Article Figures & Data
Figures
- Figure 1.
Plasmid maintenance screen. A, schematic of plasmid stability assay used for screen. Puro, puromycin. B, quantitative Western blot analysis of MCM3 and PCNA (loading control) in whole-cell extracts obtained from cells transduced with the indicated shRNAs (V2HS_262054 is an shRNA that targets MCM3 expression). The Luciferasemi203 whole-cell extract is loaded such that there is either equal total protein loaded as the V2HS_262054 whole-cell extract (lane indicated as “1.00” on the blot) or 25% or 10% total protein loaded as the V2HS_262054 whole-cell extract (lanes indicated as “0.25” and “0.10,” respectively). PCNA, proliferating cell nuclear antigen. C, colony formation assay results for triplicate HCT116 cultures transduced with either V2HS_262054, EBNA1mi1666, or Luciferasemi203 shRNAs that were grown in either growth media + puromycin (proliferation assay) or growth media + puromycin and hygromycin (plasmid stability assay). D, plot of plasmid stability ratios calculated from results shown in (C). E, plasmid stability ratios for shRNAs tested in screen. Blue bars on plot correspond to plasmid stability ratios for HCT116 cultures transduced with shRNAs that target expression of known replication factors.
- Figure 2.
DDX5 is required for plasmid stability and cell proliferation. A, quantitative Western blot analysis of DDX5 and β-actin (loading control) in whole-cell extracts obtained from cells transduced with the indicated shRNAs (V2HS_26045 and V2HS_24063 are shRNAs used in the screen that target DDX5 expression whereas the other DDX5 shRNAs shown are new shRNAs not tested in the screen). B, colony formation results from the plasmid stability assay for either duplicate (each DDX5 shRNA)- or quadruplicate (Luciferasemi203 shRNA)-transduced cultures. C, colony formation results from the proliferation assay. Quantification of colony formation results are plotted in (B) and (C).
- Figure 3.
DDX5 is required for G1–S-phase progression. A, quantitative Western blot analysis of DDX5 and β-actin (loading control) in whole-cell extracts obtained from cultures 48 hours after transfection with the indicated siRNAs. B, flow cytometric analysis of cell cycle in 48-hour posttransfection cells with either DDX5si2008 (left) or EBNA1si1666 (right). C, progression into S-phase following serum addition to serum-starved cultures previously transfected with either DDX5si2008 (circles) or EBNA1si1666 (squares) siRNAs. The fraction of cells in S-phase at each time point after serum addition was determined using flow cytometric analysis of propidium iodide incorporation and gating cells with greater than 2C but less than 4C DNA content. D, Western blot analysis of RB, G1–S cyclin expression, and DNA replication factor expression in G1 and early S-phase whole-cell extracts obtained from cells at increasing time following addition of serum to serum-starved cells previously transfected with the indicated siRNAs. E, Western blot analysis of replication factors in chromatin fractions obtained from siRNA-transfected S-phase cells over time after serum addition. The upper band detected on the CDC45 blots and marked with an asterisk in (D) and (E) is a nonspecific cross-hybridizing protein recognized by the antibody.
- Figure 4.
DDX5 is required for expression of DNA replication genes. A, quantitative Western blot analysis of DNA replication factors in whole-cell extracts obtained from asynchronous cell cultures 48 hours after siRNA transfection with the indicated siRNAs. The asterisks shown in the MCM2, MCM10, and CDC45 blots indicate the position of nonspecific proteins detected by these antibodies. B, Q-PCR analysis of DNA replication factor transcript abundance in cells either 24 or 48 hours after transfection with DDX5si2008 (red bar), EBNA1si1666 (blue bar) or mock-transfected (no siRNA—green bar). Results for each transcript are normalized to the abundance of the indicated transcript in cells transfected with the EBNA1si1666 siRNA. Error bars indicate SDs calculated from 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, heat map showing row-wise standardized expression level for DNA replication genes 24 hours after transfection of cells with the indicated siRNAs.
- Figure 5.
DDX5 localizes to promoters of DNA replication genes and functions in the loading of RNA polymerase II onto these promoters. A, Western blot (WB) analysis of E2F1 and DDX5 in DDX5 and immunoglobulin G (IgG) immunoprecipitation (IP) samples from nuclear extracts prepared from HCT116 cultures (top) and analysis of the interaction of in vitro transcribed and translated radiolabeled DDX5 with GST-E2F1 purified from bacteria and immobilized on glutathione beads (bottom). B, Q-PCR analysis of DDX5 chromatin immunoprecipitation (ChIP) samples at the indicated promoters. Cells were serum-starved, restimulated with serum for 2 hours, and then harvested for ChIP. Note that the CD4 promoter is used as negative control. Error bars show the SDs for duplicate experiments. The green bars correspond to results for the DDX5 ChIPs, whereas the black bars correspond to results for the IgG control ChIPs. All primer pairs used for Q-PCR amplify within 200 bp of the transcription start sites of the indicated genes. Q-PCR analysis of E2F1 ChIP (C), acetylated histone H3 ChIP (D), RNA polymerase II ChIP (E), and TFIIB ChIP (F) at the indicated promoters in asynchronous cells transfected with either DDX5si2008 (blue bars) or EBNA1si1666 (red bars) siRNAs. Error bars show the SDs for duplicate experiments. Neither the GAPDH nor CD4 transcripts are downregulated by DDX5 knockdown and therefore their promoters are used as negative controls in for the ChIP experiments presented in (D–F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Figure 6.
The DDX5 locus in amplified in breast cancer. A, representative plots of genome copy number variation across chromosome 17 in selected breast cancer specimens. The green vertical bar indicates the position of the ERBB2 locus and the red vertical bar indicates the position of the DDX5 locus. B, analysis of DDX5 gene copy number and mRNA abundance in 33 established breast cancer cell lines. C, frequency of DDX5 amplification in the different breast cancer subtypes. D, results of colony formation assays conducted with MDA-MB-453 cells transduced with either the DDX5mi2008 shRNA (red line) or empty vector (no shRNA, blue line) and then expanded in media containing increasing concentrations of trastuzumab. Percentage inhibition was calculated using the equation (1 − AN/A0) × 100, where AN is the measured absorbance of dye-stained cells for either DDX5mi2008 or empty vector–transduced cell cultures grown in media supplemented with a specific concentration of trastuzumab “N” and A0 is the measured absorbance of either the DDX5mi2008 or empty vector–transduced cell cultures grown in media without trastuzumab. Note that the curve for the DDX5mi2008-transduced cells is shifted to the left, indicating greater sensitivity of these cells to trastuzumab than the empty vector–transduced cells. Error bars show SDs for duplicate cell cultures.
- Figure 7.
Differential sensitivity of breast cancer cells to DDX5 depletion. A, quantitative Western blot analysis of DDX5 and β-actin and colony formation assay results for different breast cancer cell lines with amplification of the DDX5 locus following transduction of the indicated DDX5 shRNAs or EBNA1 shRNA (negative control). Representative colony formation assay cultures are shown. Plots reflect quantitated results for the colony formation assays. Error bars show SDs for triplicate cultures. B, same as in (A) except that breast cancer cell lines lacking DDX5 amplification are tested. C, Western blot (WB) analysis of DNA replication factors in the indicated cell lines transduced with either DDX5mi2008 (“D5”) or EBNA1mi1666 (“E”). The asterisk indicates a nonspecific cross-hybridizing band detected by the CDC45 antibody in the SK-BR-3 and MCF10A whole-cell extracts. D, Q-PCR analysis of the indicated transcripts in SK-BR-3 cells transduced with either DDX5mi2008 (red bars) or EBNA1mi1666 (blue bars). Results are normalized to the relative abundance of the indicated transcripts in the EBNA1mi1666-transduced cultures. Error bars indicate the SDs from duplicate experiments. E, Q-PCR analysis of RNA polymerase II ChIP samples obtained from SK-BR-3 cells transduced with either DDX5mi2008 (red bars) or EBNA1mi1666 (blue bars). Results presented are normalized to the relative abundance of the indicated promoters in ChIP samples obtained from EBNA1mi1666-transduced cultures. Error bars indicate the SDs from duplicate experiments.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 106K, This file contains supplementary information and methods, including sequences of all siRNAs employed.
- Supplementary Figures 1-11 - PDF file - 1.2MB, Supplemental Figures 1 through 11 contain additional results from the plasmid stability screen, analysis of the effect of DDX5 knockdown on cell cycle progression and BrdU incorporation by HCT116 cells, exogenous DDX5 expression studies, impact of DDX5 knockdown on expression of genes containing promoter E2F binding sites, effect of knockdown of E2F-family members on abundance of replication proteins, effect of combined DDX5 knockdown and trastuzumab treatment on MDA-MB-453 proliferation, chromosome 17 copy number plots for breast cancer cell lines presented in the manuscript either having or lacking amplification of the DDX5 locus, and co-immunoprecipitation analysis of DDX5 and E2F1 in SK-BR-3, MDA-MB-453, and MCF10A cells.
- Supplementary Figures Legends 1-11 - PDF file - 68K
- Supplementary Table 2 - PDF file - 47K, This file contains supplementary Table 2 that has a list of genes identified in the screen that scored for the plasmid stability assay.
- Supplementary Table 1 - XLS file - 71K, This file contains supplementary Table 1 that contains results from the shRNA screen, including the proliferation assay, plasmid stability assay, and plasmid stability ratios.
Volume 2, Issue 9
