Pmxs hs egr1

Lentiviral particles were packaged in human embryonic kidney (HEK) 293 T cells with psPAX2 and pMD2.G through polyethylenimine (PEI) transfection and purified as previously described (59 (link)). Purified viral particles were transduced to the NSCs with a multiplicity of infection (MOI) of 1, and shRNA-expressing cells were selected using puromycin (1 μg/ml) longer than 4 days. Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene). NSCs were infected with the resulting vectors at an approximate MOI of 1 (titering of the virus was carried out with pMXs-puro GFP-p62 by puro-based selection).

The following antibodies were used: mouse anti-MAP1LC3B (Nanotools, 0231–100/LC3-5F10), rabbit anti-ATG7 (Cell Signaling Technology, 2631S), rabbit anti-RB1CC1/FIP200 (ITK diagnostics, A301-536A), rabbit anti-EGR1 (Cell Signaling Technology, 4154S), mouse anti-RPB1 (Euromedex; PB-7C2), rabbit anti-histone H3 acetyl K27 (Abcam, ab4729), rabbit anti-histone H3 acetyl K56 (Active Motif, 39,281), rabbit anti-histone H3 trimethyl K4 (Active Motif, 39,159), mouse anti-histone H3 (Active Motif, 39,763), mouse anti-TUBA4A/tubulin (Sigma-Aldrich, T9026). pMXs-hs-EGR1 was a gift from Shinya Yamanaka (Addgene, 52,724) [52]. For EGR1 knockdown, human SMARTpool EGR1 siRNA (Dharmacon, M-006526–01-0005) was used. pBABE-puro-mCherry-EFGP-LC3B was a gift from Jayanta Debnath (Addgene, 22,418) [53]. To increase the intensity of the fluorescence, the EEF1A1/EF1α promoter was cloned into the construct using NaeI restriction sites. bafilomycin A₁ was obtained from Sigma-Aldrich (B1793). Hydroxychloroquine (HCQ) was obtained from Acros Organics (263,010,250).