Journal: ACS Central Science

Article Title: A Bioinspired Scaffold with Anti-Inflammatory Magnesium Hydroxide and Decellularized Extracellular Matrix for Renal Tissue Regeneration

doi: 10.1021/acscentsci.8b00812

Figure Lengend Snippet: In vitro cytocompatibility and cell proliferation on PLGA/ECM/Mg(OH) 2 scaffolds. (a) Fluorescence images of HRCEpC labeled with PHK26 around the edges and in the center of scaffolds (S) and collagen gel (G) on the seeding day (0 d) and after 3 days (scale bar = 200 μm). (b) Number of cells in the scaffolds on these days. Values are expressed as mean ± SD ( n = 8). * p < 0.05 and ** p < 0.005.

Article Snippet: Renal epithelial cell growth kit, human renal cortical epithelial cells (HRCEpC), and renal epithelial cell basal medium were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: In Vitro, Fluorescence, Labeling

Journal: Journal of Cellular and Molecular Medicine

Article Title: CXCL16 and oxLDL are induced in the onset of diabetic nephropathy

doi: 10.1111/j.1582-4934.2009.00761.x

Figure Lengend Snippet: Involvement of CXCL16 and CD36 in the uptake of oxLDL. (A) DiI-oxLDL uptake was analysed after the pre-treatment of podocytes with an IgG control antibody (control), a CXCL16 blocking antibody (+C16 Ab), a CD36 blocking antibody (+CD36 Ab), or both antibodies (+C16 Ab + CD36 Ab). After pre-incubation of human podocytes with the blocking antibodies, cells were incubated for 4 hrs with 100 μg/ml DiI-oxLDL (red) and subsequently washed with PBS and fixed in methanol/0.02% EDTA. In addition, cells were stained with DAPI to visualize the nuclei (blue). (B) Semi-quantitative analysis of the fluorescence intensity of DiI-oxLDL in cells pre-treated with a control IgG antibody (control), a CXCL16 antibody (C16 Ab), a CD36 antibody (CD36Ab) or with a combination of both antibodies (C16AB+CD36Ab). Mean fluorescence of DiI-oxLDL was measured with an image software from Keyence. (C) DiI-oxLDL uptake was analysed after pre-incubation of the primary tubular cell line HRCEpiC with an IgG control antibody (control), a CXCL16 blocking antibody (+C16Ab), a CD36 blocking antibody (+CD36Ab) or the combination of both blocking antibodies (+C16Ab+CD36Ab) and visualized by immunofluorescence microscopy. After pre-incubation of HRCEpiC with the different blocking antibodies, cells were incubated for 4 hrs with 100 μg/ml DiI-oxLDL (red) and subsequently washed with PBS and fixed in methanol/0.02% EDTA. The cells were stained with DAPI to visualize the nuclei (blue). (D) Fluorescence intensity of DiI-oxLDL in HRCEpiC pre-treated with an IgG control antibody (control), a CXCL16 antibody (C16 Ab), a CD36 antibody (CD36Ab) or the combination of both antibodies (C16Ab+CD36Ab) was determined with the BZ-Analyzer software (Keyence). Data represent mean ± S.D. *** P < 0.001; ** P < 0.01 considered statistically significant compared to the control. (E) Podocytes were fixed with 4% paraformaldehyde and CD36 expression was investigated using monoclonal CD36 and Cy3 coupled secondary antibodies (red). The cells were stained with DAPI to detect the nuclei (blue) of the cells. Fluorescence analyses were performed with a LSM 510 Meta confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). (F) Podocytes were fixed with methanol and incubated with CXCL16 antibodies followed by Alexa 488 coupled secondary antibodies (green). Cells were than incubated with DAPI to visualize nuclei (blue). Fluorescence analyses were performed with a LSM 510 Meta confocal laser-scanning microscope (Carl Zeiss).

Article Snippet: Primary human renal cortical epithelial cells (HRCEpiC) were supplied from Provitro (Berlin, Germany) and cultured according to the manufacturer’s instructions.

Techniques: Control, Blocking Assay, Incubation, Staining, Fluorescence, Software, Immunofluorescence, Microscopy, Expressing, Laser-Scanning Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Acute loss of iron–sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells

doi: 10.1074/jbc.RA118.001885

Figure Lengend Snippet: Lipid droplet accumulation in Fe-S–deficient and iron-deficient cells. BODIPY staining was used to assess the presence of lipid droplets in HEK293 cells. A, ISCU2D71A cells without the addition of doxycycline. B, ISCU2D71A cells treated with doxycycline for 48 h. C, cells expressing ISCUC69S treated with doxycycline for 48 h. D, parental HEK293 control cells. E, HEK293 cells treated with 100 μm DFO for 24 h. F, HEK293 cells treated with 300 μm DFO for 24 h. G–I, primary cells derived from human kidney cortex tissue (HRCE cells) were treated with 0, 50, or 150 μm DFO for 48 h and stained for lipid droplets with BODIPY. The green channel shows BODIPY staining of cellular lipids, whereas the blue channel shows nuclei stained with DAPI. Bar, 10 μm.

Article Snippet: Tissue culture HEK293 Flp-In cells (Gibco) and HRCE cells (Lonza) were cultured in T-75 flasks (BD Falcon) in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, supplemented with 25 m m d -glucose, 2 m m l -glutamine, without added pyruvate, in a humidified atmosphere of 5% CO 2 .

Techniques: Staining, Expressing, Derivative Assay

Journal: Cancers

Article Title: T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity

doi: 10.3390/cancers14081998

Figure Lengend Snippet: Co-expression of PD1-41BB does not change the in vitro safety profile of the lead PRAME-SLL-TCR. ( A ) Functional avidity of CD8+ TCR-Ts, with or without PD1-41BB, following stimulation with PD-L1-transduced T2 cells loaded with graded amounts of PRAME peptide (10 −10 M–10 −5 M). Cells were cultured at an E:T ratio of 1:1. The graph shows nonlinear regression curves of relative IFN-γ release. Peptide concentration needed for half maximal IFN-γ secretion is indicated by the dashed line. The data represent means of duplicates measured at each peptide concentration. ( B ) Left part: The recognition of 191 peptides with high sequence homology with the specific PRAME-SLL peptide by CD8+ TCR-Ts with or without PD1-41BB was analyzed. Peptides were loaded onto T2_PD-L1 cells at a concentration of 10 −6 M. IFN-γ release data for 30 peptides are shown as an example; data for all peptides are shown in . Right part: PRAME-negative, PD-L1-expressing SNB-19 cells were transfected with RNA constructs encoding either a midigene (~400 bp) coding for single peptides (SLL, 1, 26, 66) or minigene (~90 bp per peptide) constructs (MG) coding for up to five variant peptides and co-cultured with effector cells. All constructs included an epitope recognized by a positive control TCR. UT T-cells served as negative control. For read-out, supernatants were harvested after 20 h and analyzed by IFN-γ ELISA. The data represent means of duplicates. ( C ) IFN-γ released by CD8+ TCR-Ts with or without PD1-41BB co-cultured with 36 LCLs covering frequent HLA-A, -B, and -C alleles. UT CD8+ T-cells served as negative control, and PRAME-SLL-peptide-loaded HLA-A*02:01 positive LCLs were used as positive control. ( D ) IFN-γ released by CD8+ TCR-Ts with or without PD1-41BB co-cultured with normal cells derived from critical healthy tissues. As positive controls, cells were loaded with PRAME SLL-peptide. HREpC: human renal epithelial cells, HRCEpC: human renal cortical epithelial cells, RPTEC: renal proximal tubule epithelial cells, NHBE: normal human bronchial epithelial cells, NHLF: normal human lung fibroblasts, HOB: human osteoblasts, Mono: monocytes, iDC: immature dendritic cells, mDC: mature dendritic cells, iCardio: iCell Cardiomyocytes. The data represent means of duplicates. ( E ) Cytotoxicity of CD8+ TCR-Ts with or without PD1-41BB against PRAME-positive PD-L1-negative (red) and PRAME-negative PD-L1-positive (green) 3D tumor cell spheroids. Fresh PRAME-positive Mel624.38 spheroids (red) were added to the co-cultures on days 3 and 6, indicated by black arrows. For days 3 and 6 pictures, before and after adding fresh tumor cell spheroids are shown. Experiments in A–E were repeated at least three times with T-cells derived from different donors; data with cells from one representative donor are shown.

Article Snippet: Primary HLA-A*02:01-positive normal human bronchial epithelial cells (NHBE, Lonza), human renal cortical epithelial cells (HRCEpC, PromoCell, Heidelberg, Germany), human renal epithelial cells (HREpC, PromoCell), renal proximal tubule epithelial cells (RPTEC, Lonza), normal human lung fibroblasts (NHLF, Lonza), human osteoblasts (HOB, Lonza), and induced pluripotent stem cell-derived iCell Cardiomyocytes (iCardio, FujiFilm CDI, Madison, WI, USA) were cultured according to the manufacturers’ instructions.

Techniques: Expressing, In Vitro, Functional Assay, Cell Culture, Concentration Assay, Sequencing, Transfection, Construct, Variant Assay, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Derivative Assay