Cell culture, transient transfection, CRISPR/Cas9 genome editing, small interfering RNA silencing

MEF, HEK293 and NIH/3T3 cells were grown in DMEM with 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. Transient transfections were carried out using the JetPrime transfection reagent (PolyPlus) in accordance with the manufacturer’s protocol. Atg5 KO⁵⁶ and Atg14 KO⁸² MEF were kind gifts from N. Mizushima and T. Saitoh, respectively. CRISPRMOCK, CRISPRSEC62 and CRISPRFAM134B were generated by CRISPR/Cas9 genome editing in our lab as described in ref. ²⁶. RNA interferences were performed in MEF plated at 50–60% confluence. Cells were transfected with scrambled small interfering RNA or small interfering RNA (siRNA) to silence TMX4 expression (5′-GCAUGGUGUUCUUACGUUUtt-3′, 100 nM per dish, Silencer Select Pre-designed, Ambion). Cells were processed for immunofluorescence or for biochemical analyses after 48 h of transfection.

TMX4-KO MEF cells were generated as follows: the guideRNA‐Cas9 plasmids, lentiCRISPRv2‐puro system (Addgene52961) was obtained from Addgene (http://www.addgene.org). Cas9 target design tools were used to generate guide sequences (http://crispr.cos.uni-heidelberg.de/). All protocols and information can be found at the website https://www.addgene.org/crispr. The guide RNA target sequences were synthesized by Microsynth AG. Two annealed oligonucleotides (5′-CACCGCTCGCAGCGGCAGCGGCCG-3′, 5′-AAACCGGCCGCTGCCGCTGCGAGC-3′ for murine TMX4 were inserted into the lentiCRISPRv2‐puro vector using the BsmBI restriction site. LentiV2- gRNA vector was transfected in MEF cells with JetPrime (Polyplus) according to the manufacturer’s instructions. Cells were cultured in DMEM supplemented with 10% FBS. After 2 days of transfection, the medium was supplemented with 2 μg/ml puromycin (Invitrogen). Puromycin‐resistant clones were picked after 10 days and gene KO was verified by WB.