Vector Construction and Stable Cell Line Establishment

In order to better analyze the correlation between PPA1 and breast cancer progression, we hope to choose the cell line with relatively lower expression level at basal status for overexpression assays and with relatively higher expression level for knockdown assays. Meanwhile, we would like to select the cell line with high malignancy and aggressively metastatic characteristics for overexpression, and study the role of PPA1 in different molecular subtypes of breast cancer. For above considerations, we choose T47D for knockdown and MDA-MB-231 for overexpression assays.

To stably knockdown PPA1 in breast cancer cells, T47D cells were infected with lentivirus carrying pLV-H1-shPPA1-puro or pLV-H1-shRNA-scramble-puro plasmid, then treated with puromycin to obtain the stable cell line with PPA1 silencing (shPPA1) and the shRNA control. The sequences of shRNAs were: shPPA1#1: GCTACTGTGGACTGGTTTA; shPPA1#2: GGAATCAGTTGCATGAATA; shRNA control: GCTACACTATCGAGCAATT.

For the stable overexpression of PPA1 in mammalian cells, the cDNA of PPA1 was inserted into the pLV-EF1α-MCS-IRES-Bsd plasmid. MDA-MB-231 cells were infected with lentivirus carrying the recombinant plasmid and empty plasmid which was used as the control. Cells were selected using blasticidin to generate stable cell lines with PPA1 overexpression and their control.