Fig 2: The HSF1 transcriptional program is impaired by HER2 inhibition. (a) Schematic overview of HSF1-regulated gene expression. Transcriptionally activated and -repressed target genes analyzed here are indicated. (b and c) HER2 inhibition impairs the HSF1-dependent inducible heat-shock response (b) and the tumor-promoting (c) transcriptional program of HSF1. SK-BR-3 cells were treated with 2 μM CP724.714 or DMSO for 48 h and mRNA was isolated. qRT-PCR of Hsp70 (*P=0.0396), Hsp110 (***P=0.001), Hsp90α (**P=0.0049), SPTAN (*P=0.0362), CDC6 (***P=0.001), FASN (**P=0.0069) and CBX3 (P=0.067), each normalized to 36B4 mRNA. Relative values are given in (ratio (2−ddCT)). Error bars indicate ±S.E.M. of three independent experiments, repeated twice each with all in triplicates. Student's t-test, two-tailed, P-value: *P<0.05, **P<0.01 and ***P<0.001. (d) HER2 inhibition also attenuates the HSF1-mediated oncogenic program after heat shock. SK-BR-3 cells were treated with 2 μM CP724.714 or DMSO for 48 h. Heat shock (HS) at 42 °C for 1 h with 3 h recovery before mRNA isolation. qRT-PCRs of SPTAN (**P=0.0048), CDC6 (*P=0.0373) and FASN (*P=0.0161), normalized to 36B4. Relative values are given in (ratio (2−ddCT)). Error bars indicate ±S.E.M. of two or three independent experiment in triplicates. Student's t-test, two-tailed, P-value: *P<0.05, **P<0.01 and ***P<0.001

Fig 3: The expression of Cdc6 and Cdt1 upon inhibition of ER signalling. (A) Histogram and (B) Western blot showing the expression of Cdc6 in MCF7 cells treated with tamoxifen at 0, 12 and 24 hours post-treatment. (C) Histogram and (D) Western blot showing the expression of Cdt1 in MCF7 cells treated with tamoxifen at 0, 12 and 24 hours post-treatment.

Fig 4: The expression levels of Cdc6, Cdt1 and Orc1 in breast cancer patients expressing various levels of MCM genes. Histograms showing percentage of specimens that expressed different levels of (A) Cdc6, (B) Cdt1 and (C) Orc1 in six groups of specimens stratified by the number of MCM2-7 genes they overexpress.

Fig 5: Inhibition of the NADPH oxidase NOX4 abrogates H-Ras-induced DNA-damage response. (a) NOX4 and p22phox knockdown with interference RNA decrease the level of H2A.X phosphorylation in Tet-on-regulated H-RasV12 HThy-ori cells treated with doxycycline (1 μg/ml) for 48 h. Left panel: immunoblots for γ-H2A.X, p22phox and NOX4. β-Actin and total H2A.X were used as loading control. Right panel: quantification of γ-H2A.X intensity by densitometry; *P<0.05, **P<0.01 using the Student's t-test. (b) Representative micrographs illustrating γ-H2A.X staining in cells transduced with interference RNA against NOX4 or pretreated with NAC (5 m) for 30 min before addition of doxycycline (1 μg/ml) for 48 h. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole; blue). Magnification × 63. (c) Quantification of the number of γ-H2A.X foci. Histogram indicates the number of cells containing γ-H2A.X foci among all cells stained with DAPI. The data represent the average and s.d. of three independent counts. (d) Inhibition of NOX4 abrogates H-RasV12-induced CDC6 regulation. H-Ras and CDC6 were visualized by western blot in H-RasV12-inducible cells treated with doxycycline (1 μg/ml) for indicated times. β-Actin was used as loading control. All experiments were realized with siRNA pool from Dharmacon (Table 1).