Figure 2.
RNAi Screen Identifies BAF as a Critical Factor to Form a Single Nucleus
(A–C) RNAi screen to identify spindle-independent nuclear assembly factors. (A) Experimental design and hypothesized phenotypes. (B) RNAi screen targeting 1,295 genes; data points indicate fraction of micronucleated cells per siRNA as classified by supervised machine learning. Blue indicates non-targeting control siRNAs. (C) Raw data from the screen. HeLa cells were imaged following siRNA-mediated knockdown and immediately (0 hr) or 22 hr after addition of nocodazole and reversine.
(D and E) Immunofluorescence (IF) of cells 96 hr after siRNA transfection (96 hr was used for all IFs unless stated otherwise) and stained for (D) lamin B or (E) FG-repeat-containing nucleoporins (with mAb414). A single confocal section is shown.
(F–H) Super-resolution microscopy 3D analysis of nuclear morphology in BAF-depleted cells. (F) Single optical section of a cell stained for lamin B and CREST (kinetochores) by immunofluorescence and a 3D reconstruction by manual tracing of lamina and DNA signal. Micronuclei with no detectable connection to the main nucleus (gray) are shown in yellow. (G) Quantification of micronucleation as judged by super-resolution microscopy and 3D reconstruction (bars indicate mean ± SD, n = 67 cells). (H) CREST-foci per micronucleus (bars indicate mean ± SD, n = 37 micronuclei).
(I–K) Analysis of nuclear fragmentation. (I) Live cells stably expressing the nuclear import substrate IBB-EGFP were imaged 96 hr after siRNA transfection and (J) analyzed for the ratio of nuclear over cytoplasmic concentration of IBB-EGFP (dots indicate individual cells, lines indicate median and interquartile range, n = 430 [siControl] and n = 472 [siBAF]). Information on definition of significance and the underlying statistical tests for all figures are detailed in the STAR Methods. (K) BAF-depleted cells that show variable IBB-EGFP concentrations within individual nuclear fragments as shown in (I, arrow heads) were classified as micronucleated (bars indicate mean ± SD, n = 437 [siBAF] and n = 468 [siControl] cells). Scale bars, 10 μm.