Pcw57

The plasmids pCW57.1 (Addgene plasmid 41393), pDONR223 KRASWT (Addgene plasmid 81751), and pDONR223 KRASG12D (Addgene plasmid 81651) were used to generate dox-inducible KRAS constructs with Gateway LR Clonase enzyme mix (Thermo Fisher). EGFP was Gibson cloned from FgH1tUTG (a gift from Catherine Smith at UCSF) to the N-terminus of KRAS on pCW57.1 KRAS. mCherry-KRAS constructs were generated using pCW57.1 EGFP-KRAS as backbone, and the mCherry sequence of pHR-SFFV-KRAS-dCas9-P2A-mCherry (a gift from Luke Gilbert at UCSF) was used to replace EGFP by Gibson cloning. KRAS mutagenesis on the aforementioned inducible vectors was performed with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).

Candidate MET-TFs with cDNAs available in our cDNA collection were selected (Table 1). The initial screening was performed by cloning cDNAs into CSII-EF-RfA-IRES2-Puro lentiviral construct^{45 (link)}. Inducible expression of TP63, KLF4, HNF1A, HNF4A, and FOXA3 was achieved using a lentiviral expression construct with puromycin resistance (pCW57.1, a gift from David Root, Addgene plasmid # 41393). Inducible expression of OVOL2 was achieved using a construct with hygromycin resistance (pSLIK-Hygro, a gift from Iain Fraser, Addgene plasmid # 25737). Optimal transduction efficiency with low multiplicity of infection (MOI) was achieved by dual selection using hygromycin- and puromycin-resistant constructs. Lentiviruses were prepared in a mixture of the following packaging constructs: pCMV-VSV-G-RSV-Rev and pCAG-HIVgp (provided by RIKEN BRC, Japan). The nuclear red fluorescent protein (RFP)-expressing lentiviral construct (LV-RFP) was a gift from Elaine Fuchs (Addgene plasmid # 26001). To infect lentiviruses, HNDFs were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well, followed by transduction, performed on the second day by adding 8 µg/mL polybrene and 10 MOI of lentivirus into each well. The antibiotics selection was started 2 days after transduction.

pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA). Fragments of TM4SF1-AS1 and the neomycin resistance cassette were cloned into pBI-CMV2 (Takara Bio Inc., Kusatsu, Japan). Full-length cDNAs encoding Pur-α and YB-1 were amplified with PCR using cDNA derived from HSC-45 cells as a template and then cloned into pCMV6-Entry with a C-terminal Myc tag or FLAG tag (OriGene Technologies) or into pCMV6-AC GFP (OriGene Technologies). shRNAs targeting TM4SF1-AS1 were designed using the siDirect algorithm (http://sidirect2.rnai.jp/) and were cloned into Tet-pLKO-puro (a gift from Dr. Dimitri Wiederschain, plasmid #21915, Addgene, Watertown, MA, USA). TM4SF1-AS1 and 12×MS2 were amplified with PCR, respectively using pBI-CMV2 NeoR TM4SF1-AS1 plasmid and CFP betaGlobin 24×PP7 24×MS2 splicing reporter (a gift from Dr. Daniel Larson; Addgene plasmid #61762) as templates. The fragments were cloned into pCW57.1 (a gift from Dr. David Root, plasmid #41393, Addgene). ptagRFP-MS2coatProtein plasmid was a gift from Dr. Karsten Rippe (plasmid #103831, Addgene). Primer sequences are listed in Supplementary Table S1.

pEF6‐Myc‐GRP1‐S155D/T240D was provided by Dr Victor W. Hsu (Harvard Medical School, Boston, MA, USA) and mCherry‐TRPML1 plasmid was provided by Dr. Haoxing Xu (University of Michigan) and inserts sub‐cloned into the doxycycline‐inducible lentiviral plasmid pCW57.1 (Addgene Plasmid # 41393) via Gateway cloning. MEFs were transduced and selected with 3 µg of puromycin. Mitochondrial networks were evaluated 36‐h post addition of 1 µg/ml doxycycline.