Different sirna oligos

Different siRNA oligos were obtained from Gene Pharma (Shanghai, China), and the sequences were listed in Supplementary Table S1. Plasmids for Rheb overexpression were purchased from Hanbio (Shanghai, China) and the sequence is shown in Supplementary Table S1. Cells were transfected with the appropriate siRNA oligos or plasmid with Lipofectamine 3,000 (Invitrogen, CA, United States), according to the manufacturer’s protocol. After 48 h, cells were further stimulated with different reagents. The successfully transfected clones were confirmed by western blotting.

Different siRNA oligos were synthesized from Gene Pharma (Shanghai, China). Cells were transfected with siRNA oligos using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). 3XFlag-N1ICD overexpression plasmids were purchased form Addgene. NUMB isoform (p65, p66, p71, or p72) expression plasmids and SMAD3-HA overexpression plasmids were generated based on the PCDNA3.0 vector. sh-p65/p66-#1/#2, sh-p71/p72-#1/#2 and shNC lentivirus were generated with PLKO.1-puro lentiviral vector in 293T cells. RKO and HeLa cells were infected with lentivirus and selected for puromycin resistance. The siRNA sequences used are listed in Supplementary Table S1, primer sequences for plasmid construction are listed in Supplementary Table S2, shRNA sequences used are listed in Supplementary Table S3 and primer sequences for detecting NUMB specific isoforms are listed in Supplementary Table S4.