Psin ef2 sox2 pur

Conditional depletion of SOX2 was achieved using a doxycycline-inducible system obtained from Addgene (Cambridge, MA, USA) (Tet-pLKO-puro-SOX2; plasmid 47540) [50 (link)]. In addition, we also used a PLKO.1 lentivirus shRNA vector targeted against SOX2 (TRCN0000085748) together with the corresponding empty shRNA vector as a negative control (Dharmacon Lafayette, CO, USA). As an alternative method to knockdown SOX2 expression we used a specific siRNA (Supplementary Information). The lentiviral constructions to overexpress SOX2 (pSin-EF2-SOX2-Pur; addgene plasmid 16577) or GFP (used as a control) (pSin-EF2-GFP-Pur) cDNAs were kindly donated by Maria dM. Vivanco (CIC bioGUNE, Derio, Spain). Lentiviral reporter systems in which a composite SOX2/OCT4 response element (SORE6) coupled to a minimal cytomegalovirus (mCMV) drive the expression of GFP (SORE6-mCMVp-dsCopGFP-Puro) and its corresponding control lacking SORE6 (mCMVp-dsCopGFP-Puro) were previously generated and characterized [29 (link)] and were kindly donated by Dr. L.M. Wakefield (National Cancer Institute, Bethesda, MD, USA). Generation of lentiviral particles were performed as previously described [34 (link)]. Transduced cells were positively selected through a treatment with puromycin 30 mg/mL for 6 days. To achieve an inducible expression of SOX2 shRNA in vitro, cells were treated with 2 μg/mL of doxycycline (Sigma).

Pfu Ultra II Fusion HS DNA Polymerase (Stratagene, Agilent Technologies) was used to amplify the cDNA encoding RPPH1 or TUBB3 and then the cDNA was cloned into lentiviral expression vector pSin-EF2-Pur reformed from pSin-EF2-Sox2-Pur (Addgene, Cambridge, MA, USA). The oligonucleotides to suppress RPPH1 or TUBB3 expression were designed by RiboBio (Guangzhou, China). Then they were cloned into lentiviral expression vector pLKO.1-Pur (Addgene, Cambridge, MA, USA). The plasmids were verified by sequencing. Empty vector pSin-EF2-Pur and vector pLKO.1-Pur carrying a scrambled shRNA served as a control. 293T cells were incubated with the vectors described above, psPAX2 and pMD2G (Addgene) according to the manufacturer’s instructions. The supernatant containing infectious lentivirus was filtered through 0.22 μm PVDF filters after harvesting at 24 h post transfection and then added into the plate to infect SW620 and HCT8 cells. Infection efficiency was confirmed by qRT-PCR. The methods for transfection and lentiviral infection were described in a previous study^{43 (link)}. The oligonucleotide sequences for vector construction are listed in Supplementary Table 5.