FIG 4.

Validating the SSRP1 conditional knockout cell line. (A) Schematic of cell line. In a wild-type DT40 background, both endogenous loci of SSRP1 were targeted by homology constructs (indicated by red box) in order to knock down both copies of the gene. A wild-type copy of the SSRP1 gene was introduced into cells on a plasmid under the control of a Tet-repressible promoter. In the absence of doxycycline, SSRP1 is expressed (SSRP1^−/− + SSRP1). In the presence of doxycycline, SSRP1 expression is ablated (SSRP1^−/−). (B) Western blot showing that SSRP1 protein levels decrease to undetectable levels by 12 h after doxycycline addition. Relevant molecular mass markers are shown. (C) Growth curve of SSRP1 knockout and wild-type cells over 96 h after adding doxycycline. SSRP1 knockout (SSRP1^−/−) does not significantly affect cell growth and proliferation until after 48 h.