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