Trcn0000020239

Plasmid expressing shRNA against human SNAT2 (Sigma-Aldrich, TRCN0000020239 and TRCN0000020241) and MISSION® pLKO.1-puro Non-Target shRNA Control Plasmid DNA (Sigma-Aldrich, SHC016)) were purchased from Sigma-Aldrich. HEK 293T cells (150 mm dishes, 50% confluency) (ATCC, CRL-3216) were transfected with 12.5 μg shRNA expressing vector: 8 μg viral packaging plasmid (psPAX2 (Addgene,12260)): 4.5 μg viral envelope plasmid (pMD2.G (Addgene,12259)) ratio with X-tremeGENE™ 9 DNA Transfection Reagent (Roche, 6365779001) according to the manufacturer’s instructions. After transfection, cells were maintained in corneal growth media which was collected after 24, 48 and 72 h and filtered through 0.45 μM filter. The filtrate was diluted with the corneal growth media (4:1 ratio) supplemented with Hexadimethrine bromide (Sigma-Aldrich, H9268) at 10μg/mL and added to the corneal cells for 12 h followed by 12 h in the growth media without viral particles. The procedure was repeated once more and cells were allowed to recover one day in the growth media. After this, cells were subcultured and grown in media containing 1 μg/mL puromycin. After 3–4 days of the selection with puromycin cells were used for experiments. Knockdown efficiency was verified by Western blot analysis.

Propagation of lentiviral particles expressing shRNA against human SNAT2, GORASP2 and human and mouse GADD34 (TRCN0000020239, TRCN0000278406, TRCN0000003041 and TRCN0000353349, respectively; Sigma-Aldrich) or empty vector (pLKO.1) was performed in HEK293T cells. The osmolarity of media was increased by addition of NaCl for corneal epithelial cells or sorbitol for MEFs. The following reagents were purchased from commercial vendors: Sal003, nocodazole, ISRIB, brefeldin A and golgicide A (Tocris); cycloheximide, MG132, guanabenz and tunicamycin (Sigma-Aldrich). Sephin 1 was a generous gift from Anne Bertolotti (University of Cambridge, UK).