Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: Cytotoxicity activities of TIGIT BE-NK cells in vitro . A–E, A variety of different cancer cell lines (GFP expressing or red dye labeled) were inoculated into 96-well plates in quantities of 10 4 cells/well and left for 6 hours to allow cells to attach, then 10 4 PB-NK or BE-NK cells were added, and images of the same field of view were recorded at different times. A, NSCLC cell line H1299 (GFP expressing). B , NSCLC cell line A549 (GFP expressing). C , Fibrosarcoma cell line HT-1080 (GFP expressing). D , Hepatocellular cell line Huh7 (red dye labeled). E , Hepatoma cell line PLC/PRF/5 (red dye labeled). Images recorded and data analyzed by IncuCyte S3, and data are shown as mean ± SD.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: In Vitro, Expressing, Labeling

Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: TIGIT BE-NK cells specifically recognized target cells in a CD155-/CD226-dependent manner. A, The mean fluorescence intensity (MFI) of CD155 on H1299, H1975, A549, and H460 (NSCLC cell line), K652 (chronic myelogenous leukemia cell line), and CEM (acute lymphoblastic leukemia cell line) was assessed by flow cytometry. B, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. C, Tumor cells were coincubated with PB-NK or TIGIT BE-NK cells for 24 hours at an E:T ratio of 1:1, and then NK cell–mediated lysis was evaluated by luminescent cell viability assay. E:T corresponds to E:T cells. D, The mean fluorescence intensity of CD155 on U251-MG (glioblastoma cell line), Huh7 (hepatocellular cell line), HT-1080 (fibrosarcoma cell line), PLC/PRF/5 (hepatoma cell line), BxPC-3 (pancreatic adenocarcinoma cell line), T24 (urinary bladder cancer cell line), U2OS (osteosarcoma cell line), MCF7 (breast cancer cell line), HeLa (cervical carcinoma cell line), SKOV3 (ovarian cancer cell line), HCT116 (colorectal carcinoma cell line), HGC-27 (gastric cancer cell line), KG-1A, OCI-AML3 (acute myeloid leukemia cell line), and MM.1S (multiple myeloma) was assessed by flow cytometry. E, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. F, The correlation analysis of the result is shown in D and E . Simple linear regression analysis. G and H, The NK cell–mediated cytotoxicity was evaluated using a luminescent cell viability assay following coincubation of A549 ( G ) or Huh7 ( H ) cells with NK cells at a 1:1 ratio for 24 hours. I and J, Cytotoxic effects of individual and combined TIGIT, CD96, and PVRIG KO in NK cells. Data recorded and analyzed by IncuCyte S3; data are shown as mean ± SD. K and L, PB-NK, TIGIT BE-NK, or TIGIT/CD226 double–KO NK cells were cocultured with A549 or Huh7 tumor cells. NK cells were harvested at 0, 2, 15, and 30 minutes after stimulation, and p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) activity was assessed by Western blotting. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: Fluorescence, Flow Cytometry, Expressing, Lysis, Cell Viability Assay, Activity Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells

doi: 10.3390/ijms21197263

Figure Lengend Snippet: Autofluorescence of the three tested cell culture plates, differing in the material used in the bottom (plastic: polystyrene cell culture microplate Clear ® (Greiner Bio-One, Vilvoorde, Belgium); film: cell imaging microplate 25-μm film bottom (Eppendorf, Aarschot, Belgium) and glass: cell imaging microplate 170-μm coverglass bottom (Eppendorf)). Fluorescence intensity is represented in arbitrary units (AU). Plastic-bottomed plates show the highest autofluorescence values, while glass-bottomed plates showed the lowest. Film-bottomed plates showed intermediate autofluorescence that statistically differed from the two former plate types. Different letters denote values that are significantly different.

Article Snippet: Three 96-well plates were considered: polystyrene cell culture microplate μClear ® (Greiner Bio-One, Volvoorde, Belgium; Cat No 655079) (Plastic), cell imaging microplate 25-μm film bottom (Eppendorf, Aarschot, Belgium Cat No 0030741013) (Film) and cell imaging microplate 170-μm coverglass bottom (Eppendorf, Aarschot, Belgium; Cat No 0030741021) (Glass).

Techniques: Cell Culture, Imaging, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Bioengineered AAV9 and Optimised Microdystrophin Vectors Augment Phenotypic Rescue in a Murine Model of Duchenne Muscular Dystrophy

doi: 10.1111/jcmm.71078

Figure Lengend Snippet: Validation of codon‐optimised microdystrophin transgene in vitro. Transduction of AAV9K51Q‐μDys and AAV9K51Q‐CoμDys vectors in C2C12 cells (A), at an MOI: 1 × 10 5 and its quantification (B) are shown. Representative images were obtained in confocal microscope (AXR Nikon) with a 40X objective (Scale bar: 50 μm). Semi‐quantification was performed from images ( n ≥ 8 per group) obtained in ZOE‐Fluorescent cell imager. The data is represented as mean ± SD. Asterisk (*) represents statistical comparison of AAV9K51Q‐μDys and AAV9K51Q‐CoμDys treated conditions with respect to the cell control and Hash (#) refers to statistical comparison of AAV9K51Q‐CoμDys with respect to AAV9K51Q‐μDys. # p < 0.05, *** p < 0.001, **** p < 0.0001. For statistical comparison, a one‐way ANOVA was performed (GraphPad Prism 8.0.2 software).

Article Snippet: The images were obtained in the ZOE‐Fluorescent cell imager (Bio‐Rad, Hercules, CA, USA) and these images ( n ≥ 8/group) were further used for semi‐quantification analysis (ImageJ software).

Techniques: Biomarker Discovery, In Vitro, Transduction, Microscopy, Comparison, Control, Software