Extended Data Fig. 3: Quantification of condensate-based translational regulation.
From: Programmable solid-state condensates for spatiotemporal control of mammalian gene expression
(a) Fold-induction of grazoprevir-inducible translation. HEK-293 cells were co-transfected with different amounts (1-50ng) of an expression vector for SEAP-reporter mRNA containing 16 MS2-box motifs in the 3’-UTR (pYK63) and constitutive expression vectors for (left) nucleic (NLS-FUS478-AG-NS3a(H1) & NLS-MCP-2xANR; pYK209 & pYK218) or cytoplasmic condensates and anchors (NS3a(H1)-AG-LplA & MCP-2xANR; pYK91 & pYK195). Total transfection amounts were kept constant (550ng) using pcDNA3.1(+) as a filler plasmid. SEAP expression in culture supernatants were profiled at 36h after cultivation in cell culture medium containing 1µM grazoprevir or DMSO (vehicle control). (b) Kinetics of SEAP expression by grazoprevir-repressible mRNA trapping. HEK-293 cells were co-transfected with an expression vector for SEAP-reporter mRNA containing 16 MS2-box motifs in the 3’-UTR (pYK63) and constitutive expression vectors for (left) nucleic (NLS-FUS478-AG-NS3a(H1) & NLS-MCP-2xANR; pYK209 & pYK218) or cytoplasmic condensates and anchors (NS3a(H1)-AG-LplA & MCP-2xANR; pYK91 & pYK195). 18h after transfection, 0 or 1μM grazoprevir was added to culture supernatants and SEAP levels were followed for 24h. Numbers show fold-changes calculated by dividing SEAP levels of grazoprevir-treated samples by SEAP levels of samples not treated with grazoprevir for each timepoint. (c) Reversibility of translational regulation by nuclear condensates. 5x104 HEK293 cells were seeded per well in a 24-well plate and transfected with plasmids (pYK209, pYK218 and pYK63). At 24h, 48h, and 72h after transfection, the culture medium was replaced with fresh medium. Subsequently, cells were stimulated with 50nM Grazoprevir (ON) or DMSO (OFF) for 2 h, with the culture medium changed every 24 h. SEAP activity in the culture supernatant was quantified every 8 h for 72 h starting 24 h after transfection. All data are shown as the mean ± s.d. of n≥3 independent experiments (a-c). Statistical significance was assessed using two-side t-tests. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant. Exact P values are provided in source data. Filled circles show individual results (n). Numbers above bars represent fold change between two datasets (columns).
