Journal: PLoS Biology
Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins
Figure Lengend Snippet: Knock-down of endogenous Ginir affects proliferation and tumourigenic potential of fibroblasts and melanoma cells. (A) Representative RT-PCR for analyses of Ginir knock-down in NIH/3T3 cells expressing Ginir-shRNA (1, 2, and scrambled control) using G2F-G2R primers. Gapdh served as an internal control. (B) Cell proliferation analysis by MTT assay for the indicated cell lines performed over a period of 4 days. Data presented are mean ± SEM. *** P ≤ 0.0001, one tailed, by two-way ANOVA test. (C) Quantification of Ki67 antigen–expressing cells shown as percentage of positively stained cells from the total number of cells present per field. Cells from at least 10 representative fields were counted in NIH/3T3 control and Ginir shRNA1 and 2 cells. Data presented are mean ± SEM.** P ≤ 0.001 by one-way ANOVA test ( n = 3). (D) Quantitative analyses of cell cycle parameters of PI-stained cells in flow cytometry. Stable Ginir knock-down (NIHshGinir1, NIHshGinir2) cells were compared to NIH/3T3 control cells having endogenous Ginir RNA expression for percentage of cells in various phases of the cell cycle. Bars: means ± SEM; * P ≤ 0.05, ** P ≤ 0.001; two tailed; by two-way ANOVA test. (E) Representative RT-PCR data demonstrating Ginir RNA expression levels in B16F10 melanoma cells shControl and the knock-down cells B16F10-Ginir-shRNA1 and B16F10-GinirshRNA2. PCR was done using G2F-G2R primers. Gapdh RNA expression was used for normalisation. (F) Phase contrast images of B16F10 control cells and cells stably transfected with shGinir1 or shGinir2. Arrowheads point towards cells showing altered phenotypes. Magnification 10×. (G) Cell proliferation analysis of indicated cells performed by MTT assay over a period of 4 days. Data shown are mean ± SEM. * P ≤ 0.05, one tailed, Student’s paired t test ( n = 3). (H) Quantification of Ki67 antigen expression shown as percentage of positively stained cells as compared to the total number of cells per field. At least 10 fields were counted. Data shown are mean ± SEM. ** P ≤ 0.001 by one-way ANOVA test. ( n = 3). (I) Cell migration assay in the indicated cell lines. The gaps were measured after 6 hours using ImageJ software; version 1.41. Experiment was repeated at least thrice. (J) Quantitative analysis of relative wound recovery of each B16F10 Ginir knock-down cell induced by shRNA1 and shRNA2 as compared to B16F10-shGinir scrambled expressing control cells. Data represent mean ± SEM ( n = 3). *** P ≤ 0.0001 by two-way ANOVA test. (K) Representative xenograft tumours of B16F10-shControl and B16F10-shGinir1 and B16F10shGinir2 (1 and 2) cells introduced into NOD/SCID mice; tumour growth was monitored for 15 days. These experiments were performed thrice, with 3 mice in each group. (L) Tumour growth kinetics were determined by measuring tumour volume each day for a period of 15 days for the indicated cell lines. Data shown represent mean ± SEM. *** P . Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; PI, propidium iodide; RT-PCR, reverse transcription PCR; shRNA, short hairpin RNA.
Article Snippet: Nuclei were stained with PI (Invitrogen, # P1304MP) for 30 minutes.
Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA, MTT Assay, One-tailed Test, Staining, Flow Cytometry, Cytometry, RNA Expression, Two Tailed Test, Polymerase Chain Reaction, Stable Transfection, Transfection, Cell Migration Assay, Software, Mouse Assay