Table 1.
Primers, annealing temperatures, and product sizes for PCR amplification.
Figure 1.
Generation of SKOV3 cell clones.
(A) Several stable lines were created using overexpression of BRCA1 and its background vector, pcDNA3 in SKOV3 ovarian carcinoma cells. Expression levels of BRCA1 are shown in all cell clones. Parental indicates non-transfected SKOV3 cells only. Densitometric analysis from three independent immunoblots is given in (B). Error bars are SEMs. * represents P<0.05 (compared to parental).
Figure 2.
Hierarchical gene clustering generated by the Ingenuity Pathway Knowledge Base.
A heat map showing significant changes in a group of selected genes due to overexpression of wtBRCA1 in SKOV3 ovarian carcinoma cells. Three independent microarray analyses were performed. Two clusters were detected, one due to down-regulation of the genes (green) and other due to up-regulation of the genes (red). Fold change and p values of the genes in the clusters are given on the right.
Figure 3.
BRCA1 regulates the mRNA expression of FST.
(A) Proliferating stable clones of both Neo (pcDNA3-SKOV3) and wtBRCA1 (BRCA1-SKOV3) were harvested for semi-quantitative RT-PCR assay as described under ‘Methods’. Bands corresponding to the respective genes are indicated on the right. (B) The PCR bands from three independent experiments of each indicated genes were quantified by densitometry and mRNA levels were normalized to Actin. (C) Proliferating SKOV3 cells were transiently transfected either with wtBRCA1 or background pcDNA3 vector and the isolated RNAs was assayed by semi-quantitative RT-PCR. The results were quantified as above and represented by bar diagram (D). Error bars are SEMs. *P<0.05 (relative to each controls).
Figure 4.
Effect of ectopic expression of BRCA1 on FST secretion.
(A) Standard curve was generated with purified human FST as indicated in the ‘Methods’, which was used to quantitate unknown FST concentrations in the indicated samples. SKOV3 cells were transfected as before, either for BRCA1 overexpression (B) or for BRCA1 underexpression (C), and FST assays (see methods) were performed with the diluted culture medium. The same FST assay was performed with the stable clones as indicated (D) as well as with the stable cells where expression of BRCA1 was knocked down by BRCA1-siRNA (E–F). Error bars are SEMs. *P<0.05 (relative to each control). Expression of BRCA1 in the above samples is given in (G).
Figure 5.
Q-PCR analysis with the immortalized ovarian surface epithelium cells (IOSE).
BRCA1 expressions in IOSE cells was modulated either with wtBRCA1 or BRCA1-siRNA along with appropriate the controls. Total RNA was isolated from all the cells and subjected to real time PCR analysis. Overexpression of BRCA1 in IOSE 397 (A) and IOSE 7576 (B) cells induce overexpression of FST and SMAD6 but knock down the expression of ACVR2B. In contrast down-regulation of BRCA1 in IOSE 397 (C) and IOSE 7576 (D) cells, demonstrates the reverse effect. mRNA expression of GAPDH was used for normalization in each case. Error bars are SEMs. * P<0.05 (relative to each control).
Figure 6.
FST assay with IOSE cell line.
(A) IOSE 7576, IOSE 397 and IOSE 592F were grown in 10 cm tissue culture plates and the culture medium was subjected to FST assay as described under ‘Methods’. IOSE 7576 cells (B), IOSE 397 cells (C) and IOSE 592F cells (D) were transiently transfected with wtBRCA1 for 48 hr and then FST assays were performed with the culture medium. Additionally, BRCA1 expression was knocked down in both IOSE 7576 (E.) and IOSE 397 (F) cells and then subjected to FST assays. Error bars are SEMs. * P<0.05 (relative to each control).
Figure 7.
Western blot analysis for IOSE cells.
IOSE 397 (A) and IOSE 7576 (B) cells were transiently transfected either with wtBRCA1 or BRCA1-siRNA in each case. Whole cell lysates from the attached cells were fractionated in 4–12% BT gel, and subsequently immunoblotted with the indicated antibodies. Densitometric analyses of the immunoblots shown above are given in C and D respectively.
Figure 8.
Cell proliferation and cell migration assay with IOSE cells.
(A) Comparative analysis of cell proliferation for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (B) Western blot analysis showing FST levels for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (C–D) Comparative cell migration analysis for IOSE 7576, IOSE 397 and IOSE 592F cell lines. Relative fluorescence units (RFU) was measured using all of the migrated cells to the feeder tray in each sample (C), whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case is shown in (D).
Figure 9.
Effect of FST knock down on cell migration.
(A–F) Cell migration analysis was performed for IOSE 7576, IOSE 397 and SKOV3 cell lines that were transfected with either control siRNA or FST-siRNA. Relative fluorescence units (RFU) measured from all the migrated cells in each sample are shown in A–C, whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case are shown in D–F. (G) Western blot for IOSE 7576, IOSE 397 and SKOV3 cell lines confirming knock-down of FST by FST-siRNA.