Hek293t and hela cells

HeLa and HEK293T cells obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

All yeast strains used in this study were listed in Supplementary Table S1. The gene deletion mutants and genomic integration of C-terminal epitope tags were constructed by standard protocols. All yeast strains were verified by colony PCR, DNA sequencing, RT-qPCR, and/or immunoblots before being used for experiments.
The primary HUVECs were purchased from ScienCell Research Laboratories. Cells were grown in Endothelial Cell Medium supplemented with 5% of FBS, 1% of Endothelial Cell Growth Supplement and 1% of penicillin/streptomycin solution. The HeLa and HEK293T cells were obtained from the American Type Culture Collection. The HeLa and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% of penicillin/streptomycin solution. All cell lines used in this study were reauthenticated by short tandem repeat analysis after resuscitation in our laboratory.

The primary human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell Research Laboratories. Cells were grown in Endothelial Cell Medium (ECM) supplemented with 5% of FBS, 1% of Endothelial Cell Growth Supplement (ECGs) and 1% of penicillin/streptomycin solution for up to 34 passages. The primary human aortic endothelial cells (HAECs) were purchased from Procell Life Science Technology (Wuhan). The serine free ECM was purchased from ScienCell. Most experiments for PHGDH-knockdown HUVECs were performed when cells were grown in serine-free medium. The senescent cells were verified by immunoblots with p21 (1:1000; 10355-1-AP, proteintech) and Senescence-associated β-galactosidase (SA-β-gal) staining. The HeLa and HEK293T cells were obtained from the American Type Culture Collection (ATCC). The HeLa and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% of penicillin/streptomycin solution. The cell lines used in this study were reauthenticated by short tandem repeat analysis after resuscitation in our laboratory. Normally, young passage HUVECs (P10) and senescent HUVECs (P20) were used for SAHF formation (DAPI staining) and SA-β-gal staining as indicated. Other experiments were performed with young passage HUVECs (P10).

DOPC and DOPS were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ATP and ascorbic acid were purchased from Sigma-Aldrich, CHAPS from Anatrace (Maumee, OH, USA), ProteaseARREST protease inhibitor cocktail from G-Biosciences (St. Louis, MO, USA) and 1D4 peptide from Biomatik (Kitchener, ON, USA). MG132 was from ApexBio (Houston, TX, USA). HEK293T and HeLa cells were obtained from American Type Culture Collection through Cedarlane Laboratories (Burlington, Ontario, Canada). The Rho1D4 antibody initially produced in house (Hodges et al., 1988 (link); Molday and Molday, 2014 (link)) was obtained from the University of British Columbia (https://uilo.ubc.ca/industry-partners/access-ubc-technologies). The primary antibody against tubulin was from Abcam (ab15568; Toronto, Ontario, Canada) and the antibody against LAMP2 was from Santa Cruz Biotechnology (sc-5571; Dallas, TX, USA). Primary antibodies against ATP8A2 (ATP6C11 and Cdc50-7F4) have been described previously (Coleman et al., 2009 (link); Coleman and Molday, 2011 (link)). Restriction enzymes, T4 DNA ligase, Antarctic Phosphatase and Phusion polymerase were acquired from New England Biolabs (Whitby, Ontario, Canada). Primers used for mutagenesis were ordered from Integrated DNA Technologies (Coralville, IA, USA).

HEK293T and HeLa cells (American Type Culture Collection) were transfected using GenJet in vitro DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD) following the manufacturer's instructions, including the suggested total DNA according to dish size, incubation time, and the ratio of GenJet DNA for optimal transfection efficiency. For all transfection experiments, cell viability was monitored to evaluate if the expression of the above-described constructs caused cellular toxicity or damage. No evidence of toxicity was observed. Viabilities were usually around 90%.