Extended Data Fig. 8: Inducible expression of SPO11 alleles and REC8 in U2OS cells.
From: Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8
a, Experimental setup for inducible expression of SPO11 alleles and REC8 in U2OS cells. Schematic of Tet-ON system used for inducible expression of GFP-tagged genes of interest (GOI) (upper panel). The reversible tetracycline activator (Tet-ON 3G) is constitutively expressed under regulation of the EF1α core promoter. Addition of doxycycline (dox) induces binding of the transactivator 3G (blue circles) to the TRE3G promoter, inducing expression of the GOI. b, c, Representative images of fixed cells with or without addition of doxycycline. SPO11 (b) and REC8 (c) are C-terminally tagged with GFP and nuclei are counterstained with DAPI. Scale bars, 20μm. While SPO11/REC8 localize largely to the cytoplasm, all alleles enter the nucleus as well. d, Western blots probed with anti-SPO11 or anti-GFP antibody show no detectable expression of endogenous SPO11 (predicted ~44 kDa), but show robust induced expression of the GFP-tagged SPO11 alleles (predicted size ~82.5 kDa). Loading is controlled with an antibody against tubulin, and westerns were performed three times with identical results. e, Western blot probed with anti-GFP antibody shows expression of tagged REC8 (predicted ~100 kDa) upon doxycycline induction. Loading is controlled with anti-tubulin antibody.
