MISSION® pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and SerpinB3 shRNA plasmids TRCN0000373440 (KD1), TRCN0000373501 (KD2), TRCN0000052398 (KD3) and TRCN0000373500 (KD4) were purchased from Sigma. These correspond to four non-overlapping single shRNAs. Lentivirus was packaged in 293 T cells using psPAX2 and pMD2G using calcium phosphate transfection, and media containing lentiviral particles were collected. This supernatant containing lentiviral particles was concentrated using PEGit virus precipitation solution according to the manufacturer’s protocol (System Biosciences). JAM-A knockdown constructs were as follows: TRCN0000061650 (KD1) and TRCN0000061649 (KD2).
Prior to transfection, CSCs were grown adherently on 6 well plates pretreated with Geltrex (Life Technologies, A1413301). Lentivirus was added to and incubated with the cells for 24 h. Then cells were grown in their appropriate media for 24 h, after which selection with puromycin (ThermoFisher, 54–022-2100) was initiated. Transfected cells were incubated in media with puromycin (1 mg/mL stock) at 1:333 for 48 h. Stably transfected cells were maintained in their regular media plus puromycin at 1:1000.
Prior to transfection, CSCs were grown adherently on 6 well plates pretreated with Geltrex (Life Technologies, A1413301). Lentivirus was added to and incubated with the cells for 24 h. Then cells were grown in their appropriate media for 24 h, after which selection with puromycin (ThermoFisher, 54–022-2100) was initiated. Transfected cells were incubated in media with puromycin (1 mg/mL stock) at 1:333 for 48 h. Stably transfected cells were maintained in their regular media plus puromycin at 1:1000.