In vitro clonogenic survival and in vivo tumorigenic assays in MDA-MB453 tumor cells after knock-down of RecQL4 expression.

<p>(A) Western blot analysis of RecQL4 expression in parental, control shRNA (ShControl) and RecQL4 specific shRNA transduced MDA-MB453 cells (ShRecQL4-C5 & C8). RecQL4 expression was highly reduced in ShRecQL4-C5 and C8 stably selected by puromycin antibiotics. (B) Clonogenic survival assay was performed on ShRecQL4-C5 and C8 cells relative to parental and ShControl cells. Data presented at each time point were the mean value of eight cultures from two independent experiments. Bars indicate mean±SD. (C) Analysis of <i>in vivo</i> tumorigenicity of MDA-MB453 cells after RecQL4 silencing. Parental, ShControl and ShRecQL4-C5 & C8 cells were subcutaneously injected into seven immunosuppressed nude mice and tumor growth was monitored for 4 weeks. Tumor growth as a function of time is shown in the left panel. Images of tumors resected from mice are shown in the right panel. Note that 4 of 7 mice injected with either ShRecQL4-C5 or C8 cells did not show any tumor growth. Bars indicate mean±SD.</p

RecQL4 associates with survivin.

<p>(A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2-survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(−), pFlag-RecQL4-HD(−) or pFlag-RecQL4-CT(−). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag-RecQL4 protein.</p

RecQL4 protein or mRNA level in breast tumor cell lines and clinical breast cancer specimens.

<p>(A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.</p

Amplification of RecQL4 genomic locus analyzed by mBAND-FISH and quantitative real time PCR.

<p>(A) Analysis of chromosome specific mBAND-FISH in the metaphase spreads of normal primary (HMEC), immortalized (MCF-10F) and tumorigenic breast cancer cell lines. Chromosomes and chromosome regions positive for the probe are shown in the inserts. (B) FISH analysis using spectrum orange labeled BAC (Bacterial Artificial Chromosome) probe proximal to 8q24.3 chromosome locus harboring the RecQL4 gene in MDA-MB453 and MDA-MB361 cells. The BAC probe was purchased from Open Biosystems (RP11–374B7, Huntsville, Alabama, USA). (C) Upper panel: Agarose gel electrophoresis showing the abundance of RecQL4 genomic DNA detected by PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used as an internal control; Lower panel: Abundance of RecQL4 genomic DNA detected by quantitative real time PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used for normalizing the values of RecQL4.</p