Figure 1.
CK1δ inhibitor SR-3029 downregulates translation initiation. (A) CK1δ inhibition disrupts 4E-BP1 phosphorylation. Five lymphoma cell lines, Z-138, Jeko-1, Maver-1, and JVM-2 (representing mantle cell lymphoma), and OCI-LY10 (representing diffuse large B-cell lymphoma), were treated with SR-3029 or vehicle control for 24 hours. Protein levels were determined by immunoblot. P-, phosphorylated protein. (B) CK1δ inhibition disrupts eIF4F assembly. The lymphoma cell lines Z-138 and Jeko-1 were treated with SR-3029 or DMSO control at the indicated concentrations for 24 hours and then processed for the m7GTP cap binding assay using m7GTP agarose beads to pull down cap binding proteins, which were analyzed by immunoblot. The level of eIF4G reflects eIF4F assembly, whereas the level of eIF4E is constant and serves as the loading control. (C-D) CK1δ inhibition disrupts global translation. Z-138 cells were treated with DMSO vehicle control (C) or SR-3029 500 nM (D) for 6 hours. Cell lysates were fractionated on 10% to 50% sucrose density gradients by centrifugation. The x axis shows fractions collected, and the y axis shows absorbance at OD260. Shown are the ribonucleoprotein particle fraction (RNP), ribosomal subunits (40S, 60S, and 80S), and polysomes. (E) SUnSET translation assay. Bulk translation was determined in 3 MCL cell lines and 1 DLBCL cell line treated with SR-3029 at the indicated concentrations or DMSO control for 24 hours (or 6 and 24 hours in the case of OCI-LY10). Puromycin was then added at the final concentration of 1 μg/mL for 30 minutes. Cell lysates were analyzed by immunoblot using anti-puromycin or anti–β-actin antibodies. (F-G) CK1δ associates with the mRNA cap structure in lymphoma. m7GTP cap binding assays were performed in untreated Z-138 lymphoma cells. (F) Protein levels of CK1δ, CK1ε, eIF4E, 4E-BP1, and β-actin were determined in the input and pulldown fractions by immunoblotting. (G) Protein levels of mTOR, Raptor, eIF4E, P-eIF4E, and β-actin were determined in the input and pulldown fractions by immunoblotting.

CK1δ inhibitor SR-3029 downregulates translation initiation. (A) CK1δ inhibition disrupts 4E-BP1 phosphorylation. Five lymphoma cell lines, Z-138, Jeko-1, Maver-1, and JVM-2 (representing mantle cell lymphoma), and OCI-LY10 (representing diffuse large B-cell lymphoma), were treated with SR-3029 or vehicle control for 24 hours. Protein levels were determined by immunoblot. P-, phosphorylated protein. (B) CK1δ inhibition disrupts eIF4F assembly. The lymphoma cell lines Z-138 and Jeko-1 were treated with SR-3029 or DMSO control at the indicated concentrations for 24 hours and then processed for the m7GTP cap binding assay using m7GTP agarose beads to pull down cap binding proteins, which were analyzed by immunoblot. The level of eIF4G reflects eIF4F assembly, whereas the level of eIF4E is constant and serves as the loading control. (C-D) CK1δ inhibition disrupts global translation. Z-138 cells were treated with DMSO vehicle control (C) or SR-3029 500 nM (D) for 6 hours. Cell lysates were fractionated on 10% to 50% sucrose density gradients by centrifugation. The x axis shows fractions collected, and the y axis shows absorbance at OD260. Shown are the ribonucleoprotein particle fraction (RNP), ribosomal subunits (40S, 60S, and 80S), and polysomes. (E) SUnSET translation assay. Bulk translation was determined in 3 MCL cell lines and 1 DLBCL cell line treated with SR-3029 at the indicated concentrations or DMSO control for 24 hours (or 6 and 24 hours in the case of OCI-LY10). Puromycin was then added at the final concentration of 1 μg/mL for 30 minutes. Cell lysates were analyzed by immunoblot using anti-puromycin or anti–β-actin antibodies. (F-G) CK1δ associates with the mRNA cap structure in lymphoma. m7GTP cap binding assays were performed in untreated Z-138 lymphoma cells. (F) Protein levels of CK1δ, CK1ε, eIF4E, 4E-BP1, and β-actin were determined in the input and pulldown fractions by immunoblotting. (G) Protein levels of mTOR, Raptor, eIF4E, P-eIF4E, and β-actin were determined in the input and pulldown fractions by immunoblotting.

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