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: Cell Reports Methods

Article Title: Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100456

Figure Lengend Snippet:

Article Snippet: Human stem cells were subject to routine pluripotency testing using BD Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences) as recommended by the manufacturers, or by immunostaining against OCT3/4, SOX2 and NANOG using the standard immunostaining protocol as detailed below.

Techniques: Western Blot, Flow Cytometry, Immunofluorescence, Recombinant, Blocking Assay, SYBR Green Assay, XF Assay, Generated, Software