Journal: iScience

Article Title: Patient-derived tumor organoids mimic treatment-induced DNA damage response in glioblastoma.

doi: 10.1016/j.isci.2024.110604

Figure Lengend Snippet: Figure 1. Overview of the procedures used to generate GBOs from resected tumor tissue, cell-type-specific immunostaining of the GBOs and parental tumors and cytokine secretion from GBOs (A) Schematic presentation of main steps for generating GBOs from resected tumor tissue, created with bioRender.com. (B) Growth rate as the quantification of relative area of viable GBOs for five weeks. Data represent mean values GS.E.M. (n = 3 GBOs samples per patient (P)). (C) Immunofluorescence analyses of the GBO TME. GBOs express markers of GSCs (SOX2, CD9, and CD44) and TME cells, such as markers of cells of vasculature (CD31 and CD105), as well as macrophages and microglia (CD68 and Iba1). Representative images from a tissue sample from one patient are shown. Scale bar: 100 mm. (D) Selected cytokines and growth factors are secreted by GBOs in comparison to cultured GB cells from the same patient.

Article Snippet: Antibodies Mouse monoclonal to SOX2 Abcam Cat#ab171380; RRID:AB_2732072 Rabbit polyclonal to GFAP Abcam Cat#ab211271; RRID: N/A Mouse monoclonal to Iba1 Abcam Cat#ab15690; RRID:AB_2224403 Rabbit polyclonal to CD68 Atlas antibodies Cat#HPA048982; RRID:AB_2680587 Mouse monoclonal to CD44 BioRad Cat#MCA2504; RRID:AB_808430 Rabbit monoclonal to CD9 Cell signaling technology Cat#13403; RRID:AB_2732848 Mouse monoclonal to CD31 (PECAM-1) Cell signaling technology Cat#3528; RRID:AB_2160882 Rabbit polyclonal to CD105 (ENG) Atlas antibodies Cat#HPA067440; RRID:AB_2685840 Rabbit Monoclonal to CD3E Abcam Cat#ab16669; RRID:AB_443425 Mouse monoclonal to PD-1 Cell signaling technology Cat#43248S; RRID:AB_2728836 Rabbit Monoclonal to PD-L1 Cell signaling technology Cat#13684T; RRID:AB_2687655 Mouse monoclonal to MDM2 [2A10] Abcam Cat#ab16895; RRID:AB_2143534 Rabbit monoclonal to p21 [EPR362] Abcam Cat#ab109520; RRID:AB_10860537 Rabbit monoclonal to MDM2 (D1V27) Cell signaling technology Cat#86934; RRID:AB_2784534 Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Invitrogen Cat#A11008; RRID:AB_143165

Techniques: Immunostaining, Comparison, Cell Culture

Journal:

Article Title: Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Na?ve Monocyte Extravasation and Transfer of Productive Virus To Enhance Hematogenous Dissemination of HCMV ▿

doi: 10.1128/JVI.01016-06

Figure Lengend Snippet: HCMV infection of endothelial cells resulted in decreased junctional protein expression. (A) Western blot analyses of actin, VE-cadherin, and occludin protein levels were performed using equal protein loading of mock-infected and HCMV-infected (MOI, 20) HMEC lysates harvested at the indicated times after infection. (B and C) Bands were analyzed by densitometry to determine relative levels of occludin (both the 65- and 72-kDa forms) and VE-cadherin (130 kDa) (B) and the alternative forms of VE-cadherin (lower molecular mass, ∼100 kDa; represented as lower MW VE-cadherin) (C) and are expressed in arbitrary units as a ratio of x/actin. A representative experiment from three independent experiments is shown.

Article Snippet: A mouse anti-vascular endothelial-cadherin (VE-cadherin) monoclonal antibody was obtained from Immunotech (Marseille, France).

Techniques: Infection, Expressing, Western Blot

Journal:

Article Title: Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Na?ve Monocyte Extravasation and Transfer of Productive Virus To Enhance Hematogenous Dissemination of HCMV ▿

doi: 10.1128/JVI.01016-06

Figure Lengend Snippet: HCMV infection of endothelial cells promoted junctional protein internalization. HMECs were grown to confluence on fibronectin-coated coverslips and mock infected or HCMV infected (MOI, 20) for 96 h. The cells were then fixed, stained with the appropriate primary and secondary antibodies and DAPI, and examined by immunofluorescence microscopy. Magnification, ×1,000. (A and B) Expression of occludin (A) and VE-cadherin (B) was examined in mock-infected cells. (C and D) Using the same exposure time, expression of occludin (C) and VE-cadherin (D) was examined in HCMV-infected cells. (E and F) A longer exposure time was used to examine the expression of occludin (E) and VE-cadherin (F) in HCMV-infected cells. A representative experiment from three independent experiments is shown.

Article Snippet: A mouse anti-vascular endothelial-cadherin (VE-cadherin) monoclonal antibody was obtained from Immunotech (Marseille, France).

Techniques: Infection, Staining, Immunofluorescence, Microscopy, Expressing

Journal:

Article Title: Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Na?ve Monocyte Extravasation and Transfer of Productive Virus To Enhance Hematogenous Dissemination of HCMV ▿

doi: 10.1128/JVI.01016-06

Figure Lengend Snippet: HCMV-induced lateral junction disruption promotes increased endothelial cell permeability and naïve monocyte transendothelial migration. (A) TER of a confluent monolayer of endothelial cells treated with a VE-cadherin-specific blocking antibody (cl75; 25 μg/ml) or the isotype control antibody (50 μg/ml) and either mock infected, HCMV infected (MOI, 20), or cytochalasin D treated (1 μM) was determined at various times after treatment. The resistance across the empty insert was also determined. Results are plotted as means ± SD of three independent experiments performed in triplicate. HMECs that were infected or infected and treated with the isotype control, with the VE-cadherin-specific blocking antibody, or the IgG1 isotype control and mock infected or HCMV infected or treated with cytochalasin D showed a significant increase (P < 0.01) in permeability compared to mock-infected HMECs. (B) Blocking VE-cadherin-based cell adhesion promotes naïve monocyte transendothelial migration. HMECs were mock infected or HCMV infected (MOI, 20) for 24 h. Cells were washed, and medium containing the VE-cadherin-specific blocking antibody (25 μg/ml) or the IgG1 isotype control (50 μg/ml) was added to each well. Isolated and labeled human monocytes were added to each well. At 96 h after the addition of monocytes, the number of monocytes that migrated completely through the endothelial cell layer was determined. Results are plotted as means ± SD of 10 random fields of view. Magnification, ×200.

Article Snippet: A mouse anti-vascular endothelial-cadherin (VE-cadherin) monoclonal antibody was obtained from Immunotech (Marseille, France).

Techniques: Permeability, Migration, Blocking Assay, Infection, Isolation, Labeling