Experiment Overview
| Repository ID: | FR-FCM-Z6XT | Experiment name: | Flow analysis of MHC-I surface levels following DUX4 knockdown in SuSa cells | MIFlowCyt score: | 47.00% |
| Primary researcher: | Danielle Hamm | PI/manager: | Danielle Hamm | Uploaded by: | Danielle Hamm |
| Experiment dates: | 2023-03-27 - 2023-03-27 | Dataset uploaded: | Aug 2023 | Last updated: | Aug 2023 |
| Keywords: | [DUX4] [MHC-I] | Manuscripts: | |||
| Organizations: |
Fred Hutchinson Cancer Research Center, Seattle, WA (USA)
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| Purpose: | Gapmer-mediated knockdown of DUX4 to assay causal effect on population of SuSa cells with MHC-I low surface presentation levels. | ||||
| Conclusion: | Knockdown of endogenous DUX4 in SuSa cells rescued 5% of the MHC-I low population relative to control, corresponding with the percentage of cells that expressed a brief pulse of DUX4 as evidenced by prolonged expression of DUX4 targets H3.X/Y. These results demonstrated that endogenous DUX4 expression in SuSa germinoma cells suppressed IFNg-induced MHC-I expression. | ||||
| Comments: | SuSa cells were synchronized and released from confluence, lifted using trypsin, seeded onto gelatin-coated plates, and reverse transfected with either a pool of two DUX4-targeting gapmers or a non-targeting control gapmer. Cells were treated with or without IFNγ for the last 16 hours and harvested at 72 hours post-release. Cells were stained with BV605 anti-human HLA-A,B,C Antibody (BioLegend #311432), resuspended in FACS buffer (1xDPBS, 1% BSA, 5mM EDTA), and analyzed using BD LSRFortessa X-50 paired with BD FACSDiva software. Data were analyzed using FlowJo V10.5.3. | ||||
| Funding: | Not disclosed | ||||
| Quality control: | None | ||||
