Blasticidin

For infection of CRC cell lines with pMX-IRES constructs, retroviral particles were generated by transfection of HEK 293GP cells with Lipofectamine 3000 (Invitrogen). After transfection, the cell supernatants were collected and used to infect CRC cells, and the stably transfected cells were selected using puromycin and confirmed by quantitative RT-PCR. Selection for infected cells was done with 12.5 ug/ml Blasticidin (Gemini Bio Products) for over a week. SMARTpool siRNA for DKK3, Antagomir-92a, miR-92a mimic and corresponding control oligonucleotides (Dharmacon) were transfected into CRC cells by using Lipofectamine 3000 according to manufacturer's protocols. For stable infection of organoids, PMX-IRES GFP constructs were used. The retroviral particles were prepared as mentioned above and concentrated using reterconcentrator (Takara Biosciences). Organoid fragments were prepared following the protocol by (15) and combined with retroviral solution along with polybrene in a 24-well plate, sealed, and spinoculated at 1800 rpm at 37 0 C for 1 hr 45 min. Following spinoculation, plate was incubated for 6 hrs at 37 0 C. This was followed by seeding of infected organoids and 5 days post infection, GFP positive cells were sorted and resuspended in matrigel.