Fig. 2

Reduced expression of Zc3h8 leads to reduced invasive growth in vitro and in vivo. a) cV1A 03–31 mouse mammary carcinoma cells stably expressing shRNA targeting Zc3h8 or negative control were lysed and processed for western blot to detect ZC3H8 and β-TUBULIN protein levels. Densitometry using ImageJ revealed a 25–30% reduction of ZC3H8 levels in cells targeted by shRNA. RT-qPCR was also used to quantify Zc3h8 levels standardized using Gapdh. b) Cell lines above were grown in 96-well dishes for 5 days and assayed for cell proliferation using a luciferase viability assay. Significance was calculated using ANOVA at p < 0.001. c) Cells from above were grown in soft agar overlays for 14 days and imaged using light microscopy. d) Average number of soft agar colonies was measured after 14 days using ImageJ. Significance was calculated by Student’s t-test at p < 0.01. e) To test cell migration, a wound healing assay was done and the width of the gap between cells was measured over time. Significance was calculated by ANOVA at 10 h p < 0.05, at 24 h p < 0.01. f) Transwell invasion assay was done with cV1A 03–31 cells transfected a vector driving expression of shRNA for negative control, Zc3h8, or the latter cells additionally transfected with a vector driving expression of human ZC3H8 (not subject to the shRNA-mediated knockdown). Migrated cells were fixed and stained with crystal violet. g) Quantification of cells that invaded through the Matrigel transwell assay. Control and rescued cell counts were not significantly different, significance between each and the Zc3h8 siRNA was p < 0.01 by Tukey’s post-hoc analysis. h) Images of 3-D spheroid cultures of the transfected cells. i) Quantification of spheroid growth from H. Statistical difference was calculated by ANOVA at p < 0.05. j) BALB/c mice were injected subcutaneously with cV1A 03–31 mouse mammary cells transfected with silencing vector targeting Zc3h8 or negative control. Upon reaching 1 cm diameter or following 8 weeks, tissue was removed and weighed. P-value of < 0.01 was determined by Student’s t-test. k) Kaplan Meier plot detailing time of appearance of tumors. Experiments for this Figure were performed in triplicate and error bars represent standard deviation. Images are representative of typical results. For all segments, * indicates p < 0.05 and ** indicates p < 0.01