Extended Data Fig. 8: FACT inhibition disrupts NDF nuclear localization.

a, Western blot analysis of Spt16 levels in Spt16-degron iPSCs treated with DMSO or auxin for 4 h, revealing a slight reduction in Spt16 protein levels. b, Growth curve of Spt16-degron iPSCs treated with or without auxin over 24 h. The data indicate a critical dependency on Spt16, with over 90% cell death observed within 24 h. c-d, ChIP-seq data for NDF in Spt16-degron iPSCs, untreated and 4-hour post-auxin treatment. Genome browser views (c) and metagene heatmap (d) normalized to total reads across two independent experiments, suggesting mild changes in NDF chromatin enrichment upon partial Spt16 depletion. e, Western blot analysis of Spt16 levels upon curaxin treatment. f, Treatment of Spt16-GFP knock-in iPSCs with 2 µM curaxin for 1 hour rapidly alters the pattern of Spt16 puncta. g, Scatter plot of normalized numbers (left panel) and normalized intensities (right panel) of Spt16 puncta upon treatment of curaxin. Puncta intensity was normalized to total cellular fluorescence. Data show ~90% decrease in puncta number (p < 0.0001) and ~70% reduction in puncta intensity (****p < 0.0001) (DMSO 1 h: n = 50 cells; curaxin 1 h: n = 32 cells for number analysis, and n = 25 cells for intensity analysis, mean ± SD, technical replicates, repeated twice) h, Treatment of NDF-mScarlet knock-in iPSC with 2 µM curaxin for 1 hour rapidly abolished the formation of NDF puncta. i. Scatter plots display the normalized counts and normalized intensities (relative to total cellular intensity) of NDF-mScarlet puncta after treatment with either DMSO or 2 µM curaxin. The data indicate that while the number of puncta remained largely unaffected, the intensity of the puncta was significantly reduced. ****P < 0.0001. n.s. not significant. n = cells (DMSO: 49; curaxin: 31), technical replicates, repeated twice, mean ± SD, panels g and i were analyzed by nonparametric two-sided t-test.

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