Extended Data Fig. 2: The killswitch arrests the dynamics of nucleolar condensates, alters their morphology and reduces cell viability.
From: Probing condensate microenvironments with a micropeptide killswitch
a. Live cell fluorescence microscopy images of U2OS cells expressing ectopic mEGFP-fsHMGB1 variants and RFP-FIB1. The cell nucleus is highlighted with a dashed white line contour. Scale bar: 5 µm. b. (left) Model of fsHMGB1 and sequences of the KS variants within the tested mEGFP-HMGB1 proteins. (right) Quantification of circularity of nucleoli in cells expressing ectopic mEGFP-HMGB1 KS variants. P-values from Dunnett’s multiple comparison test versus fsHMGB1-full length after one-way ANOVA. P(Δkillswitch) = <0.0001, P(F-to-E&D) = < 0.0001, P(F-to-A) = 0.0009, P(F-to-G) = < 0.0001, P(0F) = 0.0972, P(F12A) = 0.0003, P(F13A) = > 0.9999, P(ΔKS-3F) = < 0.0001, P(11G3F3G) = < 0.0001, P(C16-to-A) = 0.0346, P(M-to-E&D) = < 0.0001. ****:P < 10−4, ***:P < 10−3, *:P < 0.05. c. FRAP of the indicated mEGFP-HMGB1 variants. The quantification of the corresponding mobile/immobile fraction ratios are shown in Fig. 1e. Data are mean ± s.d. d. Cell viability of U2OS cell expressing mEGFP-HMGB1 proteins. Data are mean ± s.d. P-values from one-way ANOVA Dunnett’s multiple comparison test versus fsHMGB1-full length. (Left): P(Full length) = <0.0001, P(Δkillswitch) = 0.5987, P(F-to-D&E) = 0.0320, P(M-to-D&E) = < 0.0001, P(C-to-A) = < 0.0001; (Middle): P(Full length) = <0.0001, P(Δkillswitch) = 0.9975, P(F-to-D&E) = 0.8919, P(F-to-A) = 0.9967, P(F-to-G) = < 0.5244. (Right): P(Full length) = 0.0046, P(Δkillswitch) = 0.2873, P(F-to-G) = 0.4834, P(ΔKS-3F) = 0.4818, P(0F) = 0.6900, P(11G3F3G) = 0.4578. n = three (except two for F-to-D&E from the middle plot and ΔKS-3F from the right plot) biologically independent experiments. e. Representative live cell fluorescence microscopy images of A673 cells expressing EGFP-NPM1-WT and -KS variants 24 h after doxycycline induction. The cell nucleus is highlighted with a dashed white line contour. The experiments were repeated independently twice with similar results. Scale bar: 5 µm. f. Quantification of the mobile and immobile fractions of EGFP-NPM1 proteins. Data are mean ± s.d. n = 15 cells for all samples from two biologically independent experiments. P-values from Dunnett’s multiple comparisons test versus EGFP-NPM1-WT after one-way ANOVA. P(KS) = < 0.0001, P(KS_F-to-G) = 0.76, P(KS_F-to-A) = 0.03, P(KS_0F) = 1.00. g. Mean GFP fluorescence of the bleached area. Data are mean ± s.d. n = 15 cells for all samples from two biologically independent experiments. P-values from Dunnett’s multiple comparisons test versus EGFP-NPM1-WT after one-way ANOVA.
