Extended Data Figure 10: DUXBL/TRIM24/TRIM33 complex co-localizes with H3K9me3 at DUX-induced nuclear foci.

a) Immunofluorescence analysis of DUXBL in untreated or DOX-treated ESC^DUX-FKBP. Treatments include DOX for 24 hours or DOX for 16 hours plus 8 hours with dTAG compounds. DAPI was used to visualize nuclei. Scale bar, 20 μm. Three independent experiments were performed but one representative is shown. b) High-throughput imaging (HTI) quantification of the number, total intensity and area of DUXBL foci (upper, middle and lower panel, respectively) per cell in ESC^DUX-FKBP treated as in (a). c) HTI quantification of the number, total intensity and area of DUX (HA) foci (upper, middle and lower panel, respectively) per cell in untreated or DOX-treated ESC^DUX-FKBP treated as in (a). d) HTI quantification of the number, total intensity and total area of TRIM24 foci (upper, middle and lower panel, respectively) per cell in ESC^DUX-FKBP treated as in (a). For (b,c, and d), center lines indicate mean values; n=2000. p values are shown from one-tailed unpaired t-tests. Percentages of cells above the threshold (dotted line) are indicated. At least, two independent experiments were performed but one representative experiment is shown. e, f) Plot showing the ratio for HDAC (e) and HP1 (f) between the fluorescence intensity found at TRIM24 foci and the mean nuclear intensity per cell detected in DOX-treated LTR-RFP reporter ESC^DUX. Center lines indicate mean values. n=2000. Two independent experiments using two ESC^DUX clones were performed but one representative is shown. g) HTI quantification of the number, total intensity and area of H3K9me3 foci (right, middle and left panel, respectively) per cell in untreated or DOX-treated LTR-RFP reporter ESC^DUX. Center lines indicate mean values. n=2000; p values are shown from one-tailed unpaired t-tests. Three independent experiments using at least two ESC^DUX clones were performed but one representative is shown.