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

Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

Article Title: The entomotoxin Jack Bean Urease changes cathepsin D activity in nymphs of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae).

doi: 10.1016/j.cbpb.2020.110511

Figure Lengend Snippet: Fig. 1. DmCatD expression profile (mRNA and protein) in fifth instar nymphs of D. maxima. The anterior (AM) and posterior midgut (PM), the fat body (FB), the Hemocytes (He) and the hemolymph (Hemo) from fed nymphs were processed for qPCR (A) and Western blot (B), as described in Materials and Methods. For qPCR, total RNA was extracted and the DmCatD transcripts were quantified using specific primers and 18S ribosomal RNA as normalizer. ***P < 0.0001 vs fat body, hemocytes and posterior midgut (n = 5). For Western blot, approximately 50 μg of proteins were loaded in each lane, separated by 15% SDS-PAGE and probed with the anti-CatD antibody. Whole cell MCF7 lysate (0.2 μg) was employed as a positive control. Densitometric analyses of three independent blots were performed upon the intensity of the pro-DmCatD and DmCatD bands detected in each lane and the amount of total proteins loaded and stained with Ponceau S.

Article Snippet: Rabbit polyclonal anti-human cathepsin D antibody (sc-10725) and MCF7 whole cell lysates (Santa Cruz Biotechnology, Palo Alto, CA, USA); Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody and 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Carlsbad, CA, USA); Tissue-Tek embedding medium Optimal Cutting Temperature (OCT, Miles, Elkhart, IN, USA); Fluorsave (Calbiochem, Darmstadt, Germany); electrophoresis protein standards (New England Biolabs, Ipswich, MA, USA); MMLV reverse transcriptase (Promega, Heidelberg, Germany); PCR Primers (Sigma Genosys, Houston, TX, USA); MasterPure RNA Purification Kit (Epicenter Biotechnologies, Madison, WI, USA); Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were from indicated commercial sources.

Techniques: Expressing, Western Blot, SDS Page, Positive Control, Staining