Retrovirus vectors psm2 encoding shrnas

For knockdown of Nestin expression, retrovirus vectors (pSM2) encoding shRNAs were purchased from Open Biosystems (Huntsville, AL, USA). Oligonucleotides for shRNA-mediated targeting of human lamin A/C and Cdk5 were cloned into pLL3.7 (Addgene). The target sequences of shRNAs against Nestin, lamin A/C (LAC), and Cdk5 were listed in Supplementary Table 1. Full-length Nestin and its deletion mutants were cloned into the pcDNA3.1-Myc vector (Invitrogen). DsRed, Flag-tagged Nestin, and the GFP-tagged Nestin truncation mutants were constructed using Invitrogen’s Gateway System. pBABE-H2B-GFP (26790) and pBABE-puro-GFP-WT-lamin A (17662) were obtained from Addgene. WT GFP-lamin A/C and GFP-lamin A/C-S392D were provided by Dr. J.E. Eriksson (Åbo Akademi University, Turku, Finland). GFP-Lamin A/C-S392A mutant was constructed based on overlap extension PCR. BMS-265246, roscovitine, MK-2206, and 3-MA were purchased from Selleck. Z-VEID-FMK and LMB were purchased from Calbiochem. MG132 and CHX were purchased from Sigma.

For knockdown of Nestin expression, retrovirus vectors (pSM2) encoding shRNAs were purchased from Open Biosystems (Huntsville, AL, United States) (Yang et al., 2017 (link)). In addition, we used lentivirus provided by Andy Peng Xiang to upregulate Nestin. Myc-tagged Nestin was constructed using Invitrogen’s Gateway System. For overexpression of HIF1-α, plasmid was constructed using Invitrogen’s Gateway System. MG132 and CHX were purchased from Sigma. Non-target control (NTC) was used as the control group.

The truncation mutants of Keap1 were generous gifts from B. Xia (University of Medicine and Dentistry of New Jersey, New Brunswick, NJ)^{6 (link)}. A series of Nestin truncation mutants including Nestin (1-1621), Nestin (8-313), Nestin (314-640), Nestin (641-1621), Nestin (1295-1621) were constructed in our laboratory. The encoding cDNAs were PCR amplified and subcloned into the pcDNA3.1-Myc vector (Invitrogen) using appropriate restriction enzyme digests. The detailed information of the plasmids was listed in Supplementary Table 3. Two Nestin mutation plasmids, Nestin-ΔESGE (deletion mutant) and Nestin-ESGA (harboring a site-specific mutation that 1417E was replaced by 1417A within the Nestin open-reading frame) were constructed by PCR and fused into the BamHI/XhoI sites of pcDNA3.1-Myc. For knockdown of Nestin and Nrf2 expression, retrovirus vectors (pSM2) encoding shRNAs were purchased from Open Biosystems (Huntsville, AL, USA). Myc-Nestin and Flag-Nrf2 overexpression vectors were constructed using Invitrogen’s Gateway System.