drp1 wt  (Thermo Fisher)

Figure Legend Snippet: CRISPR-Cas9-mediated deletion of DRP1 in NK cells . A. Schematic representation of DRP1-mediated mitochondrial fission and its inhibition by Mdivi-1 or CRISPR-Cas9-mediated knockout (KO); B. Cytotoxic activity (% K562 cells killed) of NK cells cultured under hypoxia (1% O₂) with or without Mdivi-1 treatment (n = 6); C. Mitochondrial occupancy in hypoxic NK cells with or without Mdivi-1, as measured by confocal microscopy (n = 4). Representative images shown. Scale bar = 5 µm; D. Schematic of the DRP1 gene with target sites for the three guide RNAs used for CRISPR editing; E. Representative Sanger sequencing chromatograms (left) for each guide RNA and corresponding KO efficiency scores (right) at day 3 and week 3 post-editing, analyzed with Synthego’s ICE tool; F. Flow cytometric quantification of DRP1 protein expression in DRP1 KO vs DRP1 WT NK cells (n = 3); G. Growth curves of DRP1 WT vs DRP1 KO NK cells (n = 3). * p < 0.05; ** p < 0.01; ns = not significant.

Techniques Used: CRISPR, Inhibition, Knock-Out, Activity Assay, Cell Culture, Confocal Microscopy, Sequencing, Expressing

Figure Legend Snippet: Mitochondrial, transcriptomic, and metabolic profiling of DRP1 KO NK cells under hypoxia . A. Mitochondrial occupancy in DRP1 KO NK cells cultured in normoxic (21% O₂) or hypoxic (1% O₂) conditions, assessed by 3D confocal imaging, with each point representing a single cell. Representative images shown. Scale bar = 5 µm; B. Mitochondrial membrane potential measured by TMRM staining in DRP1 KO NK cells under normoxia or hypoxia, analyzed by flow cytometry (n = 3); C. mtROS levels after normoxic/hypoxic culturing as determined by mitoSOX staining (n = 3); Data in panels D-F are based on bulk RNA-seq (n = 3): D. Volcano plot showing differentially expressed genes between DRP1 KO NK cells cultured in hypoxia (1% O₂) versus normoxia (21% O₂); significantly upregulated and downregulated genes are highlighted; E. Hallmark gene set enrichment analysis displaying the top up- and downregulated pathways in hypoxic versus normoxic DRP1 KO NK cells; F. Gene set enrichment analysis (GSEA) plots demonstrating increased expression of the hypoxia gene signature, no significant reduction in mTORC1 signaling and no significant upregulation of mitochondrial dynamics genes in hypoxic DRP1 KO NK cells; G-H. SCENITH™-based analysis of metabolic activity in DRP1 KO NK cells under normoxia and hypoxia, showing corresponding protein translation level (G) and metabolic phenotypes (H) (n = 3).

Techniques Used: Cell Culture, Imaging, Single Cell, Membrane, Staining, Flow Cytometry, RNA Sequencing, Expressing, Activity Assay

Figure Legend Snippet: DRP1 KO cytotoxic effector potential in cell lines and microtumors . A. Cytotoxic activity of DRP1 KO CD70-CAR-IL-15 NK cells against HeLa, Panc-1, and Raji target cells cultured in normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, cytotoxicity was measured by xCELLigence (HeLa, Panc-1; n = 7) and flow cytometry (Raji; n = 7), upper panels show endpoint % killing and lower panels show longitudinal normalized cell index over 24 h; B. Representative confocal images of patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid-CAF microtumors Top: fluorescent signal from CAFs. Bottom: corresponding segmentation mask used for quantification; C. Quantification of normalized green CAF signal 36 h post CAR NK cell treatment, relative to untreated control (n = 3 technical replicates). * p < 0.05; ** p < 0.01; ns = not significant; D. Unsupervised clustering of bulk RNA-sequencing of three PDAC patient-derived organoids revealed distinct transcriptional patterns per patient across six transcriptional modules, each representing a dominant biological program.

Techniques Used: Activity Assay, Cell Culture, Flow Cytometry, Derivative Assay, Control, RNA Sequencing