed using ImageJ software. One-way ANOVA, Tukey’s multiple comparison tests ( n = 6). p < 0.01 **, p < 0.001 *** and p < 0.0001****.

Article Snippet: Human astrocytes (ScienCell Research Laboratory, Carlsbad, CA, USA; Cat No. #1800, Lot No. 9063) were cultured exactly as we have described previously [ , , , , , , , , , ].

Techniques: Binding Assay, Control, Comparison, Staining, Software

Journal: Biomedicines

Article Title: The Effects of Antipsychotics in Experimental Models of Krabbe Disease

doi: 10.3390/biomedicines11051313

Figure Lengend Snippet: Psychosine induces changes in astrocytes, but not microglia, markers attenuated by Haloperidol and Clozapine. Treatment of cerebellar slices with psychosine decreases ( a , b ) GFAP and ( a , c ) Vimentin fluorescence at 100 nM and 1000 nM psychosine. Haloperidol 10 µM attenuated psychosine-induced decrease in ( d , e ) GFAP and ( d , f ) Vimentin fluorescence at 100 nM psychosine. Clozapine 10 µM attenuated the psychosine-induced decrease in ( g , h ) GFAP and ( g , i ) Vimentin fluorescence at 100 nM psychosine. Psychosine in the presence or absence of ( j , l ) Haloperidol or ( k , m ) Clozapine did not alter ( j , k ) Iba1 fluorescence or ( l , m ) microglia morphology in white matter tracts or whole cerebellar slices areas. Confocal images at 10× and 20× magnification, scale bar 100 µm and 10 µm, respectively. Data are shown as mean +/− SEM, Kruskal-Wallis test, Dunn’s multiple comparisons tests, #### p < 0.0001, **** p < 0.0001 and ** p < 0.01 ( n = 5).

Article Snippet: Human astrocytes (ScienCell Research Laboratory, Carlsbad, CA, USA; Cat No. #1800, Lot No. 9063) were cultured exactly as we have described previously [ , , , , , , , , , ].

Techniques: Fluorescence

Journal: iScience

Article Title: MircroRNA-92b as a negative regulator of the TGF-β signaling by targeting the type I receptor

doi: 10.1016/j.isci.2023.108131

Figure Lengend Snippet:

Article Snippet: Human renal glomerular endothelial cells (HRGECs) (ScienCell, Carlsbad, CA, USA; Cat. #4000) were cultured in Endothelial Cell Medium (ScienCell, Cat.#1001) and human mesangial mells (HMCs) (ScienCell, Cat. #4200) were cultured in Mesangial Cell Medium (ScienCell, Cat. #4201).

Techniques: Recombinant, Control, Plasmid Preparation, Software, Modification, Staining, Extraction, Bicinchoninic Acid Protein Assay, Western Blot, cDNA Synthesis, SYBR Green Assay, Immunohistochemistry, Isolation

Journal: Journal of the American College of Cardiology

Article Title: Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury

doi: 10.1016/j.jacc.2017.07.762

Figure Lengend Snippet: (A) Human primary cardiomyocytes were exposed to 2.6 mM hydrogen peroxide (H2O2) at time T = 0 h and throughout the course of the study. Treatment of cells 30 min after the start of intoxication with 3E10-Fv alone ( ) or HSP72 alone ( ) did not affect the increase in cell death, while treatment with Fv-HSP72 ( ) significantly attenuated apoptosis (p = 0.0006). Addition of 3E10-Fv prior to Fv-HSP72 treatment ( ) inhibited Fv-HSP72 efficacy, but still significantly attenuated apoptosis at 12 h (p = 0.002). (B) To study cardiomyocyte exposure overnight, cells were intoxicated with 1.4 mM H2O2 at time T = 0 h and throughout the course of the study. Fv-HSP72 treatment at 30 min after the start of intoxication significantly reduced the percentage of cell death at 17 h compared to the H2O2-only control (p = 0.0003), while treatment with 3E10-Fv alone or HSP72 alone showed no statistically significant inhibition. Percentages reflect normalization of the average fluorescent signal at the last reading divided by the maximum signal obtained after total cell lysis in 3 of the No H2O2 control wells. Number of wells receiving each treatment cited in parentheses. *statistically significant; ***highly significant. Error bars represent SE of the mean. Abbreviations as in Figure 1.

Article Snippet: Human primary cardiomyocytes (Cat. #6200, ScienCell, Carlsbad, California) were thawed and the 1 ml (1 × 10 6 cells/vial) suspended in 19 ml of phenol-red free cardiac myocyte media (includes 5% fetal bovine serum, 1% cardiac myocyte growth supplement, 1X penicillin/streptomycin; Cat. #6201-prf, ScienCell) prior to seeding into white, opaque bottom 96-well tissue culture plates at 5,000 cells per well (Cat. #3917, Corning Life Sciences, Tewksbury, Massachusetts).

Techniques: Control, Inhibition, Lysis

Journal: Journal of the American College of Cardiology

Article Title: Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury

doi: 10.1016/j.jacc.2017.07.762

Figure Lengend Snippet: A single-chain variable fragment (Fv) of the 3E10 antibody transports heat shock protein (HSP)72 into cardiomyocytes through the equilibrative nucleoside transporter 2 (ENT2) channel, where the HSP inhibited apoptosis in a rabbit model of ischemia-reperfusion injury. Either moiety alone is not cardioprotective.

Article Snippet: Human primary cardiomyocytes (Cat. #6200, ScienCell, Carlsbad, California) were thawed and the 1 ml (1 × 10 6 cells/vial) suspended in 19 ml of phenol-red free cardiac myocyte media (includes 5% fetal bovine serum, 1% cardiac myocyte growth supplement, 1X penicillin/streptomycin; Cat. #6201-prf, ScienCell) prior to seeding into white, opaque bottom 96-well tissue culture plates at 5,000 cells per well (Cat. #3917, Corning Life Sciences, Tewksbury, Massachusetts).

Techniques:

Journal: Oncology Letters

Article Title: Repositioning of duloxetine to target pancreatic stellate cells

doi: 10.3892/ol.2021.13005

Figure Lengend Snippet: Discovery of a potential pathway for the identification of novel compounds for pancreatic stellate cells via pathway analysis. (A) Kyoto Encyclopedia of Genes and Genomes pathway analysis of cancer-associated fibroblasts vs. normal fibroblasts from the public database. The top 10 altered pathways are ranked according to their P-values. (B) Lipid accumulation was calculated as the resultant fluorescence intensity after treatment with drugs (10 µM each) related to the ‘neuroactive ligand-receptor interaction’ pathway. The control group was treated with 0.025% dimethyl sulfoxide. (C) Structure of duloxetine. *P<0.05, ***P<0.001, ****P<0.0001 vs. control group.

Article Snippet: Human Pancreatic Stellate Cells (HPaSteC cells, cat. no. 3830; ScienCell Research Laboratories) were maintained in Stellate Cell Medium (cat. no. 5301; ScienCell Research Laboratories), according to the manufacturer's instructions.

Techniques: Fluorescence, Control

Journal: Oncology Letters

Article Title: Repositioning of duloxetine to target pancreatic stellate cells

doi: 10.3892/ol.2021.13005

Figure Lengend Snippet: Duloxetine induces PSCs into a quiescent state. (A) Cell viability of PSCs in vitro . Each IC 50 value is presented in the graph. (B) Representative photomicrographs of LipiDye staining of PSCs taken 0, 24 and 48 h after duloxetine treatment. DAPI was used for nuclear staining. After 48 h, the medium was washed off, fresh Dulbecco's modified Eagle's medium with 10% FBS was added, and the culture was continued. Magnifications, ×200 (upper panels) and ×400 (lower panels). Scale bar, 100 µm. (C) mRNA expression levels of αSMA and ECM proteins in PSCs. mRNA expression levels were normalized to GAPDH expression and are presented as the fold-change in gene expression relative to control PSCs. (D) Western blotting of αSMA and ECM proteins from whole cell lysate of PSCs. Values indicate densitometric ratios normalized to α-tubulin. **P<0.01, ***P<0.001, ****P<0.0001 vs. control group. PSCs, pancreatic stellate cells; αSMA, α-smooth muscle actin; ACTA2, actin α2 smooth muscle; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hPSC, human pancreatic stellate cells; COL1A1, α-1 type-1 collagen; POSTN, periostin.

Article Snippet: Human Pancreatic Stellate Cells (HPaSteC cells, cat. no. 3830; ScienCell Research Laboratories) were maintained in Stellate Cell Medium (cat. no. 5301; ScienCell Research Laboratories), according to the manufacturer's instructions.

Techniques: In Vitro, Staining, Modification, Expressing, Gene Expression, Control, Western Blot

Journal: Oncology Letters

Article Title: Repositioning of duloxetine to target pancreatic stellate cells

doi: 10.3892/ol.2021.13005

Figure Lengend Snippet: Duloxetine suppresses pancreatic cancer cell line and organoid growth. (A) Proliferation assay for SUIT-2 cells in vitro . (B) Representative photomicrograph of an organoid derived from a human surgical specimen. Each organoid was treated with duloxetine at various concentrations (10, 15 and 20 µM). Graph shows the quantification of grown organoids in five random fields at ×100 magnification. Scale bar, 100 µm. *P<0.05, ****P<0.0001 vs. 0 µM group.

Article Snippet: Human Pancreatic Stellate Cells (HPaSteC cells, cat. no. 3830; ScienCell Research Laboratories) were maintained in Stellate Cell Medium (cat. no. 5301; ScienCell Research Laboratories), according to the manufacturer's instructions.

Techniques: Proliferation Assay, In Vitro, Derivative Assay

Journal: Oncology Letters

Article Title: Repositioning of duloxetine to target pancreatic stellate cells

doi: 10.3892/ol.2021.13005

Figure Lengend Snippet: Duloxetine suppresses tumor-stromal interactions by attenuating secretomes from PSCs. (A) Representative photomicrographs of invading and migrating SUIT-2 cells in monoculture and indirect coculture with drugs or supernatants following hematoxylin and eosin staining. Magnification, ×100. Scale bar, 100 µm. (B) Graphs show the number of invading and migrating SUIT-2 cells. (C) Western blotting of extracellular matrix proteins in PSC supernatant and drug-treated PSC supernatant. (D) Relative mRNA expression of growth factors and cytokines associated with tumor-stromal interactions in PSCs treated with duloxetine. The expression levels of each gene were normalized to GAPDH. (E) Western blotting of PP2A and related proteins in whole cell lysates of PSCs treated with duloxetine at various concentrations (1, 5 and 10 µM). Values indicate densitometric ratios normalized to α-tubulin. *P<0.05, **P<0.01, ****P<0.0001 vs. control group. PSCs, pancreatic stellate cells; PSN-SN, PSC supernatant; Dulo-SN, supernatant from PSCs treated with duloxetine; PP2A, protein phosphatase 2A; COL1A1, α-1 type-1 collagen; POSTN, periostin; CTGF, connective tissue growth factor; HGF, hepatocyte growth factor; IL-1β, interleukin-1β; IL-6, interleukin-6; p, phosphorylated.

Article Snippet: Human Pancreatic Stellate Cells (HPaSteC cells, cat. no. 3830; ScienCell Research Laboratories) were maintained in Stellate Cell Medium (cat. no. 5301; ScienCell Research Laboratories), according to the manufacturer's instructions.

Techniques: Staining, Western Blot, Expressing, Control