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

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: The deficiency of FARS2 impairs cell motility, proliferation, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs). (A) Representative images of scratch-wound assays of HUVECs 0 and 6 h at 48 h after transfection with a control (siCtrl) or FARS2 -specific (si- FARS2 ) siRNA. Scale bar = 200 μm. (B) Quantification of the healed wound area from (A) . Data are prepresented as the mean and SEM ( n = 10). *** P < 0.001 via ANOVA. (C) A CCK8-based cell proliferation assay of HUVECs at the indicated time-points after transfection with siCtrl or si- FARS2 . The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. **** P < 0.0001. (D) Representative images of transwell-based migration assays of HUVECs 48 h after transfection with siCtrl or si- FARS2 . Scale bar = 200 μm. (E) Quantification of the number of migrated cells from (D) . *** P < 0.001. (F) Representative images of tube network assays of HUVECs 48 h after transfection with siCtrl or si- FARS2 . Scale bar = 200 μm. (G,H) Quantification of the branching points (G) and tube lengths (H) from (F) . The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. **** P < 0.0001.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Transfection, Control, Proliferation Assay, Migration

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: FARS2 silencing causes mitochondrial dysfunction in human umbilical vein endothelial cells (HUVECs). (A) The oxygen consumption rate (OCR) in HUVECs transfected with siCtrl or si- FARS2 . The HUVECs were seeded 48 h after transfection with siRNAs and 12 h before analysis using a Seahorse XF24 analyzer. The OCR was measured continuously throughout the experimental period, both at baseline and in the presence of the indicated drugs. (B) Non-mitochondrial respiration, basal respiration, maximal respiration, proton leak, ATP production, and spare respiratory capacity in control and FARS2-deficient HUVECs. The measurements were made in triplicate (mean and SEM). ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) The effects of FARS2 knock-down on intracellular reactive oxygen species production by HUVECs. The measurements were made in triplicate (mean and SEM). * P < 0.05, (D) Quantification of total ATP levels in HUVECs 48 h after transfection with the indicated siRNAs. The measurements were made in triplicate (mean and SEM). ** P < 0.01.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Transfection, Control, Knockdown

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: The deficiency of FARS2 impairs angiogenesis by disrupting the Notch and Wnt signaling pathways. (A) The expression levels of genes involved in the Notch/Wnt pathways in control and fars2 zebrafish morphants, as determined by qRT-PCR analyses ( n = 6–10 individual embryos). *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant. (B) The relative mRNA expression levels of Notch/Wnt pathway-related genes. The human umbilical vein endothelial cells (HUVECs) were transfected with the indicated siRNAs for 48 h and then harvested for qRT-PCR analysis. The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) Western blot analyses of NOTCH1 and β-catenin protein levels. The HUVECs were transfected with the indicated siRNAs for 48 h prior to analysis. (D) Quantification of the western blotting data described in (C) . The measurements were made in triplicate (mean and SEM). * P < 0.05, ** P < 0.01.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Protein-Protein interactions, Expressing, Control, Quantitative RT-PCR, Transfection, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: Developmental angiogenesis requires the mitochondrial phenylalanyl-tRNA synthetase. An overview of the mechanisms by which the deficiency of mitochondrial phenylalanyl-tRNA synthetase impairs angiogenesis by disrupting the Notch/Wnt pathways in zebrafish and human umbilical vein endothelial cells.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: