Centrosomal staining for CHK2 antibodies persists in cells expressing CHK2 targeting shRNAs. U2OS cells were stably transduced with various shRNAs directed against CHK2 or GFP. (A) Western blots showing the expression level of endogenous CHK2 in the transduced cell lines. (B and D) Cells were fixed and immunostained with the indicated anti-CHK2 antibodies (green) and costained for centrosomes (red) and DNA (blue). Representative fields of the immunofluorescence stainings obtained with the indicated cell lines are shown. (C and E) The fluorescence intensity of the CHK2 antibodies H-300 and P-Thr 68 was quantified and represented as a ratio of the control γ-tubulin signal at centrosomes. This ratio was set at 100% in control untransduced cells. Results are representative of 3 independent experiments where the intensity at the centrosomes was monitored in 200 cells. Error bars represent the standard deviation from the mean of 3 experiments. To validate the method of quantification of the fluorescence intensity signal at centrosomes, stable transduced GFP-PACT U2OS cells were generated. (F) A representative field showing the centrosomal localization of the GFP-PACT fusion protein. 48 h after 20 ng/ml doxycycline addition, cells were fixed and stained with anti-γ-tubulin antibody (red) and DAPI (blue). GFP-PACT was visualized by direct fluorescence. (G, H) GFP-PACT expression was induced with 2.5, 5 or 20 ng/ml doxycycline and cells were stained as in (F). (G) The expression of GFP-PACT at each doxycycline concentration was analyzed by Western blotting using anti-GFP antibody. The arrow indicates the bands corresponding to GFP-PACT and the asterisk designates non-specific signal. (H) The fluorescence intensity at centrosomes of GFP-PACT was quantified as in C and E and set at 100% in cells induced with 20 ng/ml doxycycline. Graphs represent the mean of intensity ± s.d. of 3 independent experiments.