Shc001

The FUNDC1 knockdown HeLa cells (sh-F1) and rescued HeLa cells (sh-F1/WT, sh-F1/△LIR), PGAM5 knockout HeLa cells, Tet-inducible Bcl-xL expression HeLa cells, GFP-Parkin HeLa cell, and GFP-LC3 HeLa cells were maintained in our lab. The HeLa cell stably expressed mt-Keima was kindly gifted by Dr. Xin Pan from the Academy of Military Medical Sciences, Beijing.
The pLKO.1 vector was obtained from Sigma-Aldrich (SHC001). Plasmids expressing hairpin siRNA were constructed by inserting pairs of annealed DNA oligonucleotides into pLKO.1 according to the manufacturer's instructions. The targeting sequence to IRP1 was 5′-CCATAAGACCTTTATCTAT-3′. A nontargeting sequence, 5′-ACTACCGTTGTTATAGGTG-3′, was used as a scramble control (sc). To generate the stable knockdown cells, HeLa cells were transfected with pLKO.1-IRP1 or pLKO.1-sc. 24 h after transfection, 0.5 μg/ml puromycin (Sigma-Aldrich, P9620) was added to the medium for selection. Stable clones were analyzed by western blotting to confirm IRP1 knockdown.

The PMLI gene fragment was obtained by PCR amplification from pcDNA3.1-PMLI (a kind gift from Dr Mounira Chelbi-Alix, University PARIS V) using forward primer SpeI/PML F′: 5′-CATCTAACTAGTATGGAGAGCCTGCACCCGCCC-3′ and reverse primer PML R′/BamHI 5′-CATCTAGGATCCTCAGCTCTGCTCGGAGGCC-3′ (Eurofins MWG Operon). The fragment obtained was cloned into the pLKOpuro vector using the SpeI and BamHI restriction sites (SHC001, Sigma, St Louis, MO, USA). An HA-tag was introduced in the C-terminal end of PMLI by PCR using QuikChange Site-Directed Mutagenesis Kit (200518, Stratagene, Cedar Creek, TX, USA) and the following primers: HA-tag-sense 5′-CCTCCCAGCAGAGCTACCCTATCGATGTTCCAGATTACGCTTGAGGATCCACCGG-3′ and HA-tag-antisense 5′-CCGGTGGATCCTCAAGCGTAATCTGGAACATCGTATGGGTAGCTCTGCTGGGAGG-3′. The deletions of PMLI NLS and PMLI NES were also performed by PCR using QuikChange Site-Directed Mutagenesis Kit using ΔNLS-sense 5′-GCCCCAGGAAGGTCGGGAAGGAGGCAAG-3′ ΔNLS-antisense 5′-CTTGCCTCCTTCCCGACCTTCCTGGGGC-3′ or ΔNES-sense 5′-ACATTAACAGGCTGTGGGAAGTGCCCGGGGC-3′ ΔNES-antisense 5′-GCCCCGGGCACTTCCCACAGCCTGTTAATGT-3′. The lentiviral particles used to infect DU145 and PC3 with pLKO-PML constructs were produced according to the manufacturer's recommendations. The lentiviral packaging mix was also purchased from Sigma (SHP001).