human nk cell line nk 92mi  (ATCC)

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: Chem_NK cells exhibit enhanced mitochondrial metabolism and acid resistance. A Pie graph showing expression of protein products of significantly regulated genes. Heatmap showing metabolic genes identified by proteomics analysis. B Dot plot showing the top enriched GO biological processes related to metabolism in Chem_NK cells based on DEGs, as determined by DAVID analysis. The number of genes enriched within each GO term is represented by dot size. Metabolic changes were confirmed in KEGG analysis. C OCR of C_NK and Chem_NK cells. D–F Parameters extracted from the OCR. ( D ) Basal and maximal respiration, ( E ) ATP production, and ( F ) SRC. G TMRE staining indicating the ΔΨm in C_NK and Chem_NK cells. Quantification of Mean Fluorescence intensity (MFI) is shown on the right. H Cytotoxicity of C_NK and Chem_NK cells against MDA-MB-231 cells was assessed by the CFSE/7-AAD assay at an E: T ratio of 10:1 in the presence of LA (lactate 20 mM, pH 6.0), NaL (lactate 20 mM, pH 7.5), and HCl (non-lactate, pH 6.0) for 4 h. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t -test for panels C–G and a two-way ANOVA with Tukey’s multiple comparisons test for panel H. Values represent the mean ± SD. ns, non-significant. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Staining, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: Chem_NK cells maintain high mitochondrial metabolism under acidosis, which confers acid resistance. A OCR of C_NK and Chem_NK cells after exposure to pH 6.0 for 1 h. B–D Parameters extracted from the OCR. ( B ) Basal and maximal respiration, ( C ) ATP production, and ( D ) SRC. E TMRE staining indicating the ΔΨm after exposure to pH 6.0 for 1 h in C_NK and Chem_NK cells. MFI is shown on the right. F Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of Oligo (1 µM) or Rot/AA (0.5 µM) for 4 h, with a 30 min pre-treatment with Oligo and Rot/AA. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t -test for panels B–E and a two-way ANOVA with Tukey’s multiple comparisons test for panel F. Values represent the mean ± SD. ns, non-significant. ** p <0.01, *** p <0.001, **** p< 0.0001.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: Mitochondrial metabolism in Chem_NK cells supports their migration and tumor infiltration in acidosis, which contributes to antitumor activity. A Schematic illustration showing the sequential process of NK cell migration, target recognition, and killing. B Representative time-lapse images of C_NK or Chem_NK cells (outlined with white dotted lines) exhibiting motility and interacting with MDA-MB-231 cells (green) at pH 6.0. Nuclei were stained with DAPI (blue). Red arrows indicate points of contact between NK cells and target cells. Scale bar, 20 μm. Quantitative killing rates (%) of target cells are shown in the accompanying graph. (C_NK: n=64; Chem_NK: n=62) C Cell trajectory plots of C_NK and Chem_NK cells co-cultured with MDA-MB-231 cells at pH 6.0. Corresponding quantification of migration velocity is shown (C_NK: n=55; Chem_NK: n=46). Each dot represents an individual cell. D Immunofluorescence staining of NK-92MI cells (green, CFSE) and nuclei (blue, DAPI) in the indicated tumor sections. Scale bar, 100 μm. E The percentage of C_NK and Chem_NK cells that migrated into tumor tissue determined by flow cytometry (n=5). F Migration assay of C_NK and Chem_NK cells incubated in medium at pH 6.0 for 12 h, with or without Oligo (1 µM) or Rot/AA (0.5 µM). NK cells were pre-treated with inhibitors for 30 min prior to the assay. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t -test for panels B, C, and E and a two-way ANOVA with Tukey’s multiple comparisons test for panel F. Values represent the mean ± SD. ns, non-significant. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Activity Assay, Staining, Cell Culture, Immunofluorescence, Flow Cytometry, Incubation, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: Chem_NK cells resist acidosis-induced mitochondrial fragmentation via the PKA–DRP1 axis. A Schematic of the PKA–DRP1 regulatory axis. PKA-mediated phosphorylation of DRP1 at S637 inhibits mitochondrial fission, while phosphorylation at S616 promotes fission. B – C Western blot analysis of NK cells cultured at pH 7.5 and 6.0. B Detection of phosphorylated PKA substrates. C DRP1 phosphorylation status showing levels of p-DRP1 S637 and p-DRP1 S616 . Quantification graphs are shown on the right. D TEM images of mitochondria in NK cells cultured at pH 7.5 and 6.0. Red boxes indicate regions shown in the magnified insets, and red outlines highlight individual mitochondria. Scale bars, 1 μm. Quantification of mitochondrial length from TEM images is shown on the right. E Schematic of the 4T1 orthotopic breast cancer mouse model. C_NK or Chem_NK cells were intratumorally injected to evaluate p-DRP1 S637 expression in tumor-infiltrating NK cells. F Flow cytometric analysis of dissociated tumor tissues comparing p-DRP1 S637 expression between C_NK cells and Chem_NK cells ( n = 5). MFI is shown on the right. All experiments were performed at least in triplicate. Statistical analyses were performed using a two-way ANOVA with Tukey’s multiple comparisons test for panel B–D and an unpaired Student’s t -test for panel F. Values represent the mean ± SD. ns, non-significant. * p <0.05, ** p <0.01.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Phospho-proteomics, Western Blot, Cell Culture, Injection, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: Chem_NK cells preserve functional activity in acidosis through the PKA–DRP1 axis. A Schematic of the PKA–DRP1 regulatory axis and the effect of H-89 treatment. H-89, a pharmacological inhibitor of PKA, suppresses PKA activity and thereby reduces DRP1 S637 phosphorylation, resulting in increased mitochondrial fission. B Western blot analysis of p-DRP1 S637 in NK cells cultured at pH 6.0 with or without H-89. Quantification is shown on the right. C OCR of Chem_NK cells after exposure to pH 6.0 for 1 h with or without H-89. D – F Parameters extracted from the OCR. ( D ) Basal and maximal respiration, ( E ) ATP production, and ( F ) SRC. G Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of H-89. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. H Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of H-89 for 4 h. H-89 (20 µM, 30 min) was applied before all experiments. All experiments were performed at least in triplicate. Statistical analyses were performed using a two-way ANOVA with Tukey’s multiple comparisons test for panel B, G, and H and an unpaired Student’s t -test for panel D–F. Values represent the mean ± SD. ns, non-significant. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Functional Assay, Activity Assay, Phospho-proteomics, Western Blot, Cell Culture, Migration, Incubation, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

doi: 10.1186/s12964-026-02786-3

Figure Lengend Snippet: The PKA–DRP1 axis preserves effector function of NK cells in acidosis. A OCR of C_NK cells after exposure to pH 6.0 for 1 h with or without Forskolin. B – D Parameters extracted from the OCR. ( B ) Basal and maximal respiration, ( C ) ATP production, and ( D ) SRC. E Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of Forskolin. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. F OCR of C_NK cells after exposure to pH 6.0 for 1 h with or without P110. G–I Parameters extracted from the OCR. ( G ) Basal and maximal respiration, ( H ) ATP production, and ( I ) SRC. J Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of P110. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. K Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of P110 for 4 h. Forskolin (50 µM, 30 min) or P110 (0.5 µM, 24 h) was applied before the indicated experiments. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t -test for panel B, C, D, F, H, and I and a two-way ANOVA with Tukey’s multiple comparisons test for panel E, J, and K. Values represent the mean ± SD. ns, non-significant. *p <0.05, **p <0.01, ****p <0.0001.

Article Snippet: The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Incubation, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: NK Cell‐Derived Small Extracellular Vesicles Armed With CLDN4‐Targeting Peptides Potentiate Radiotherapy in Gastric Cancer

doi: 10.1002/jev2.70200

Figure Lengend Snippet: ROS induction and radiosensitising effects of NESC in NCI‐N87 cells. (a) Western blot analysis of perforin, granzyme B and FasL in NK cell lysate and NK‐sEV. (b) Cell viability of NCI‐N87 cells incubated with three concentrations of NK‐sEVs or NESC (5, 10 and 20 µg). Viability of PBS‐treated cells was considered as a base value. Data was shown as mean ± s.d. ( n = 3). (c) Bar plot showing the KEGG enrichment analysis results, including the pathway names, related genes and pathway classifications. (d and f) Confocal images (f) and quantified data (d) of ROS generation in NCI‐N87 cells treated with different regimens. (blue: DAPI, green: DCFH‐DA). (e and g) Confocal images (g) and quantified data (e) of DNA breaks in NCI‐N87 cells treated with different regimens. (blue: DAPI, red: γ‐H2AX). G1: PBS, G2: NESC (200 µg/mL), G3: 2 Gy and G4: NESC (200 µg/mL) + 2 Gy. p values were calculated by an unpaired t ‐test (* p < 0.05, ** p < 0.01).

Article Snippet: Human NK cell line NK‐92MI, human GC cell lines AGS, MGC803, MKN45, NCI‐N87, SGC7901, SNU‐1 and normal human gastric epithelial cell line GES‐1 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Incubation