Fig 1.
SURF6 knockdown does not disturb morphology of HeLa cells.
Western blot analysis (A, B) of SURF6 in protein extracts of HeLa cells with si-mediated knockdown of SURF6. Anti-tubulin staining was used for normalization. Confocal microscopy of fixed/permeabilized HeLa cells stained with anti-SURF6 (C, G, K, O), anti-B23 (D, H), anti-fibrillarin (L, P) and DAPI (E, I, M, Q) prior to imaging. (F, J, N, R) merged images.
Fig 2.
SURF6 overexpression does not disturb morphology of HeLa cells [31].
Western blot analysis (A, B) of SURF6 in protein extracts of HeLa cells with overexpression of SURF6. Anti-tubulin staining was used for normalization. Confocal microscopy of fixed/permeabilized HeLa cells stained with anti-SURF6 (C, G, K, O), anti-B23 (D, H), anti-fibrillarin (L, P) and DAPI (E, I, M, Q) prior to imaging. (F, J, N, R) merged images.
Fig 3.
Pre-rRNA processing pathways in mammalian cells [30].
Fig 4.
SURF6 knockdown and overexpression change the profile of pre-rRNA in HeLa cells as well as in HCT116 p53+ and HCT116 p53- cells.
(A-F) Nothern blots of RNA extracts from HeLa, HCT116 p53+ and HCT116 p53- cells with knockdown or overexpression of SURF6 and from control cells. Membranes were hybridized with biotinylated ITS1, ITS2 and 7SK (used for loading control and normalization) probes prior to ECL visualization. Probes hybridization sites on rRNAs are shown at the right side of the panel. (G, H) Quantification of bands intensity corresponding to different precursors in HeLa and HCT116 cells, respectively (results of densitometry analysis). Mean values are shown as bar graphs with standard deviations. Stars indicate statistically significant differences (n = 3). (I, J) Results of the RAMP analysis of changes in pre-rRNA profiles of HeLa and HCT116, respectively. Bars show mean fold changes (with standard deviations) in the ratios between indicated precursors. Stars indicate statistically significant differences (n = 3, p<0,05). Stars above bars on the panel H indicate statistically significant differences between HCT116 p53+ and p53- cells. Stars at the bottom of the bars show statistically significant differences between either HCT116 p53+ or p53+ and the control (n = 3).
Fig 5.
Effects of SURF6 knockdown and overexpression on the HeLa cell cycle.
(A, B, E, F) Quantitation of DNA content of cells stained with propidium iodide using flow cytometry. Representative histograms show changes in proportions of cells in different stages of the cells cycle in comparison to control cells. (C) Distribution of cells at different stages of the cell cycle at various times after SURF6 knockdown. (D, G) Mean values of proportion of cells in different stages of the cell cycle with standard deviations (n = 3). Stars indicate statistically significant differences between control HeLa and cells with knockdown or overexpression of SURF6 (p<0,05). (H) MTT proliferation assay in HeLa cells with SURF6 knockdown or in control HeLa cells. Data are presented as bar graphs of mean values (n = 3) with standard deviations. Significant difference (p<0,05) is indicated with stars.
Fig 6.
SURF6 knockdown affects cell cycle distribution of HCT116 human intestinal carcinoma cells.
(A) Western blot analysis of SURF6 and p53 levels in HCT116 p53+ and HCT116 p53- cell lines treated with anti-SURF6 siRNA or non-targeting siRNA as a control. Anti-actin staining was used for normalization. (B) Ratio between p53 and actin in HCT116 p53+ SURF6+ and HCT116 p53+ SURF6- cells. Data presented as mean ± SEM. Statistical analysis for p53 expression corresponds to a biological triplicate. (C-F) Quantitation of DNA content of cells from (A) stained with propidium iodide and analyzed by flow cytometry. Representative histograms show changes in proportions of cells in different stages of the cells cycle in comparison to control cells. (G) Mean percentages of cells in each phase of the cell cycle in four independent experiments. Stars indicate statistically significant differences between control cells HCT116 p53+/SURF6+ or HCT116 p53-/SURF6+, and respective cell lines with siRNA-mediated SURF6 knockdown. (H) MTT assay results obtained in HCT116 p53+/p53- control cells and respective cell lines with SURF6 knockdown and plotted as bar graphs. Mean OD ± SEM values at 570 nm were plotted on the vertical axis. Stars indicate statistically significant differences between control HCT116 and cells with knockdown of SURF6 in their viability/proliferation (p<0,05).