Extended Data Figure 9: DUXBL/TRIM33/TRIM24 colocalize at DUX-induced nuclear foci.

a) Immunofluorescence analysis of DUX (HA) and endogenous TRIM24 in untreated or DOX-treated ESC^DUX−2XHA. Scale bars, 20 μm. b) Immunofluorescence analysis of endogenous TRIM24 in endogenous 2CLC observed in LTR-RFP reporter WT ESC. Scale bars, 20 μm. For (a,b), DAPI was used to visualize nuclei and two independent experiments were performed but one representative is shown. c) High-throughput imaging (HTI) quantification of the number, total intensity and area of TRIM24 foci (upper, middle and lower panel, respectively) per cell in untreated or DOX-treated LTR-RFP reporter WT or DUXBL^KO ESC^DUX. d) Western blot analysis of TRIM24 performed in WT and TRIM24^KO ESC^DUX. e) Western blot analysis of TRIM33 performed in WT and TRIM33^KO ESC^DUX lysates. f) HTI quantification of the number, total intensity and area of DUXBL foci (upper, middle and lower panel, respectively) per cell in untreated or DOX-treated LTR-RFP reporter WT or TRIM33^KO ESC^DUX. For (c and f) Percentages of cells above the threshold (dotted line) are indicated. Center lines indicate mean values. n=2000; p values are shown from one-tailed unpaired t-tests. Two independent experiments using at least two WT, DUXBL^KO ESC^DUX or TRIM33^KO ESC^DUX clones were performed but one representative is shown. g) Graph showing 2CLC residency time evaluated by using the LTR-RFP reporter in DOX-treated WT and TRIM24^KO ESC^DUX. p value is shown from a two-tailed unpaired t-test. Plot was generated by using data from two independent ESC lines per genotype. h) Western blot analysis of TRIM24 performed in WT and TRIM24^FKBP ESC^DUX. i) Western blot analysis of TRIM24 and DUXBL performed in untreated and DOX/dTAG-treated TRIM24^FKBP and DUXBL^KO; TRIM24^FKBP ESC^DUX for 16 hours. For (d,e,h,i), tubulin levels were used as loading control and two independent experiments were performed but one representative is shown. j) Graph showing 2CLC residency time evaluated by using the LTR-RFP reporter in dTAG-treated WT and DUXBL^KO; TRIM24^FKBP ESC^DUX. p values are shown from two-tailed unpaired t-tests. Plot was generated by using data from two independent ESC lines per genotype.