Vectors and cell lines

HTC75, 293T and U2OS cells were cultured in DMEM with 10% fetal bovine serum. 293T was used to package virus and HTC75 was used to construct stable cell lines. TPP1^ΔOBRD (∆T), truncated 1–330 amino acids of human TPP1, was cloned into phage-based tet-on lentiviral vector which have a C-terminal HA-flag tag and G418 resistance. TPP1 mutant overexpressed with 100 ng/ml DOX treatment. The target sequences of ATRX shRNA1 (sh590, shATRX-1) and ATRX shRNA2 (sh592, shATRX-2) were previously reported³³ and were cloned into pCl-mU6 retroviral vectors with puromycin resistance. The target sequences of shGFP and DAXX shRNA1/2 (shDAXX-1 and shDAXX-2) were previously reported and were cloned into pCl-H1 lentiviral vectors using blasticidin (BSD)³⁵. The sequences of shRNAs are:

ATRX shRNA1: CGACAGAAACTAACCCTGTAA

ATRX shRNA2: CCGGTGGTGAACATAAGAAAT

shGFP:5′-CACAAGCTGGAGTACAACT-3′

DAXX shRNA1: 5′-AAGGAGTTGGATCTCTCAGAA-3′

DAXX shRNA2: 5′-GGTAAAGATGGAGACAAGA-3′

The knockout cell lines were established using a transient transfection system (PX330 vectors) with guide RNA targeting DAXX or TERT⁵¹.

The gRNA sequences are:

DAXX: GATGTTGCAGAACTCCGCCG AGG

hTERT: GGCAGTCAGCGTCGTCCCC GGG