Extended Data Fig. 10: Nucleolar mispartitioning and dysfunction in cells expressing mutant proteins with disease-associated frameshifts.

(a) Cavitation of the nuclear inclusion formed by frameshift mutant FOXC1, FOXF1, RAX, HMGB3, PHOX2B and SOX2. Representative images of U2OS cells co-expressing RFP-Fibrillarin and EGFP-tagged mutant proteins are shown. Scale bar = 5 µm. Right: fluorescence intensity profiles of EGFP (green) and RFP (purple) quantified from the region highlighted with yellow a dashed line on images on the left. (b) Relative fluorescence intensity of bleached EGFP-tagged proteins before and after photobleaching with identical laser settings. Data displayed as mean ± SD. (c) qRT-PCR analysis of rRNA species in U2OS cells expressing the indicated wild type and mutant proteins. rRNA levels are normalized against a control RNAPII transcript (GAPDH, actin, or Cyclophilin), and the values are normalized against the rRNA/control transcript level measured in the cells expressing wild type proteins. Data displayed as mean +/− SD. P-values are from two-tailed Student’s t-tests, n = 3 biologically independent experiments. (d) Actinomycin D control experiments for measuring rRNA levels. U2OS cells were treated with Actinomycin D (30 nM for 2 h), and rRNA levels were quantified using qRT-PCR. rRNA levels are normalized against a control RNAPII transcript (actin, GAPDH or Cyclophilin), and the values are normalized against the rRNA/control transcript level measured in the control (i.e. untreated) cells. Data displayed as mean +/− SD. P-values are from two-tailed Welch’s t-test, n = 3 biologically independent experiments. On the right, light microscopy images of control and Actinomycin D-treated cells are shown. Note the dimming of the dark spots in the nucleus (corresponding to nucleoli).