FIGURE 2 from p35 is a Crucial Player in NK-cell Cytotoxicity and TGFβ-mediated NK-cell Dysfunction
Year: 2023
Type: other
Abstract: CDK5 knockdown inhibits NK92 survival while p35 knockdown increases NK92 cytotoxicity. A, qRT-PCR results using RNA isolated from NK92 cells transduced with p35 shRNA (shp35) or CDK5 shRNA (shCdk5) compared with shGFP control. GAPDH was used as the housekeeping gene. Graphs display mean ± SD, n = 3 technical replicates. B, Western blot analysis of protein from NK92 cells transduced with p35 shRNA (shp35) or negative control GFP shRNA (shGFP). C, Phase contrast microscopy of NK92 cells transduced with CDK5 shRNA or one of two p35 shRNA clones, compared with shGFP control. Images are 10x magnification; scale bar, 100 μm. D, 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. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. E, 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. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours to measure the effect of p35 knockdown on cytotoxicity. Cells were stained with PI and gated on the labeled cancer cells to measure cancer cell death. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4372286358