Figure 1.
Flow diagram of the strategy used to identify kinases interfering with MCF-7 cell density and up-regulation of CYP1A1 activity in human TCDD-exposed cells. Negative controls (siNeg) are non-targeting siRNA (NT1)-transfected cells and Dharmafect1-exposed cells (without siRNA) (wells A12, B12 and G12, H12). Two positive controls are constituted of cells transfected with siRNA targeting AhR (siPos, G1, H1).
Figure 2.
Validation of screen protocol.
A, Scatter plot showing the global distribution of control siRNA in function of EROD activity and methylene blue data. Three experiments were shown in the same plot. For each plate, individual values of negative controls were expressed in percentage in function of the mean value of the four controls, which was set at 100%. B, Visualization of global distribution of all siRNA of the screen as explained for A. Negative controls: wells A12, B12 and G12, H12.
Figure 3.
Putative kinases affecting TCDD-induced EROD (CYP1A) activity.
A, Hit distribution on quantile-quantile plot and B, boxplots of z-scores for the different types of probes (sample, Pos or Neg). C, a screen-wide image plot to visualize the position in plates of hits. Here, the picture corresponds to z-scores calculated from 3 experiments for individual siRNA. One experiment corresponds to 30 plates. Pos for siRNA targeting AhR, Neg for negative controls constituted of non-targeting siRNA (NT1)-transfected cells and Dharmafect1-exposed cells (without siRNA) and Sample for 3×712 siRNA targeting mRNA of human kinases. Hits corresponded to the top 150 z-scores.
Table 1.
List of 22 kinases potentially implicated in the up-regulation of EROD activity in response to TCDD.
Table 2.
Gene ontology of the 22 kinases potentially implicated in the up-regulation of EROD activity in response to TCDD.
Figure 4.
Hit confirmation and selectivity of siRNA.
A, Knock-down of ITPKA, PNCK, CaMKIα and AhR in MCF-7 cells. In this confirmation step, 2 siRNA among 3 of the library were selected for ITPKA and PNCK and effects were compared to those of 2 siRNA targeting CaMKIα and AhR (Positive controls) and to negative control (siNT1). 72 h after transfection, cells were exposed to 5 nM TCDD for 6 h and EROD activity was measured as explained in Materials and Methods. Knock-down efficiencies were evaluated by RT-qPCR assays for ITPKA (B) and PNCK (C). Evaluation of off-target effects of siRNA targeting ITPKA or PNCK through mRNA quantification of ITPKB, ITPKC (D) and CaMKIα (E). mRNA level data are expressed in function of the values of mRNA levels found in siNT1-transfected cells exposed to TCDD, arbitrarily set to 1; they correspond to the means ± S.E.M. of three independent experiments. * p-value<0.05 compared to NT1-transfected cells.
Figure 5.
Involvement of PCNK in CaMKIα phosphorylation.
MCF-7 cells were transfected with siRNA targeting PNCK or control siRNA (NT1) and 72 h later, cells were exposed to the ionophore ionomycin, a strong activator of the Ca2+/CaM/CaMKs pathway. Next, Western-blots were performed to analyze the phosphorylation state of CaMKIα on Thr177. Hsc70 was used as loading control. The data shown are representative of two independent experiments.