Mission rnai system

SPIN90 KD were generated by infecting HeLa cells with small interfering RNA (siRNA) against SPIN90 using the Mission RNAi system (Sigma, St. Louis, MO, USA), as described previously^{27 (link)}. HEK293T cells were transfected with various constructs for binding assays and GST pulldown assays. COS-7 cells were used for immunofluorescence staining and live-cell imaging. Lipofectamine 3000 or LTX supplemented with plus reagent was used for transfections. Transfected cells were cultured for 24 h, followed by serum starvation for 16 h. HeLa-control, HeLa-SPIN90-KD, HEK293T, and COS-7 cells were authenticated by morphological analysis and gene expression profiling in 2015 and were tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Rockland, ME, USA) in 2017. Cell lines were usually used from passages 3 to 8.

To generate cell lines in which ALCAM expression had been stably knocked down, we used the MISSION^® RNAi system and lentiviral pLKO-puro vector (Sigma Aldrich, St Louis, MO, USA). The sequences of the shRNAs were as follows: shALCAM1, 5’-CCGGCAGCCATGATAATAGGTCATACTCGAGTATGACCTATTATCATGGCTGTTTTTG-3’ and shALCAM2, 5’-CCGGCTTCGATCTAGCCCGTCATTTCTCGAGAAATGACGGGCTAGATCGAAGTTTTTG-3’. Non-target shRNA Control (Sigma Aldrich) was used as negative control shRNA. Lentivirus was produced by transfection of the lentiviral vector and the Lentiviral Packaging Mix (Sigma Aldrich) in 293T cells. Daoy and ONS-76 cells were then infected with the lentivirus expressing the shRNAs. Knockdown of ALCAM was confirmed by flow cytometry and qPCR after puromycin selection. Before performing the in vitro and in vivo assays, the 10–15% cells with lower ALCAM expression were sorted from the heterogeneous Daoy cell population in which ALCAM had been knocked down (expressing different levels of the protein) by FACS using PE-conjugated mouse anti-human ALCAM antibody.