Generation of stable cell lines by lentiviral transduction

To generate recombinant lentiviruses expressing EB3-tdTomato, pLVX-EF1α-EB3-tdTomato plasmid was co-transfected with the following third-generation packaging plasmids (gifts from Didier Trono): pMDLg/pRRE, pRSV-Rev and pMD2.G (Addgene, 12251, 12253 and 12259, respectively) (Dull et al., 1998) into HEK293T cells. Viral supernatants were collected from medium 24-48 h post transfection, filtered by a 0.22-μm filter, and were used to infect the inducible GFP-PCNT (854-1960) TP53^−/− RPE-1 cells with 8 μg/ml polybrene. Next, 18-24 h post infection, the viruses were removed, and the cells were expanded. To minimize the impact on MT dynamics, the cells expressing low levels of EB3-tdTomato (gated and collected by FACS) were used for experiments. To generate lentiviruses expressing mScarlet-i-H2A and miRFP670-CETN2 fusion proteins, the same lentiviral packaging procedure was performed as above, except for using the targeting vectors pLVX-EF1α-mScarlet-i-H2A and pLVX-EF1α-miRFP670-CETN2, respectively. The resulting viral supernatants were then used to infect the cells of interest. Sometimes, FACS was further performed to obtain cells with the desired more uniform expression of the fusion proteins.