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. 2021 Jun 18;11:12893. doi: 10.1038/s41598-021-92223-x

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021

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NRAS expression is downregulated post HML-2 downregulation, and its overexpression is sufficient to restore cellular proliferation to AT/RT cells. (a) Downregulation of HML-2 transcription with shRNA results in a decrease in NRAS expression. Transcription level of HML-2 env and NRAS measured with qPCR 48 h post transfection with shRNA targeting HML-2 env in CHLA 02. (b–g) Co-transfection with NRAS overexpression plasmid can overcome the effects of HML-2 downregulation. (b) Expression of env, gag, and NRAS transcripts 48 h post transfection in CHLA 04 cells with both CRISPRi + gRNA and NRAS plasmids. (c) Cell number in CHLA 04 AT/RT cells transfected with CRISPRi + gRNA and either a CAT plasmid or an NRAS construct and 5 days post-transfection. (d) Viable cell percentage 5 days post transfection with CRISPRi and either CAT or NRAS plasmids. Image of cells transfected with CRISPRi plus HML-2 gRNA and either CAT (e) or NRAS (f). (g) Quantification of cell aggregate size 5 days post-transfection with CRISPRi + gRNA and either CAT or NRAS plasmids. Graphs generated with Prism v9. [Error bars represent SEM. Statistics in Supplemental Table S6].