FIGURE 4 from p35 is a Crucial Player in NK-cell Cytotoxicity and TGFβ-mediated NK-cell Dysfunction
Year: 2023
Type: other
Abstract: Overexpression of kinase-dead CDK5 increases NK92 cytotoxicity while overexpression of p35 decreases cytotoxicity. A, Western blot analysis of protein from NK92 cells overexpressing CDK5 (OE CDK5), kinase-dead mutant CDK5 (OE CDK5-K33T), p35 (OE p35), or empty vector control (OE Ctrl). B, NK92 cells were plated in complete media with a starting count of 250,000 cells. Live cell count was obtained after 48 hours cell culture via trypan blue staining and manual counting on a hemocytometer. ns, not significant. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. C, NK92 cells were stained with Annexin V and PI and analyzed via flow cytometry. Percentage of cells staining positive for Annexin V and/or PI were graphed. Graphs display mean ± SD, n = 3 biological cultures. D, NK92 cells were fixed in 70% ethanol and stained with PI, then analyzed via flow cytometry. G0–G1, S, and G2–M phases of the cell cycle are indicated. E, NK92 cells (OE Ctrl, OE CDK5, OE CDK5-K33T) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at different E:T ratios, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. ****, P < 0.0001. Graph displays mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells (OE Ctrl, OE p35) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at 5:1 E:T ratio, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. *, P < 0.05. Graph displays mean ± SD, n = 3 biological cocultures, unpaired two-tailed t test.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4372184667