Journal: Nature Communications

Article Title: MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

doi: 10.1038/ncomms6413

Figure Lengend Snippet: ( a ) Western blot analysis of total and phospho-eIF4E1(S209) as well as total and phospho-ERK after 24 h treatment with an MEK inhibitor AZD6244 (AZD 200 nM), compared with untreated and vehicle controls in HLY-1, GM02184 and Pfeiffer cell lines. Densitometry analysis is shown in . ( b ) Western blot analysis of phospho-MNKs (antibody detects p-MNK1 and p-MNK2), total MNK1, total MNK2, total and phospho-eIF4E1(S209) after 1 h treatment with a p38 inhibitor VX702 (200 nM). Densitometry analysis is shown in . ( c ) Western blot analysis of total and phospho-eIF4E1 and MCL-1 post 4 h treatment with a p38 inhibitor VX702. ( d ) Densitometry analysis of phospho-eIF4E1(S209) and ( e ) MCL-1 band intensity of western blot in , relative to GAPDH following 4 h treatment with vehicle (black bar) or 200 nM VX702 (white bar). Mean±s.d., n =3, * P -value of Student’s t -test <0.05. ( f ) Trypan blue exclusion assay of HLY-1, GM02184 and Pfeiffer cells after 72 h treatment with vehicle (black bar) or 200 nM VX702 (white bar). Mean±s.d., n =3, * P -value of Student’s t -test <0.001. ( g ) Representative CFSE analysis of HLY-1 cells 48 and 72 h post treatment with 200 nM VX702 (blue line) or vehicle (DMSO; red line). P0 denotes initial population, while P1, P2 and P3 denote subsequent daughter cell populations. Three independent experiments are shown in . ( h ) Western blot showing total and phospho-eIF4E1(S209) as well as MCL-1 with or without 4 h treatment with 200 nM VX702 in HLY-1 cells expressing empty vector (EV), MNK1-wildtype (M1WT), MNK1-phosphomimetic (M1TD), MNK1-phosphonull (M1 AA), and MNK2-wildtype (M2WT). ( i , j ) Densitometry analysis showing relative band intensity of ( i ) phospho-eIF4E1(S209) or ( j ) MCL-1 to GAPDH in HLY-1 cells treated with vehicle (black bar) or 200 nM VX702 (white bar) for 4 h of immunoblot in (mean±s.d., n =3, * P -value of Student’s t -test <0.05). ( k ) Summarizing figure illustrating the p38-regulated MNK-dependent eIF4E1 phosphorylation in DLBCL cells. Cartoon depicts two potential modes of disrupting the p38-MNK-eIF4E1 axis in DLBCL, that are, via the use of p38 or MNK inhibitors. Full immunoblots are shown in .

Article Snippet: VX702 (p38 inhibitor) was purchased from Cayman Chemical, and MEK inhibitor AZD6244 was purchased from CalBiochem.

Techniques: Western Blot, Trypan Blue Exclusion Assay, Expressing, Plasmid Preparation, Phospho-proteomics

Journal: Nature Communications

Article Title: MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

doi: 10.1038/ncomms6413

Figure Lengend Snippet: ( a ) On activation by p38, MNKs phosphorylate eIF4E1 at S209. ( b ) In the absence of MNK kinase activity, eIF4E1 cannot be phosphorylated, while in the ( c ) absence of MNK protein expression or its physical suppression, eIF4E1 protein expression is downregulated. ( d ) The unphosphorylated eIF4E1 (at S209) form is stimulatory for eIF4E3 protein upregulation. Increased abundance of eIF4E3 in a cellular context enhances the ability for eIF4E3 to bind cap. The relative abundance of either eIF4E1 or eIF4E3 is determined by MNKs. The accessibility to mRNA cap structure by both eIF4Es mandates a distinct cellular translatome that dictates pro- or anti-oncogenic phenotype.

Article Snippet: VX702 (p38 inhibitor) was purchased from Cayman Chemical, and MEK inhibitor AZD6244 was purchased from CalBiochem.

Techniques: Activation Assay, Activity Assay, Expressing