Psingle tts shrna vector

The most effective sequence of siRNA for Notch1 (5′-GAACGGGGCUAACAAAGAUUU-3′) was determined by western blotting, and sense and antisense oligonucleotides were synthesized accordingly. Annealed oligonucleotides were subcloned into the pSingle-tTS-shRNA vector (Clontech) between the HindIII and XhoI sites. After confirming the sequence, the vector was transfected into KATOIII cells using Lipofectamine LTX (Invitrogen), and the transfected cells were selected using 200 μg/ml G418. Single clones were selected and were stimulated with 10³ ng/ml Dox. Then, the cells were harvested and subjected to western blot for Notch1. The clone in which Notch1 was most effectively suppressed was named N1KOK3 and was employed for further studies. KATOIII cells stably transfected with the empty vector were used as a control (Mock).

The shRNA-TGM2 (5′-ATG GTC AAC TGC AAC GAT GA-3′, nucleotides 909–929 of TGM2 transcript variant 1, seq. ID: NM_004613.3) was drawn on the region 657–1101 common to all variants of TGM2 described in GenBank database. The sequence was inserted into the pSingle-tTS-shRNA Vector (Clontech laboratories Inc., Mountain View, CA, USA) for doxycycline-inducible expression of the shRNA. The plasmid was transfected into K562 cells using the Lipofectamine transfection reagent (Invitrogen, Thermo Fisher Scientific, Milan, Italy), and cells resistant to G418 were selected. Following the addition of doxycycline (DOX) in the medium (3 µg/mL), the expression level of all the TGM2 variant transcripts was evaluated with RT-qPCR using the primer couple TGM2-ALL (F: 5′-GAC TCG GAA GAG GAG CGG CA-3′; R: 5′-CTC AGC ACT GTG CAG GCC AC-3′) that annealed to all the TGM2 transcript variants described so far.