br Materials and methods br

Materials and methods
Results Recently, a quantitative proteomic analysis of C2C12 mouse myoblasts cell line totally deprived of protein kinase CK2 catalytic subunits (CK2α/CK2α′(−/−)), suggested that the cellular expression of HSP27, a chaperone with multiple functions related to proteostasis, cytoskeleton organization, apoptosis, and oxidative stress, is strongly downregulated [18]. To validate this analysis, HSP27 expression has been compared in C2C12 wild-type and two different CK2α/α′(−/−) clonal cell lines by Western blotting. Fig. 1a shows that HSP27 expression in C2C12 myoblasts genetically deprived of both protein kinase CK2 catalytic subunits (CK2α/α′(−/−)), is strongly reduced with respect to the wild-type cells. The same analysis was performed on two different clonal cell line knocked out only for the regulatory β subunit of CK2 (CK2β(−/−)), which plays a role in substrate targeting. Fig. 1a shows that the lack of the regulatory β subunit alone leads to the same HSP27 downregulation observed in CK2α/α′(−/−). These results demonstrate a direct correlation between tetrameric CK2 and HSP27 expression. Notably, the expression of other well-known heat shock proteins such as HSP90, HSP70 and HSP105 is not affected in our knockout cell lines (Fig. 1a). Knockout 4SC-202 sale are adapted to live without CK2 and our quantitative proteomic analysis revealed a broad genetic reprogramming of CK2α/α′ (−/−) [18] and CK2β(−/−) (manuscript in preparation) cell lines. Thus, HSP27 downregulation could be the result of this selection process. Therefore, it has been assayed if a transient CK2 downregulation would also affect HSP27 expression. Fig. 1b confirms that also a transient downregulation of CK2β subunit leads to a decrease of HSP27, strengthening the link between HSP27 and CK2 expression. Noteworthy, the downregulation of Akt, a CK2 direct substrate [24] is, instead, without any effect on HSP27 expression suggesting that the molecular pathway linking CK2 and HSP27 expression does not involve this kinase (Fig. 1b). To assess whether the observed effect on HSP27 expression is related to the enzymatic activity of CK2 holoenzyme or to its ability to physically interact with other proteins, ATP site-directed CK2 inhibitors, that do not affect the total amount of the kinase, have been exploited. C2C12 cells were treated with three different specific and structurally unrelated CK2 inhibitors, namely CX-4945, tetra-bromo-deoxyribofuranosyl-benzimidazole (TDB), and Quinalizarin (QZ). The effect of these inhibitors on endogenous CK2 activity has been monitored assaying the phosphorylation rate of the CK2 specific target S129 of Akt by a phosphospecific antibody [24], and the residual CK2 kinase activity in cell lysates with the CK2 specific peptide substrate R3AD2SD5. Regardless the type of CK2 inhibitor used, a decrease of protein kinase enzymatic activity parallels the drop of HSP27 expression as illustrated in Fig. 1c and in a dose-response curve of CX-4945 on HSP27 expression in C2C12 cells (Fig. S1) confirming a direct relationship between CK2 activity and HSP27 expression. In accordance with the results of Fig. 1a, CX-4945 is without any effect on the expression of other HSPs (HSP105, HSP90, HSP70) in C2C12 cells (Fig. S1). To confirm that the correlation between CK2 activity and HSP27 expression is a general phenomenon rather than cell-specific, HEK293T cells (human embryonic kidney cell line) and two different tumour cell lines, HeLa (human cervical cancer cells) and HepG2 (human hepatocyte carcinoma cell line), have been treated with the two CK2 inhibitors, CX-4945 and QZ. Both the inhibitors promote a decrease in HSP27 expression in all the cell lines analysed in a parallel manner with the inhibition of CK2 kinase activity (Fig. 2a). The corresponding dose-response curves of CX-4945 on HSP27 expression are also shown in Fig. S1 where it is possible to see a slight effect of CK2 inhibition on other HSPs, i.e. HSP105 in HeLa and HSP70 in HepG2 (Fig. S1).