Generation of Plasmids for Stable Expression in HeLa Cells

Plasmids for stable expression in HeLa cells were generated as follows: DNA fragments encoding monomeric enhanced GFP with A206K mutation (mGFP), monomeric RFP (mRFP), mRuby3 (codon-optimized from Addgene #74252), HaloTag7 (Promega, N2701), or SNAP-tag (New England BioLabs, N9181S) were inserted into the retroviral plasmids pMRX-IP (harboring a puromycin-resistant marker; Kitamura et al., 2003 (link); Saitoh et al., 2003 (link)), pMRX-IB (harboring a blasticidin-resistant marker; Morita et al., 2018 (link)), or pMRX-No (without a drug resistance marker) by the seamless ligation cloning extract (SLiCE) method (Motohashi, 2017 (link)). Then, DNA fragments encoding rat LC3B, the signal sequence of Drosophila BiP (residues 1–18) and KDEL (for pMRX-IB-Halo-mGFP-KDEL), or the presequence of Neurospora crassa Fo-ATPase subunit 9 (residues 1–69; for pMRX-IB-pSu9-Halo-mGFP) were inserted into the pMRX-IP-, pMRX-IB-, pMRX-No-based plasmids by the SLiCE method. mRFP-GFP-LC3B in pMXs was described previously (Jiang et al., 2014 (link)). Primers used in this study are listed in Supplementary file 1. Plasmids containing the Halo constructs used in this study can be found in addgene: 184899 (Halo-LC3), 184901 (Halo-mGFP-rLC3), 184902 (Halo-mGFP), 184904 (Halo-mGFP-KDEL), and 184905 (pSu9-Halo-mGFP).