Shc002

The human embryonic kidney cells (HEK-293T), the mouse macrophage cell lines RAW264.7 (ATCC TIB-71) and J774 (ATCC TIB-67) were obtained from ATCC (Manassas, VA, USA) and maintained in DMEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2. Fyn kinase-silenced and control stable cell lines were produced by transducing the macrophage cell lines with either MISSION shRNA lentivirus Fyn particles (TRCN000023380) or MISSION non-mammalian target shRNA control particles (SHC002) (Sigma). The transduced cells were selected with 3 μg/ml Puromycin for 10 days.
Transient transfections of HEK293 cells were performed using the FugeneHD (Roche Applied Science, Basel, Switzerland) according to the manufacturer’s protocol.

Ectopic over expression of eIF4E in AT2 cells: Primary rat lung alveolar epithelial cells (AEC) were transduced with lentivirus expressing eIF4E (pEF1α-HA-eIF4E-IRES-eGFP) or control (pEF1α-eGFP) (MOI = 10) using polybrene (final concentration 8 μg/ml) on day 2 of culture. On day 3 of culture, treatment with vehicle control or TGF-β1 (2.5 ng/ml) was initiated. Cell extracts were prepared for immunoblot analysis after 7 days of transduction or cultures were continued until day 14 and fixed in 4% PFA for immunostaining.
shRNA knockdown of eIF4E: RLE-6TN cells (4 × 10⁴) were seeded into 24 well dishes, and transduced with lentivirus expressing eIF4E shRNA (TRCN0000077474, ThermoForma/Open Biosystems) or non-silencing shRNA (SHC002, Sigma) on day 1 (MOI = 1), followed by initiation of TGF-β1 (2.5 ng/ml) treatment on day 2. After 2 days of TGF-β1 treatment (day 4), cell extracts were prepared for immunoblot analysis of α-SMA expression. A similar procedure was used for inhibition of eIF-4E in RLE-6TN cells by transducing cells with virus expressing constitutively active 4E-BP1 (pEF1α-3HA-4EBP1 TTAA-IRES-GFP) or control virus (pEF1α-eGFP).

pLKO.1-puro lentiviral vectors expressing SOX2 shRNA and control shRNA (SHC002) were purchased from Sigma-Aldrich. For generation of lentiviral particles, HEK293FT cells were co-transfected with the shRNA lentiviral plasmid (pLKO.1-puro) and ViraPower Lentiviral packaging mix (pLP1, pLP2, pLP-VSV-G; Life Technologies) using Lipofectamine 2000 and the culture supernatants containing lentivirus were harvested at 48 h after transfection. For lentiviral transduction, cells were treated with the shRNA-expressing lentivirus in the presence of 5 μg/mL polybrene (Sigma-Aldrich, MO). To ensure shRNA-mediated silencing of SOX2 expression, the mRNA levels of SOX2 and GAPDH were determined by RT-PCR analysis.
Retroviral plasmids carrying the pMX-SOX2 were kindly provided by Jeong Beom Kim (UNIST, Republic of Korea) and individually co-transfected with packaging plasmids (gag-pol and VSV-G) into Plat-GP cells using Lipofectamine-Plus reagent. Plat-GP cells (RV-103, Cell Biolabs, Inc., San Diego, CA) were maintained in DMEM with high glucose, 10% FBS, 10 μg/mL blasticidin, and 1× penicillin-streptomycin. Virus-containing supernatants were collected two days after transfection, passed through a 0.45 μm filter, and stored at −80°C.

Lentivirus-expressing shRNA targeted to mouse OSCP was packaged with lentivirus shRNA construct (clone TRCN0000076166: 5′-CCGGGCTTCCTGAGTCCAAACCAAACTCGAGTTTGGTTTGGACTCAGGAAGCTTTTTG-3′, Sigma-Aldrich), packaging vector psPAX2 (Addgene) and envelope vector pMD2.G (Addgene). Lentivirus-expressing nontarget shRNA control (SHC002, Sigma-Aldrich) was used as a control. Mouse primary neurons were cultured for 3 days before infection with lentivirus at a multiplicity of infection (m.o.i.). of 5. The virus containing medium was removed after 2 h and fresh culture medium was replaced to continue culturing. Neurons were treated and collected for experiments after a further 7 days in culture.