Figure 5.
The N- and C-terminal ends of STAG1 regulate expression in different genomic compartments
(A and B) Relative expression of Stag1, Nanog, LINE1-T, and pre-rRNA by qRT-PCR in mESCs after treatment with the siRNA panel. Shown are (A) total and (B) nascent RNA levels. Data are represented as mean ± SEM and statistical analysis as before. Data are from three independent experiments.
(C) Representative confocal images of IF to NCL and nascent RNA in siRNA-treated mESCs labeled with EU-488. Nuclei were counterstained with DAPI. Scale bars, 2 μm.
(D) Imaris quantification of the MFI of nascent RNA (EU) within the nucleoli from (C), as defined by a mask made to the NCL IF signal. Quantifications and statistical analysis were done as above. Data are from four independent replicates. n > 50/condition, except for siSA1 5p where n > 35.
(E) Imaris quantification of the number of NCL foci in siRNA-treated mESCs. Quantifications and statistical analysis were done as above. Data are from n > 100 independent cells/condition in 2 independent replicates. See also Figure 2K.
(F) Analysis of global levels of nascent translation by measuring HPG incorporation using flow cytometry and analyzed using FloJo software. Shown is the quantification of the change in EU incorporation relative to si scr-treated cells. Data are from four independent replicates.
(G) Chromatin immunoprecipitation using an N-terminal Stag1 antibody (Ab4455) in SA1NG−FKBP mESCs treated with DMSO or dTAG. Green arrow indicates residual C-terminal truncated STAG1 isoforms. Shown also are WB for the core cohesin subunits RAD21 and SMC3 and NCL. See also Figure S5.