Fig. 2

IPW inhibits DCIS growth in vitro and in vivo. A MCF10A and 10AshIPW cells were seeded in a 96-well plate (5000 cells/well, n = 6/group), and cell viability was evaluated at days 1, 3 and 5 by MTS assay. B LncRNA IPW or empty vector was ectopically expressed in DCIS.com cell line using lentivirus, and IPW expression was examined in DCIS (control) and IPW-expressed DCIS.com cells (n = 4/group) by qRT-PCR. C DCIS.com cells were transduced with either empty vector or IPW. 5000 cells were seeded in 96-well plate (n = 6/group) and relative cell viability was examined by MTS assay at days 1, 3 and 5. D DCIS.com and DCIS-IPW cells were seeded in six-well plates (n = 500 cells/well, n = 5/group), and number of colonies were counted at day 10. All data were analyzed by unpaired two-tailed Student’s t test. E DCIS.com (left panel) and SUM225 (right panel) cells transduced with empty vector or IPW were subjected to cell cycle analysis using flow cytometry. % of cells at G1, S and G2 phase are shown (n = 3/group). Cells were synchronized and stained with PI followed by visualization by FACS. The data were analyzed using ANOVA and Tukey’s multiple comparison test. F, G DCIS.com and SUM225 wells were transduced with either empty vector or IPW were examined for p21 mRNA and protein expression. p21 mRNA expression was evaluated qRT-PCR (n = 3/group; upper panels) and protein expression by western blot (bottom panels). The statistical inference for qRT-PCR was calculated by unpaired two-tailed Student’s t test. H DCIS.com cells were ectopically expressed with either empty vector or IPW by lentivirus transduction and evaluated by mammosphere assay. 1000 cells were seeded/well in mammosphere media (n = 8/group). Number of spheres were calculated at D5 under microscope. Scale bar 50 µm I 20,000 cells were seeded in 24-well plate for 3 days. At the end of incubation, cells were incubated with and 4 µM of Caspase3/7 green detection reagent for 1 h at 37⁰C. Then, cells were rinsed with PBS to wash off the unbound reagent followed by trypsinization and analysis by flow cytometry. J DCIS.com (control) and DCIS-IPW cells were implanted in the mammary fat pad of nude mice (n = 5, 1 × 106 cells/mouse), and tumor growth was monitored by bioluminescence (BLI). Representative IVIS images are shown in right panel. Animals were killed when the tumor volume is 1000mm3 or when the animals became morbid. K H&E staining was performed for tumors isolated at the endpoint in Study (upper and middle panels). Tumor sections were also stained for human cytokeratin by immunohistochemistry (lower panel). Representative images are shown. Scale bar 100 µm. All statistical inference between the two points was evaluated by unpaired two-tailed Student’s t test. Data are represented as mean + S.E.M. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)