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Fig. 6 | Breast Cancer Research

Fig. 6

From: LncRNA IPW inhibits growth of ductal carcinoma in situ by downregulating ID2 through miR-29c

Fig. 6

Toyocamycin selectively inhibits low IPW-expressing DCIS. A Flowchart of the natural compound library screening. DCIS.com and DCIS-IPW cells were seeded in 96-well plates (5000 cells/well) and treated individually with 390 natural compounds at 5uM final concentration. Cells were treated for 48 h, and colonies were stained with crystal violet. Then, the stain was dissolved in 10% acetic acid, and absorbance was obtained at 595 nm absorbance in spectrophotometer. B DCIS.com and DCIS-IPW cells were treated with toyocamycin at indicated concentrations for 48 h, and relative cell viability was examined by MTS assay (5000 cells/well, n = 3/group). Statistical inference was made by unpaired two-tailed Student’s t test. C DCIS.com and DCIS-IPW cells (500 cells/well, n = 3/group) were treated with toyocamycin at indicated concentrations for 10 days in 6-well dish. Colonies formed at day 10 were stained with crystal violet, and the dye was dissolved in 10% acetic acid followed by measuring absorbance at 595 nm. Statistical inference was calculated by unpaired two-tailed Student’s t test. D Luciferase-labelled DCIS.com cells (1 × 106 cells/mice, n = 6/group) were implanted into nude mice through m.f.p injection, and they were treated with either toyocamycin at 2 mg/kg body weight or control (DMSO) once a week. Animals were randomized before starting drug treatment. Tumor growth was monitored by IVIS bioluminescence. Representative animal pictures from control and toyocamycin treated group are shown on right side. E H&E staining was performed in tumors from DCIS implanted mice in Figure D. Representative image from each group is shown. Scale bar 100 µm. F: DCIS.com cells were treated with control or toyocamycin (1uM) for 24 h, and ID2 mRNA and protein expression was examined by qRT-PCR (n = 3/group) and western blot, respectively. Actin was used as internal control. Statistical inference was made by using Student’s t test. G–J DCIS.com cells were treated with control (DMSO) or toyocamycin (1uM) for 24 h and p21 (G), OCT4 (H), SOX2 (I) and NANOG (J) mRNA expression was examined by qRT-PCR (n = 3/group). Actin used as internal control and statistical inference was made by using two-tailed Student’s t test. K DCIS.com cells were treated with either control (DMSO) or toyocamycin (1uM) for 24 h and lysates were prepared. p21, OCT4, SOX2 and NANOG protein expressions were determined by western blot. Actin expression was used as a loading control. Statistical inference between two groups was determined by unpaired two-tailed Student’s t test, and data are represented as mean + S.E.M. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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