Extended Data Fig. 2: Validation of SLC34A2 and XPR1 protein levels and viability defects upon shRNA induction.
a) Validation of SLC34A2 in cell lines using immunohistochemistry. N = 1 experiment. b) Five days after viral transduction of the indicated sgRNA in the indicated cell lines stably expressing Cas9, cells were harvested and XPR1 levels were analyzed by immunoblotting. Note that irrelevant lanes were cropped out for clarity, but that this image represents a single blot at a single exposure. N = 1 technical replicate of at least N = 5 representative experiments. c) Three days after induction of shRNA, protein levels were measured in cellular lysates by protein simple. Protein levels normalized to vinculin and the untreated (-Dox) conditions are expressed below each band. Note that shXPR1 reagents effectively suppress XPR1 protein levels but shSeed reagents do not. N = 1 technical replicate of at least N = 5 representative experiments. d) Colony formation assay to measure the long-term (14 day) penetrance and viability effect of suppression of XPR1 using shRNA in IGROV1 and OVISE cells. N = 3 technical replicates of at least N = 2 representative experiments. e) Growth curves of SLC34A2-expressing cell lines after suppression of XPR1. In 96-well plates, confluency of the indicated cell lines was assessed every 4 hours. N = 3 technical replicates of at least N = 2 representative experiments. f) Six days after induction of shXPR1 in the indicated cell lines, cells were stained with DAPI to distinguish live and dead cells and Annexin V to distinguish non- and pre-apoptotic cells. N = 2 flow cytometric analyses of at least 10,000 cells, representative of N = 2 experiments.
