Flag p21 wt

The human Chk1 cDNA (NCBI accession number NM_001114122.2) was amplified from plasmid pF1K-Chk1 (Promega; FXC03424) and cloned into downstream of the Flag tag sequence in the pcDNA3-Flag vector (Invitrogen) to generate pcDNA3-Flag-Chk1. The human p21 cDNA (NM_001291549.1) was amplified from Flag-p21-WT (Addgene plasmid # 16240; a gift from Dr. M. C. Hung)48 (link). The cDNA fragments of human PVRL4 (NM_030916.2), PRODH (NM_016335.4), LY6D (NM_003695.2), DAO (NM_001917.4), and EPN3 (NM_017957.2) were amplified from a cDNA sample prepared from U2OS cells. The cDNA fragment of human GPR172B (NM_001104577.1) was amplified from a plasmid containing PAR2 (GPR172B) cDNA (kindly provided by Dr. Y. Takeuchi). The resultant fragments were digested with appropriate restriction enzymes and cloned into downstream of the HA tag sequence in the pcDNA3-HA vector. To generate Chk1 mutant (S345A), PCR was performed using mutagenic primers. The sequences of primers used for plasmid constructions are listed in Supplementary Table 1.

For Tet‐inducible overexpression of p16^INK4a and p21^Cip1 in human primary fibroblasts we used a two‐component transposon system (Figure S1). First, the puromycin resistance gene with a preceding 2A site was integrated into the transposable region of the tet‐controllable transposon plasmid pTOV‐11. This DNA fragment was amplified from the vector pSpCas9n(BB)‐ 2A‐Puro (PX462) V2.0 (Addgene #62987). This was followed by integration of the sequence into the target vector via restriction digestion by BamHI and Crf9I. Integration of the p16^INK4a or p21^CIP1 cDNA into the target vector followed in another cloning step. The cDNA was amplified from vectors pBabepuro3‐p16Flag (Addgene #24934) and Flag p21 WT (Addgene #16240) and integrated into the transposon vector via NotI and SalI restriction sites.