Plenti6.3 to v5 dest

The open reading frames of the ABCD1, ABCD2, or ABCD3 genes were cloned into the lentiviral vector pLENTI 6.3/TO/V5-DEST (Invitrogen). Hek-293T cells were cotransfected with one of the generated plasmids and the lentiviral packaging plasmids pMD2G, pMDL/RRE, and pRSV/REV using jetPRIME (Polyplus) to produce lentiviruses. Virus-containing media were collected 48 and 72 h after transfection, filtered through 0.45 μm filters, and used to transduce ΔABCD1ΔABCD3 clone A, ΔABCD1ΔABCD3 clone B, or ΔABCD3ΔACOX1 cells. Blasticidin-resistant cells were selected with 3 μg/ml Blasticidin (InvivoGen) for 7–10 days. After selection, cells were allowed to recover for at least 1 week prior to experiments.

Lentivirus vectors expressing mature let-7e, anti-miRNA-oligo of let-7e (AMO-let-7e) or NC sequence were constructed by Invitrogen (Invitrogen). Briefly, the precursor sequence of let-7e and its antisense fragment were synthesized by Invitrogen. The synthesized fragments were annealed and inserted into the pcDNA™ 6.2-GW/EmGFP-miR vector (Invitrogen). Then, lentivirus plasmid was constructed by BP and LR recombination into pLenti6.3/TO/V5-DEST vector (Invitrogen) through the Gateway recombination technology. The constructed pLenti6.3/TO/V5-DEST plasmid and an optimized mix of the three packaging plasmid (pLP1, pLP2 and pLP/VSVG; Invitrogen, China) were cotransfected into 293FT procedure cell line by liposome reagent to package lentivirus. The virus liquid was collected and concentrated 48 hrs after cotransfection, and the titre of the lentivirus liquid was determined.

To generate the lentiviral constructs pLenti6.3/TO/V5-DEST-IGFBP2 (Addgene, #191006), pLenti6.3/TO/V5-DEST-IGFBP2-Clover (Addgene, #191008), and pLenti6.3/TO/V5-DEST-mTurquoise2 (Addgene, #191010), entry clones were LR subcloned with the Gateway destination vector pLenti6.3/TO/V5-DEST (Invitrogen) using LR Clonase II (Invitrogen, 11791), as per the manufacturers’ protocol. pENTR221-IGFBP2-Clover (Addgene, #191007) was synthesized by BioCat (Heidelberg, Germany), while pENTR2b-mTurquoise2 (Addgene, #191014) was generated by first polymerase chain reaction (PCR)–amplifying mT2 from pmTurquoise2-N1 [Addgene, #54843 (64 (link))] using primers 5′-GGCTGGCGCCGGTACCGCCACCATGGTGAGCAAGGGCG-3′ and 5′-GGGTCTAGATATCTCGAGTCATTACTTGTACAGCTCGTCCATGCCGAGAG-3′. This PCR fragment was then digested with Xho I/Kpn I (New England Biolabs), in parallel with digestion of the pENTR2b (Invitrogen, A10463) backbone with the same restriction enzymes. These fragments were then ligated using T4 DNA ligase (Thermo Fisher Scientific, EL0011). All plasmids were validated by analytical digests and sequencing.

For the functional complementation assay, cells were infected with viral particles for
stable overexpression of either ERAL1 or GFP (pLenti/ERAL1, pLenti/GFP). The pLenti/ERAL1
vector was constructed using the Gateway technology (Invitrogen). Specifically, the ERAL1
ORF PCR product containing attB sites was cloned to the entry plasmid pDONR 221
(Invitrogen) using the BP Clonase enzyme mix (Invitrogen) and further cloned to the
destination vector pLENTI 6.3/TO/V5-DEST (Invitrogen) using the LR Clonase enzyme mix
(Invitrogen). The pLenti/GFP vector was a kind gift of Prof. Dr. Noam Zelcer (Department
of Biochemistry, Academic Medical Center, University of Amsterdam). For virus production,
HEK293 cells at ∼50% confluency were transfected with the ERAL1 or GFP overexpressing
plasmids, together with the lentiviral packaging plasmids pMD2G, pMDL/RRE, pRSV/REV using
the DNA and siRNA transfection reagent jetPRIME® (VWR) in the normal DMEM culture medium.
The following day the medium was refreshed, and viral supernatant was collected and
filtered 48 and 72 h post-transfection. Patient fibroblasts at ∼70% confluency were
infected with the viral supernatant, grown under blasticidine selection (10 µg/ml) for
2 weeks, and further expanded in blasticidine-free medium for four more passages. Cells
were regularly checked for GFP expression. Overexpression of ERAL1 was confirmed by
Western blot analysis.

An oxygen-resistant HIF-1α, HIF1α(PP), with P402A and P564A substitutions [36 (link)], was cloned into pLenti6.3/TO/V5-DEST through homologous recombination reactions (Invitrogen, Carlsbad, CA, USA). Similarly, HIF-2α(PP) with P405A and P531A substitutions and a 3xFLAG at the amino terminus was cloned into the same vector. To produce lentiviruses, 293FT cells (Invitrogen) derived from a human embryonic kidney cell line were transfected with a lentiviral vector and Virapower packaging mix (Invitrogen) using Lipofectamine 2000. Lentiviral supernatant was harvested 3 days after transfection and filtered through a 0.45-μm sterile syringe filter (VWR, Radnor, PA, USA). The filtered virus was aliquoted and stored at −80°C. Viral titers were determined according to the manufacturer’s instruction.