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. 2022 Jun 3;25(7):104516. doi: 10.1016/j.isci.2022.104516

Figure 3.

Figure 3

Quantitative analysis of translational responses and nascent phosphorylation

(A) Experimental design for global analysis of translational changes induced by a CK2 inhibitor silmitasertib (10 μM).

(B) A histogram of log2 fold changes [H/M (silmitasertib/DMSO) ratios] of protein synthesis induced by silmitasertib. Averaged log2 H/M ratios based on proteins quantified in at least two of the three replicates are shown. Results from individual replicates are shown in Figure 3B. A subset of proteins whose mRNAs contain a TOP motif is shown in dark blue. CK2 substrates determined by an in vitro kinase reaction (Sugiyama et al., 2019) are shown in red. All quantified proteins are shown in gray. The dashed lines indicate median values of the three groups (all, TOP motif, and CK2 substrates) The p-value was computed using the two-sided Wilcoxon rank-sum test.

(C) qRT-PCR validation of pSNAP result. (Left panel) Representative polysome profiles of HeLa cells treated with DMSO or silmitasertib. (Right panel) Polysomal association of mRNAs encoding for the selected ribosomal proteins that were repressed upon CK2 inhibition according to pSNAP.

(D) CK2 inhibition led to decreased phosphorylation levels of nascent forms of known CK2 substrates. (Left panel) A histogram of log2 fold changes [H/M (silmitasertib/DMSO) ratios] of phosphorylation sites induced by silmitasertib. Averaged log2 H/M ratios based on class I phosphosites quantified in at least one of the three replicates are shown. CK2 substrates determined by in vitro kinase reaction (Sugiyama et al., 2019) are shown in red. All quantified proteins are shown in gray. The p-value was computed using the two-sided Wilcoxon rank-sum test. (Right panel) Change of NPC and phosphorylation levels due to silmitasertib treatment versus DMSO treatment. The indicated phosphorylation sites of G3BP1, HSP90AA1, and HSP90AB1 are known CK2 substrates. Levels of the NPCs were quantified from NPC-derived unphosphorylated peptides. The bars represent averaged values from two or three replicates.