Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Oncology Letters

Article Title: MKP-1 overexpression is associated with chemoresistance in bladder cancer via the MAPK pathway

doi: 10.3892/ol.2020.11741

Figure Lengend Snippet: MKP-1 and MAPK expression in RT112 cells transfected with NC and MKP-1 siRNA. (A) Relative MKP-1 expression in the siNC and siMKP-1 groups was examined using reverse transcription-quantitative PCR. **P<0.01. (B) Representative microscopic images of siNC and siMKP-1 treated cells captured in both 2D and 3D environments under a phase contrast microscope. (C) MKP-1 expression of the siNC and siMKP-1 groups, as determined via western blotting. GAPDH was used as the internal control. (D) Phosphorylated and total ERK1/2 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (E) Phosphorylated and total p38 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (F) Phosphorylated and total JNK protein expression levels of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. MKP-1, mitogen activated protein kinase phosphatase-1; NC, negative control; siMKP-1, MKP-1 small interfering RNA; siNC, small interfering negative control.

Article Snippet: 3D Cell Culture Gel (cat. no. P720M-10) was purchased from Col-Tgel Med ( http://www.101bio.com/P720_3D_cell_culture_gel.php ).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Microscopy, Western Blot, Negative Control, Small Interfering RNA

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia concentration is increased in cancer cell–conditioned medium and TIF. Ammonia concentration in the tumor-conditioned medium, collected after 48 hours of incubation, was measured using a Dimension Ammonia assay (Siemens; n = 3). Cells were cultured at different densities: lymphoma (0.5 × 10 6 , 1.0 × 10 6 , 1.5 × 10 6 /mL; A ), multiple myeloma (1 × 10 6 , 2 × 10 6 , 4 × 10 6 /mL; B ), and breast cancer cell lines (0.5 × 10 6 , 1 × 10 6 , 1.5 × 10 6 /mL; C ). In A–C , empty medium incubated for 48 hours (without the cells) at 4°C or 37°C is presented as a control. D, Schematic presentation of the TIF and SCF isolation from mice. TIF was collected from tumors not exceeding 1,500 mm 3 . SCF was isolated at the same time from the contralateral tight as a control tissue fluid. E–G, The concentration of ammonia in TIF and SCF isolated from Raji tumor–bearing NSG ( n = 5; E ), MM.1s tumor–bearing SCID mice ( n = 5; F ), and breast cancer–bearing mice (EMT6 in BALB/c mice, n = 5; E0771 in BALB/c mice, n = 5; 4T1 in BALB/c mice; n = 5; MCF7 in NSG mice, n = 4; MDA-MB-231 in NSG mice, n = 3; G ). P values were calculated using a paired t test. Data show individual values and means ± SEM. n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/x56a982 .

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Concentration Assay, Incubation, Cell Culture, Control, Isolation, In Vitro

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia inhibits natural cytotoxicity and ADCC of NK cells and CAR NK cells. A, The viability of NK cells incubated with different concentrations of NH 4 Cl for 4 hours was assessed using propidium iodide staining and flow cytometry ( n = 3). B, Natural cytotoxicity of NK cells against K562 cells in the presence of different concentrations of NH 4 Cl ( n = 3). K562 cells were stained with CFSE and incubated with NK cells in different concentrations of ammonia. C, RTX-dependent cell cytotoxicity of NK cells against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 4). Raji cells were stained with CFSE and incubated with NK cells and 100 μg/mL RTX in different concentrations of ammonia. D, Daratumumab (Dara)-dependent cell cytotoxicity of NK cells against Daudi cells in the presence of different concentrations of NH 4 Cl ( n = 5). Daudi cells were stained with CFSE and incubated with NK cells and 1 μg/mL daratumumab in different concentrations of ammonia. In B–D , cytotoxicity was assessed after 4 hours using flow cytometry and plotted as the percentage of propidium iodide–positive CFSE-positive target tumor cells. E, Trastuzumab-dependent cell cytotoxicity of NK cells against MCF7 cells in the presence of different concentrations of NH 4 Cl ( n = 4). Cytotoxicity was assessed using RTCA for 12 hours. The right panel presents the normalized cell index at the 12-hour time point. F, CD19 CAR NK cell cytotoxicity against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 3). Cytotoxicity was determined after 18 hours in a luciferase-based killing assay, with Raji cells stably expressing luciferase as target cells. G, PD-L1 CAR NK cell cytotoxicity against MDA-MB-231 cells in the presence of 5 mmol/L NH 4 Cl ( n = 3). Cytotoxicity was assessed using RTCA for 24 hours. P values were calculated using two-way ANOVA with Tukey post hoc test. Data show individual values and means ± SEM. n values are the numbers of biological replicates in in vitro experiments.

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Incubation, Staining, Flow Cytometry, Luciferase, Stable Transfection, Expressing, In Vitro