Figure 4. Acetylation of SPRTN is critical for SPRTN-mediated DPCs repair and is dependent on SPRTN deubiquitination.

(A) HEK293T cells transfected with Flag-SPRTN were treated with FA (2 mM, 2 h). Cell lysates were subjected to immunoprecipitation with Flag beads and immunoblotted with the indicated antibodies.

(B) The alignment of the amino-acid sequence of SPRTN among different species. BOVIN: Bos Taurus; XENTR: Xenopus tropicalis; CAEEL: Caenorhabditis elegans.

(C-D) HEK293T cells transfected with WT, K230R or K230Q Flag-SPRTN were treated with or without FA (2 mM, 2 h). Cells were lysed as in (A) and immunoblotted with the indicated antibodies.

(E-F) Sprtn^F/F; Cre-ER^T2 MEFs stably expressing control vector (Vec), human WT, K230R or K230Q SPRTN were treated with MeOH or 4-OHT for 48 h, followed by CPT (1 μM, 30 min). Top1cc signals were detected by immunofluorescence. Nuclei were visualized with DAPI (blue). Representative images are shown in (E). Quantification of foci signals is shown in (F) (** p<0.01). Scale bars, 10 μm.

(G) Cells from (E) (treated with MeOH or 4-OHT for 48 h) were treated with FA (250 μM, 1 h) and DPCs were isolated. DPCs were measured as the ratio of crosslinked DNA compared to total DNA.

(H) Survival assays for cells from (E) (treated with MeOH or 4-OHT for 48 h) exposed to FA or CPT. Error bars indicate mean ± SD of three independent experiments (** p<0.01).

(I) Control or VCPIP1-knockdown HEK293T cells transfected with Flag-SPRTN were treated with or without FA (2 mM, 2 h). Cells were lysed as in (A) and immunoblotted with the indicated antibodies.

See also Figures S3–S4.