Figure 2.

SPRTN-Deficient Mammalian Cells Fail to Repair DPCs and Are Hypersensitive toward DPC-Inducing Agents

(A) Schematic representation of the KCl/SDS precipitation assay used to measure DPC repair. Cells are lysed in denaturing conditions (1% SDS), followed by sonication and precipitation of cellular protein by the addition of KCl. Crosslinked DNA co-precipitates with the protein, whereas free DNA remains in the supernatant. The precipitate is washed several times before quantification of soluble and crosslinked DNA.

(B) SPRTN-deficient MEFs fail to repair formaldehyde-induced DPCs. Sprtn^F/−, Sprtn^F/+ (untreated or treated with 4-hydroxy tamoxifen [4-OHT] for 48 hr), Fancd2^+/+, and Fancd2^−/− MEFs were treated with 200 μM formaldehyde (FA) for 1 hr to induce DPCs and lysed directly or allowed to repair. DPCs were measured as the ratio of crosslinked DNA compared to total DNA. Error bars indicate SEM of two independent experiments.

(C and D) Knockdown of SPRTN results in formaldehyde sensitivity in human cells. Relative cell numbers were determined 6 days after U2OS cells transfected with SPRTN or control siRNA were treated with the indicated doses of formaldehyde, aphidicolin, or mytomicin C. Error bars represent SD of two to four replicates.

See also Figure S2.