(A) Downregulation of the DKC1 complex upon retinoic acid (RA)-induced differentiation of mouse ES cell line D3. Western blot analyses of whole cell extracts prepared from D3 cells (mESC D3 WCE) collected at indicated days post LIF withdrawal and RA treatment using antibodies against the DKC1 complex (DKC1, GAR1, and NOP10), XPC, OCT4, the NOP58/fibrillarin (FBL) complex, TFIIB, and β-actin as loading control. (B) shRNA-mediated knockdown of the DKC1 complex in mouse ES cells. Whole cell extracts of mouse D3 cells infected with control non-target (NT) lentiviruses or with lentiviruses targeting XPC (shXPC) or DKC1 (shDKC1-1 and shDKC1-2) are analyzed by western blotting. MOI = 25. Asterisk denotes non-specific signals. (C) ES cell colony morphology and alkaline phosphatase (AP) activity are maintained in control non-target shRNA infected D3 cells (NT), but are compromised in XPC (shXPC) and DKC1 depleted cells using two independent shRNAs (shDKC1-1 and shDKC1-2). (D) DKC1 and/or XPC depletion in ES cells compromised pluripotency gene expression. Quantification of Nanog, Oct4, Sox2, Klf4, and Fgf4 mRNA levels in single and double knockdown of XPC and DKC1 in D3 cells are analyzed by qPCR and normalized to β-actin (Actb). For double knockdown experiments, a cumulative MOI = 50 is used. Data from representative experiments are shown. Error bars represent standard deviation (n = 3). (E) DKC1 and/or XPC depletion in ES cells induces spontaneous differentiation towards primitive ectoderm and trophectoderm. Quantification of mRNA levels of primitive ectoderm marker Fgf5, mesoderm marker T, primitive endoderm marker Gata6, and extraembryonic trophectoderm marker Cdx2 in single and double knockdown of XPC and DKC1 in D3 cells are analyzed as in (D). Primer sequences can be found in Supplementary file 1.