Extended Data Fig. 1: nSREBP2 forms condensates at the target gene locus upon cholesterol depletion.
a,b, Schematic of human SREBP2 domain structure (a) and the process of the activation of nSREBP2 (b). When cells are depleted of sterols, SCAP transports pSREBP2 from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal nuclear segment (nSREBP2) from the membrane. nSREBP2 enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. c, Total cholesterol content was determined in CHO-K1 cells. After cholesterol depletion for 16 h, CHO-K1 cells were harvested for extracting cholesterol and measuring protein concentration (n = 3 independent biological replicates). Data were shown as mean ± s.d. Statistical significance was calculated with one-way ANOVA followed by Turkey’s multiple comparison. d, Immunofluorescent staining showing nSREBP2 formed puncta under cholesterol depletion condition in SV589 cells. e, Immunofluorescent staining showing nSREBP2 formed puncta under cholesterol depletion condition in HeLa cells. f, Immunofluorescence staining showing that SREBP2 with knocking-in Halo-Tag formed puncta under cholesterol-depleted condition. g, Time-lapse imaging of the fusion behavior of nSREBP2-mEGFP. Images were captured with 5 s between frames. h, Western blotting showing that the expression level of overexpressing nSREBP2-mEGFP was the same as the endogenous level of nSREBP2 under cholesterol-depleted condition. Right panel, quantification of the exogenous and endogenous nSREBP2 expression level (n = 3 independent biological replicates). Data were shown as mean ± s.d. Statistical significance was calculated with unpaired two-tailed Student’s t-test. i, Fluorescence recovery after photo-bleaching imaging of nSREBP2-mEGFP condensates. j, Recovery curve of nSREBP2-mEGFP condensates in FRAP in (i). Data were shown as mean ± s.d. (n = 3 independent biological replicates). k, DNA FISH and immunofluorescent image showing SREBP2 puncta colocalized with INSIG1 gene locus in CHO-K1 cell nucleus under cholesterol depletion condition. Line scan analyses of the merged pictures in the zoomed images in (k) are depicted individually. d-g,i,k, Scale bars for the large images and zoomed images are 10 μm (f), 3 μm and 300 nm. Imaging data represents three independent experiments with similar results.
