Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Expression levels of miR-145-5p are decreased in HG podocytes. Podocytes were incubated with 5 mM (NG group) and 25 mM (HG group) glucose for different time periods (12, 24, 48 and 72 h). The expression levels of miR-145-5p were measured by reverse transcription-quantitative PCR. Data are presented as the mean ± SD, and as the fold change relative to the control group (n=3). **P<0.01 vs. NG. miR-145-5p, microRNA-145-5p; HG, high glucose; NG, normal glucose.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Expressing, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Effect of miR-145-5p on HG-induced podocyte apoptosis. (A) Podocyte apoptosis was detected by flow cytometry following HG (25 mM) stimulation for 48 h. Podocytes treated with NG (5 mM) were used as the negative control group. (B) Quantification of the percentage of apoptotic cells. (C) TUNEL staining of apoptotic podocytes. Magnification, ×200. (D) Percentages of apoptotic cells identified by TUNEL staining. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and as the fold change relative to the control group (n=3). *P<0.05 and **P<0.01 vs. the scrambled control. HG, high glucose; miR-145-5p, microRNA-145-5p; NG, normal glucose.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Flow Cytometry, Negative Control, TUNEL Assay, Staining, Comparison, Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: miR-145-5p is a direct target of Notch1 in podocytes. (A) Venn diagram showing the 267 genes identified as potential targets of miR-145-5p using three prediction tools (B) Predicted binding sequences of miR-145-5p with the 3′-UTR of Notch1. (C) Relative luciferase activity was analyzed after WT or mut 3′-UTR reporter plasmids were co-transfected with miR-145-5p mimic or miR-145-5p inhibitor in podocytes. (D) Reverse transcription-quantitative PCR detection of Notch1 mRNA expression in podocytes transfected with miR-145-5p mimic or miR-145-5p inhibitor. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group (n=3). *P<0.05 and **P<0.01 vs. the respective NC or scrambled control. HG, high glucose; miR-145-5p, microRNA-145-5p; NC, negative control; MUT, mutant; NG, normal glucose; UTR, untranslated region; WT, wild-type.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Comparison, Control, Negative Control, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Overexpression of miR-145-5p suppresses the HG-induced activation of the Notch signaling pathway. (A-C) RT-qPCR detection of (A) NICD, (B) Hes1 and (C) Hey1 mRNA expression in control (NG) and transfected podocytes under HG conditions. (D) Western blotting detection of NICD, Hes1 and Hey1 protein expression in podocytes. (E) Semi-quantification of the protein levels of NICD, Hes1 and Hey1. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the scrambled control. miR-145-5p, microRNA-145-5p; HG, high glucose; NG, normal glucose; RT-qPCR, reverse transcription-quantitative PCR; NICD, intracellular domain of Notch; Hes1, hes family bHLH transcription factor 1; Hey1, hes related family bHLH transcription factor with YRPW motif 1.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Over Expression, Activation Assay, Quantitative RT-PCR, Expressing, Control, Transfection, Western Blot, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: mRNA and protein expression levels of caspase-3, Bcl-2 and Bax in podocytes. (A-C) RT-qPCR detection of (A) caspase-3, (B) Bcl-2 and (C) Bax mRNA expression in podocytes. (D) Western blotting detection of cleaved caspase-3, Bcl-2 and Bax protein levels in podocytes transfected with miR-145-5p mimics and inhibitors. (E) Semi-quantification of protein levels of cleaved caspase-3, Bcl-2 and Bax. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group. GAPDH was used as an internal control (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the scrambled control. miR-145-5p, microRNA-145-5p; NG, normal glucose; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Comparison, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Radiation Research

Article Title: The malignancy of liver cancer cells is increased by IL-4/ERK/AKT signaling axis activity triggered by irradiated endothelial cells

doi: 10.1093/jrr/rraa002

Figure Lengend Snippet: IL-4 released from 2 Gy–irradiated endothelial cells contributed to increasing the malignancy of liver cancer cells. ( a ) The secretion of IL-4, IL-2, IL-13 or IL-16 in 2 Gy–irradiated HUVECs. After HUVECs were irradiated with 2 Gy, and then cultured for 24 h, ELISA was performed to detect the secretion of interleukins, using conditioned medium obtained from HUVECs. ( b ) qRT-PCR analysis of the mRNA expression levels of IL-4, IL-2, IL-13 and IL-16 in the presence or absence of siRNAs targeting IL-4, IL-2, IL-13 and IL-16, respectively, in 2 Gy–irradiated HUVECs. ( c – e ) Migratory and invasive properties of HepG2 cells after treatment with conditioned medium from 2 Gy–irradiated HUVECs pretreated with siRNAs targeting IL-4, IL-2, IL-13 and IL-16. These properties of the cells were measured using Transwell chambers (magnification, ×200) (A = conditioned medium from unirradiated HUVECs pretreated with siCont; B = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siCont; C = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siIL-4; D = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siIL-2; E = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siIL-13; F = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siIL-16). ( f ) Western blot analysis of the expression levels of N-cadherin and Slug after treatment of HepG2 cells with conditioned medium from 2 Gy–irradiated HUVECs pretreated with siRNA targeting IL-4 for 48 h. Experiments were performed in triplicate, and the data shown are representative of a typical experiment. ( g and h ) Quantification of the sphere-forming ability of HepG2 cells after treatment with conditioned medium from 2 Gy–irradiated HUVECs pretreated with siRNA targeting IL-4 (A = conditioned medium from unirradiated HUVECs pretreated with siCont; B = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siCont; C = conditioned medium from 2 Gy–irradiated HUVECs pretreated with siIL-4). These cells (300 cells per well) were grown in DMEM/F12 supplemented with B27, N2, EGF and bFGF in 24-well ultralow attachment plates for 7 and 14 days, and the size of the spheres was determined. The size of 12 randomly selected spheres per group ( n = 12/group) was measured. The average size of each sphere was quantified with the standard deviation and is shown in the representative graph. β-actin was used as the loading control. The results from three independent experiments are expressed as the means ±1 SEM. (* P < 0.05).

Article Snippet: A mouse monoclonal antibody against N-cadherin (cat. no. 610920) was purchased from BD Bioscience (San Jose, CA, USA).

Techniques: Irradiation, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation

Journal: Journal of Radiation Research

Article Title: The malignancy of liver cancer cells is increased by IL-4/ERK/AKT signaling axis activity triggered by irradiated endothelial cells

doi: 10.1093/jrr/rraa002

Figure Lengend Snippet: Activation of the JAK/STAT3 pathway in liver cancer cells induced by 2 Gy–irradiated ECM. ( a ) Western blot analysis of the phosphorylation of STAT3 in HepG2 cells treated with 2 Gy–irradiated ECM. ( b ) Effects of AG490 on the protein expression levels of phospho-STAT3. HepG2 cells treated with 2 Gy–irradiated ECM were grown in the presence or absence of AG490 (5 μM) for 24 h, and western blot analysis was performed. ( c – e ) Effects of AG490 on the migratory and invasive properties of HepG2 cells enhanced by treatment with 2 Gy–irradiated ECM. Cells were treated with 2 Gy–irradiated ECM in the presence or absence of AG490 (5 μM) and grown for 48 h. These properties of HepG2 cells were measured using Transwell chambers (magnification, ×200). ( f ) Effects of AG490 on the protein expression levels of N-cadherin and Slug in HepG2 cells treated with 2 Gy–irradiated ECM for 48 h. (f– g ) Effects of AG490 on the sphere-forming abilities of HepG2 cells enhanced by treatment with 2 Gy–irradiated ECM. Cells (300 cells per well) were grown in DMEM/F12 supplemented with B27, N2, EGF and bFGF on 24-well ultralow attachment plates for 7 and 14 days, and the size of the spheres was determined. The size of 12 randomly selected spheres per group ( n = 12/group) was measured. The average size of each sphere was quantified with the standard deviation and is shown in the representative graph. β-actin was used as the loading control. Western blotting was performed in triplicate, and the data shown are representative of a typical experiment. The results from three independent experiments are expressed as the means ±1 SEM. (* P < 0.05).

Article Snippet: A mouse monoclonal antibody against N-cadherin (cat. no. 610920) was purchased from BD Bioscience (San Jose, CA, USA).

Techniques: Activation Assay, Irradiation, Western Blot, Expressing, Standard Deviation

Journal: Journal of Radiation Research

Article Title: The malignancy of liver cancer cells is increased by IL-4/ERK/AKT signaling axis activity triggered by irradiated endothelial cells

doi: 10.1093/jrr/rraa002

Figure Lengend Snippet: Activation of AKT and ERK induced by 2 Gy–irradiated ECM. (a) Western blot analysis of the phosphorylation of AKT and MAPKs in HepG2 cells treated with 2 Gy–irradiated ECM. ( b and c ) Effects of LY294002, PD98059, SP600125 and PD169316 on the migratory and invasive properties of HepG2 cells treated with 2 Gy–irradiated ECM. Cells were grown in the presence or absence of each inhibitor for 48 h, and migration and invasion assays were performed using Transwell chambers. ( d and e ) Effects of LY294002 and PD98059 on the protein expression levels of N-cadherin and Slug. HepG2 cells treated with 2 Gy–irradiated ECM were grown in the presence or absence of LY294002 (5 μM) or PD98059 (5 μM) for 48 h, and western blot analysis was performed. ( f and g ) Effects of LY294002 and PD98059 on the sphere-forming ability of HepG2 cells enhanced by treatment with 2 Gy–irradiated ECM. Cells (300 cells per well) were grown in DMEM/F12 supplemented with B27, N2, EGF and bFGF in 24-well ultralow attachment plates for 7 and 14 days, and the size of the spheres was determined. The size of 12 randomly selected spheres per group ( n = 12/group) was measured. The average size of each sphere was quantified with the standard deviation and is shown in the representative graph. β-actin was used as the loading control. Western blotting was performed in triplicate, and the data shown are representative of a typical experiment. The results from three independent experiments are expressed as the means ±1 SEM. (* P < 0.05).

Article Snippet: A mouse monoclonal antibody against N-cadherin (cat. no. 610920) was purchased from BD Bioscience (San Jose, CA, USA).

Techniques: Activation Assay, Irradiation, Western Blot, Migration, Expressing, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: MPP3 Is Required for Maintenance of the Apical Junctional Complex, Neuronal Migration, and Stratification in the Developing Cortex

doi: 10.1523/JNEUROSCI.5627-12.2013

Figure Lengend Snippet: Disruption of adherens junctions in Mpp3 cKO cortex. A–C, p120 catenin levels and apical membrane localization are essentially unaffected in E12.5 Mpp3 cKO cortex. At areas with a loss of p120 catenin, there is cellular disorganization in the ventricular zone (C). B and C are magnification of boxes 1 and 2 in A, respectively. D, E, Likewise, at areas in which Nectin1 is unaffected, cellular organization is indistinguishable from control cortex, but at areas with a loss of Nectin1, a clear disruption of cellular organization was observed (E). F, G, No difference in level and localization of adherens junction protein β-catenin between control (F) and Mpp3 cKO (G) at the level of the third ventricle in which Cre recombinase is not expressed. H, I, Whereas β-catenin displays a punctate pattern at the apical membrane in control cortex (H), in the Mpp3 cKO, there is a loss and delocalization of β-catenin (I). J–O, The same differences were observed between control and Mpp3 cKO for adherens junction proteins p120 catenin (J, K), N-cadherin (L, M), and M-cadherin (N, O). P, Q, Clear loss of Nectin1, which directly interacts with MPP3, during loss of MPP3. R, S, The PAR complex member PAR3 has a punctate distribution in the control cortex (R), but in the Mpp3 cKO cortex, there is a prominent loss of PAR3 from the apical membrane (S). Scale bars, 20 μm.

Article Snippet: Le Bivic, Developmental Biology Institute of Marseille Luminy, Marseille, France), Nectin1 (1:500; MBL), β-catenin (1:100; BD Biosciences), p120 catenin (1:100; BD Biosciences), N-cadherin (1:500; BD Biosciences), M-cadherin (1:500; BD Biosciences), PAR3 (1:200; Millipore), phospho-Histone H3 (pH3; 1:500; Millipore), BrdU (1:250, Abcam), Ki67 (1:100; BD Biosciences), CUX1 (1:200; Santa Cruz Biotechnology), CTIP2 (1:100; Abcam), Calretinin (1:500; Millipore Bioscience Research Reagents), Sox2 (1:300; Millipore Bioscience Research Reagents), Tbr1 (1:200; Abcam), Tbr2 (1:200; Abcam), and Tuj1 (1:200; Covance).

Techniques:

Journal: The American Journal of Pathology

Article Title: Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

doi: 10.1016/j.ajpath.2016.07.018

Figure Lengend Snippet: Primary Antibodies Used for Immunofluorescence Assays

Article Snippet: This design provided converging lines of evidence to ensure validity and reproducibility. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Host/clone Company Catalog no. Dilution Catenin β-1 (CTNNB1) Rabbit/E247 Abcam (Cambridge, MA) ab32572 1:100 Keratin, type II cytoskeletal 8 (KRT8) ∗ Rabbit/EP1628Y Abcam (Cambridge, MA) ab53280 1:200 Cadherin-1 (CDH1) Mouse/36 BD Biosciences (San Jose, CA) 610181 1:50 Olfactory marker protein (OMP) Goat (polyclonal) Wako (Richmond, VA) 544-10001 1:100 Ubiquitin, conjugated and unconjugated Mouse/Ubi-1 Millipore (Billerica, MA) MAB1510 1:400 Ubiquitin (linkage-specific, K63) Rabbit/EPR8590-448 Abcam (Cambridge, MA) ab179434 1:100 Allograft inflammatory factor 1 (AIF1) Chicken (polyclonal) Abcam (Cambridge, MA) {"type":"entrez-nucleotide","attrs":{"text":"Ab139590","term_id":"62160171","term_text":"AB139590"}} Ab139590 1:200 Mouse lysosomal membrane glycoprotein 1 (LAMP1) Rat/monoclonal † Developmental Studies Hybridoma Bank (Iowa City, IA) 1D4B 1:50 Mouse lysosomal membrane glycoprotein 2 (LAMP2) Rat/monoclonal † Developmental Studies Hybridoma Bank (Iowa City, IA) ABL-93 1:50 SQSTM 1/p62 Rabbit (polyclonal) Lifespan Biosciences, Inc. (Seattle, WA) LS-B4617 1:200 Open in a separate window ∗ This antibody also recognizes keratin, type I cytoskeletal 18 (KRT18).

Techniques: Immunofluorescence, Marker

Journal: The American Journal of Pathology

Article Title: Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

doi: 10.1016/j.ajpath.2016.07.018

Figure Lengend Snippet: Dual immunofluorescence for K63-ubiquitin and CDH1. K63-ubiquitin is localized as intraepithelial accumulations in the airway epithelium. The accumulations are associated with diacetyl exposure and are more frequently identified in bronchi than terminal bronchioles. A: Photomicrograph of immunofluorescence double label for K63-ubiquitin (red) and CDH1 (green) in the airway epithelium of the lung of a male Dcxr knockout mouse exposed to 200 ppm diacetyl. K63-ubiquitin forms large puncta similar to those identified by staining for total ubiquitin. B: Morphometric quantification of K63-ubiquitin positive cells in the epithelium of terminal bronchioles of wild-type (open bars) and knockout (closed bars) mice. K63 ubiquitin accumulations were only significantly increased in terminal bronchioles of wild-type mice inhaling 100 ppm. C: Morphometric quantification of K63-ubiquitin positive cells in the epithelium of bronchi. K63-ubiquitin puncta were rare except in diacetyl-exposed mice, and cells containing puncta were significantly increased relative to controls at all exposure concentrations. Knockout mice (closed bars) were significantly more susceptible than wild-type mice (open bars) at 200 ppm when the sexes were pooled for the statistical analysis. ∗P < 0.05, ∗∗∗P < 0.001 compared with air controls of the same genotype; †P < 0.05 compared with wild-type mice. Scale bar = 20 μm (A).

Article Snippet: This design provided converging lines of evidence to ensure validity and reproducibility. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Host/clone Company Catalog no. Dilution Catenin β-1 (CTNNB1) Rabbit/E247 Abcam (Cambridge, MA) ab32572 1:100 Keratin, type II cytoskeletal 8 (KRT8) ∗ Rabbit/EP1628Y Abcam (Cambridge, MA) ab53280 1:200 Cadherin-1 (CDH1) Mouse/36 BD Biosciences (San Jose, CA) 610181 1:50 Olfactory marker protein (OMP) Goat (polyclonal) Wako (Richmond, VA) 544-10001 1:100 Ubiquitin, conjugated and unconjugated Mouse/Ubi-1 Millipore (Billerica, MA) MAB1510 1:400 Ubiquitin (linkage-specific, K63) Rabbit/EPR8590-448 Abcam (Cambridge, MA) ab179434 1:100 Allograft inflammatory factor 1 (AIF1) Chicken (polyclonal) Abcam (Cambridge, MA) {"type":"entrez-nucleotide","attrs":{"text":"Ab139590","term_id":"62160171","term_text":"AB139590"}} Ab139590 1:200 Mouse lysosomal membrane glycoprotein 1 (LAMP1) Rat/monoclonal † Developmental Studies Hybridoma Bank (Iowa City, IA) 1D4B 1:50 Mouse lysosomal membrane glycoprotein 2 (LAMP2) Rat/monoclonal † Developmental Studies Hybridoma Bank (Iowa City, IA) ABL-93 1:50 SQSTM 1/p62 Rabbit (polyclonal) Lifespan Biosciences, Inc. (Seattle, WA) LS-B4617 1:200 Open in a separate window ∗ This antibody also recognizes keratin, type I cytoskeletal 18 (KRT18).

Techniques: Immunofluorescence, Knock-Out, Staining