Plko 1 shgfp

For CR overexpression, pLV-CALB2 was used [23 (link)]; and for CR downregulation CALB2 shRNAs [7 (link)]. pLKO.1-shGFP (plasmid #30323) was obtained from Addgene. Lentivirus particles were produced as described before [7 (link)]. Briefly, HEK293T cells were co-transfected by the CaPO₄ method with 3 μg of the envelope plasmid pMD2.G-VSVG (Addgene plasmid #12259), 8 μg of the packaging plasmid psPAX2 (Addgene plasmid #12260) and 10 μg of the transfer plasmid. Lentivirus in the supernatant of HEK293T cells was harvested 48 h and 72 h after transfection. The supernatant was filtered (0.45 μm) and resuspended in DMEM containing 10% FBS and 1% PS solution. To stably express Renilla Luciferase in MSTO-211H cells the GFP cassette in pLVTHM was replaced with a cDNA coding for the Renilla luciferase pGL4.74[hRluc/TK] (Promega). Briefly, the plasmid was digested with HindIII, filled with Klenow enzyme, and then digested with XbaI. The fragment was inserted into the PmeI and SpeI sites of the backbone of pLVTHM to produce the final plasmid pLV-hRluc. Lentivirus were produced using the same envelope plasmid as above and the packaging plasmid pCMV-dR8.91 (kind gift from Prof. D. Trono, EPFL, Switzerland).

The cDNA molecules coding for various human MEIS2 isoforms were generated by reverse transcription using total RNA prepared from the human neuroblastoma cell line BE(2)-C, verified by sequencing, and cloned into the lentiviral expression vector pCDH-CMV-MCS-puro (SBI, Mountain View, CA, USA). The Retro-X Tet-Off Advanced Inducible Gene Expression System (Clontech, Mountain View, CA, USA) was used to generate BE(2)-C cells with inducible MEIS2d expression in the absence of doxycycline. Myc-tagged human MEIS2d was generated by PCR using pCDH-puro-MEIS2d, verified by sequencing, and subcloned into pRetroX-Tight-pur. pLKO.1-based lentiviral constructs expressing shRNA to MEIS2 (clone ID: TRCN0000016043, TRCN0000016044, TRCN0000016045, TRCN0000016046, and TRCN0000016047) were purchased from Open Biosystems (Huntsville, AL, USA) and to FOXM1 (clone ID: TRCN0000273984, TRCN0000015543, TRCN0000015544, TRCN0000015545, and TRCN0000015546) from Sigma-Aldrich (St. Louis, MO, USA). pLKO.1-shGFP (Addgene, Cambridge, MA, USA) was used as a control. Retroviruses and lentiviruses were produced in 293FT cells using the packaging plasmids pHDM-G and pMD.MLVogp (retroviruses) or pLP1, pLP2, and pLP/VSVG (lentiviruses).

Lentivirus plasmids containing pLKO.1-shc-Met (TRCN0000040043, TRCN0000040044), pLKO.1-shEGFR (TRCN0000039633, TRCN0000039636) and pLKO.1-shIGF1Rβ (TRCN0000000422, TRCN0000000426) were purchased from GE Dharmacon. The c-Met overexpression plasmid (RC217003) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The pLKO.1-shGFP (Addgene plasmid #30323), the lentiviral packaging plasmid psPAX2 (Addgene plasmid #12260) and the envelope plasmid pMD2.G (Addgene plasmid #12259) were available on Addgene (Cambridge, MA). The generation of gene stable knocking down cell lines was performed as described previously [51 (link)]. Briefly, pLKO.1-sh-GFP or pLKO.1-shRNA lentivirus plasmids were co-transfected into 293T cells with psPAX2 and pMD2-G. Viral supernatant fractions were collected at 48 hours after transfection and filtered through a 0.45 μm filter followed by infection into cells together with 10 μg/mL polybrene. At 16 hours after infection, the medium was replaced with fresh medium containing 1 μg/ml puromycin and cells were incubated for another 6 days.

The negative control plasmid pLKO.1-shGFP was purchased from Addgene (Massachusetts, USA). The shNUCKS sequences (Table S1) were inserted into the pLKO.1 vector. As described previously, the cDNA sequence of human NUCKS was purchased from Youbio Biological Technology Co., Ltd. (Changsha, China), which was cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vector provided by GeneCreate (Wuhan, China) [27 ]. The Beclin1 shRNA plasmid was purchased from Sigma-Aldrich (TRCN0000299864), and the plasmid GFP-LC3B [28 (link)] was a gift from Prof. Ning Gao at the Third Military Medical University, China. Lentiviruses were generated by co-transfecting the 293FT cell line with the packaging plasmids pLP1, pLP2, and pLP/VSVG and the corresponding shRNA plasmids. Lipofectamine 2000 was used for all transfections according to the manufacturer’s instructions. Virus-containing supernatants were harvested and used for cell infections at a final concentration of 4 mg/ mL polybrene. At 24 h after the final round of infection, the cells were cultured in the presence of 2 mg/mL puromycin, and the drug-resistant cells were selected and pooled for subsequent experiments.

Deguelin (>97% purity) and other chemical reagents, including Tris, NaCl, SDS, and DMSO, for molecular biology and buffer preparation, were purchased from Sigma‐Aldrich (St. Louis, MO, USA). z‐VAD‐fmk (cat#S7023), Necrostatin‐1 (cat#S8037), and GSK'872 (cat#S8465) were purchased from Selleckchem (Houston, TX, USA). Lentivirus plasmids containing pLKO.1‐shBmi1 (#1, TRCN0000020154; #2, TRCN0000020155; #3, TRCN0000020156; #4, TRCN0000020157; #5, TRCN0000020158) were purchased from Thermo Scientific (Rockford, IL, USA), pLKO.1‐shNoxa (V3SH11240‐224893462) was purchased from GE Dharmacon (Lafayette, CO, USA). The Bmi1 expression construct pT3‐EF1a‐Bmi1 (#31783), the luciferase reporter pGL3‐Noxa‐N1 (#26112), pLKO.1‐shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), and the envelope plasmid pMD2.G (#12259) were available on Addgene (Cambridge, MA, USA). The pGL3‐Basic and the Renilla luciferase reporter construct pRL‐SV40 (Promega, Madison, WI, USA) was used as previously described.43

Cisplatin and chemical reagents, including Tris, NaCl, SDS and DMSO, for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). Lentivirus plasmids containing pLKO.1-shEZH2 (#1, TRCN0000040073; #2, TRCN0000040074; #3, TRCN0000040075; #4, TRCN0000040076; #5, TRCN0000040077), pLKO.1-shSUZ12 (#1, TRCN0000038724; #2, TRCN0000038725; #3, TRCN0000038726; #4, TRCN0000038727; #5, TRCN0000038728), and pLKO.1-shEED (#1, TRCN0000021204; #2, TRCN0000021205; #3, TRCN0000021206; #4, TRCN0000021207; #5, TRCN0000021208) were purchased from Thermo Scientific. The pCMV-HA vector (cat#631604) was purchased from CloneTech. The EZH2 expression construct pCMV-HA-hEZH2 (Addgene plasmid #24230), the luciferase reporter Puma-Luc (Addgene plasmid #16591), the pBV-Luc construct (Addgene plasmid #16539), pLKO.1-shGFP (Addgene plasmid #30323), the lentiviral packaging plasmid psPAX2 (Addgene plasmid #12260) and the envelope plasmid pMD2.G (Addgene plasmid #12259) were available on Addgene (Cambridge, MA). The Renilla luciferase reporter construct pRL-SV40 (Promega) was used as previously described [59 (link)].