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: eLife

Article Title: Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1

doi: 10.7554/eLife.38564

Figure Lengend Snippet: SV589 cells were transfected with the indicated plasmids encoding Flag-tagged fragments of NPC1 as described in Materials and methods. Cells were fixed and double stained with 0.8 µg/ml of rabbit monoclonal anti-Flag IgG ( green ) together with 1 µg/ml of mouse monoclonal anti-LAMP-2 IgG ( red ), and images were merged ( yellow ). LAMP-2 is a marker for lysosomes. Immunofluorescence microscopy was performed as described in Materials and methods. Scale bar, 20 µm.

Article Snippet: Antibody , Mouse monoclonal IgG against LAMP-2 , BD Biosciences, Franklin Lakes, NJ , 555803, RRID: AB_396137 , .

Techniques: Transfection, Staining, Marker, Immunofluorescence, Microscopy

Journal: eLife

Article Title: Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1

doi: 10.7554/eLife.38564

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal IgG against LAMP-2 , BD Biosciences, Franklin Lakes, NJ , 555803, RRID: AB_396137 , .

Techniques: Modification, Mutagenesis

Journal: iScience

Article Title: High glucose macrophage exosomes enhance atherosclerosis by driving cellular proliferation & hematopoiesis

doi: 10.1016/j.isci.2021.102847

Figure Lengend Snippet: Assessment of exosomes produced by macrophages cultured in a high-glucose environment (A) Representative size and concentration distribution of BMDM–NG-exo or BMDM–HG-exo purified from BMDM conditioned cell culture supernatants after a 24 h period of culture. Measurement of particle concentration (B) and particle mode size (C) using nanoparticle tracking analysis. (D) Quantification of protein concentration by qubit assay. (E) Calculation of secreted particles amounts per million of BMDM in high glucose or low glucose conditions. (F) Western blot analysis of GM130, Calnexin, Alix, Flotillin, and CD81 and CD9 in exosome-free media (EFM), cell lysate, and BMDM–derived exosomes (representative of three independent experiments). An equal volume (37.5 μL) of cDGUC fraction samples was loaded for exosomes analysis and 10 ug of protein was loaded for the cell lysate sample. (G) Electron micrograph of purified exosomes from BMDM cells. Scale bars: 100 nm. (H) Merged images showing internalization of PKH26–labeled BMDM exosomes (red) by naive culture BMDM counterstained with DAPI (blue). BMDM were co-incubated with 2x10 9 PKH26-labeled exosomes for 2 h at 37°C and washed repeatedly to remove unbound exosomes. All images were acquired using a using a Nikon microscope system with 20× objectives. Scale bars: 100μm. (I) Quantification of the fluorescence intensity.∗p< .05; ∗∗p< .01 as determined by unpaired Student's t test analysis. Data are represented as mean ± SEM.

Article Snippet: Immunoblotting: Mouse monoclonal anti-GM130 , BD Biosciences , Cat# 610823, RRID: AB_398142.

Techniques: Produced, Cell Culture, Concentration Assay, Purification, Protein Concentration, Western Blot, Derivative Assay, Labeling, Incubation, Microscopy, Fluorescence

Journal: iScience

Article Title: High glucose macrophage exosomes enhance atherosclerosis by driving cellular proliferation & hematopoiesis

doi: 10.1016/j.isci.2021.102847

Figure Lengend Snippet:

Article Snippet: Immunoblotting: Mouse monoclonal anti-GM130 , BD Biosciences , Cat# 610823, RRID: AB_398142.

Techniques: Western Blot, Binding Assay, Flow Cytometry, Recombinant, SYBR Green Assay, Staining, Modification, Lysis, Plasmid Preparation, Live Cell Imaging, Cell Culture, Qubit Protein Assay, RNA Sequencing Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells

doi: 10.3390/ijms232012201

Figure Lengend Snippet: Effect of exosomes from the three corneal cell types on hCECs proliferation. hCECs were incubated with 800–1000 μg exosomes isolated from medium of hCFs, hCECs and hCEnCs conditioned for 48 h. HBS was used as control (CTRL). Cells were fixed and Ki-67 was labeled by indirect immunofluorescence. Panel ( A ) shows representative images of Ki-67 expression in hCECs. Nuclei were counterstained with Hoechst 33258 reagent (blue). Scale bars: 100 μm. ( B ) The number of Ki-67 positive cells was calculated and plotted on graph. Between four and eight photos per condition were used for the counts. The data is expressed as the mean ± SEM from three independent experiments on two different populations. ** p < 0.01, **** p < 0.001, ns: not significant.

Article Snippet: Cells were then permeabilized with 0.2% Triton X-100 for 10 min and incubated with the following primary antibody: mouse monoclonal antibody against Ki-67 (1:200, #556003, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Incubation, Isolation, Labeling, Immunofluorescence, Expressing