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

Journal: MedComm

Article Title: Targeting the Cdc2‐like kinase 2 for overcoming platinum resistance in ovarian cancer

doi: 10.1002/mco2.537

Figure Lengend Snippet: Cisplatin induces CLK2 upregulation via p38. (A) Western blot analysis of CLK2 expression in A2780 cells treated with various signaling pathway inhibitors or dimethyl sulfoxide (DMSO) in the presence or absence of cisplatin. Only p38 inhibitor attenuated the expression of CLK2. (B) CLK2 mRNA level in A2780 cells treated with cisplatin in the presence or absence of p38 inhibitor was measured by qRT‐PCR. Data was shown as mean ± SD. p Values were determined by Student's t ‐test. (C) A2780 cells treated with phosphate‐buffered saline (PBS), or cisplatin, or cisplatin plus p38 inhibitor (p38i), or cisplatin and p38 overexpression (p38‐Myc) were incubated with 20 µg/mL cycloheximide (CHX) for the indicated periods and then analyzed by Western blot. The results were normalized to the levels of GAPDH. (D) Quantitation of CLK2 protein levels was based on the Western blot result. Data were shown as mean ± SD. (E) Coimmunoprecipitation assay showing the presence of a complex containing CLK2 and p38. CLK2 antibody coprecipitating p38 (top panel). p38 antibody coprecipitating CLK2 (bottom panel). Input, protein expression in cell lysates detected by Western blot. IgG, negative control. IP, expression of compound coprecipitated by CLK2 or p38 antibody. n.s., no significance.

Article Snippet: To determine the half‐life of CLK2, A2780 cells were treated either with PBS, cisplatin (6.67 μM) or p38i (10 μM, VX702, #A8687; APExBio) plus cisplatin.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Saline, Over Expression, Incubation, Quantitation Assay, Co-Immunoprecipitation Assay, Negative Control

Journal: MedComm

Article Title: Targeting the Cdc2‐like kinase 2 for overcoming platinum resistance in ovarian cancer

doi: 10.1002/mco2.537

Figure Lengend Snippet: p38 and ring finger protein 8 (RNF8) mediate CLK2 protein ubiquitination. (A) Western blot analyses of CLK2 expression in A2780 and Skov3 cells which were treated with cisplatin (16.7 µM) in the presence or absence of p38 inhibitor (10 µM). Cells were incubated with MG132 (10 µM) for the indicated periods. (B) Ubiquitination assays of CLK2 in human embryonic kidney (HEK293T) cells transfected with Flag‐CLK2, Myc‐p38, and HA‐ubiquitin or its mutants (K48R, K48o, K63R, K63o) plasmids. IP, expression of compound coprecipitated by Flag antibody. WCL, whole cell lysate. (C) Ubiquitination assays of CLK2 in HEK293T cells transfected with Myc‐p38, HA‐Ub‐K63o, and CLK2‐WT or its mutants (T343A, K192R) plasmids. IP, expression of compound coprecipitated by CLK2 antibody. WCL, whole cell lysate. (D) Coimmunoprecipitation assay showing the endogenous (top panel) presence of a complex containing CLK2 and RNF8 and the exogenous (bottom panel) presence of a complex containing CLK2 and Flag‐RNF8. Input, protein expression in cell lysates detected by Western blot. IgG, negative control. IP, expression of compound coprecipitated by CLK2, RNF8, or Flag antibody. WCL, whole cell lysate. (E) Ubiquitination assays of CLK2 in HEK293T cells transfected with CLK2, Flag‐RNF8, and HA‐ubiquitin or its mutants (K48o, K63o) plasmids. IP, expression of compound coprecipitated by CLK2 antibody. WCL, whole cell lysate. (F) Ubiquitination assays of CLK2 in HEK293T cells cotransfected by various combinations of plasmids expressing CLK2, Myc‐p38, Flag‐RNF8, and HA‐Ub‐K63o. IP, expression of compound coprecipitated by CLK2 antibody; WCL, whole cell lysate.

Article Snippet: To determine the half‐life of CLK2, A2780 cells were treated either with PBS, cisplatin (6.67 μM) or p38i (10 μM, VX702, #A8687; APExBio) plus cisplatin.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Incubation, Transfection, Co-Immunoprecipitation Assay, Negative Control

Journal: Journal of Cell Communication and Signaling

Article Title: Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-β signalling

doi: 10.1007/s12079-019-00525-7

Figure Lengend Snippet: a Scatter diagram of apoptosis analysis by Annexin V-FITC/PI staining. b Analysis of percent apoptosis rates. The rate of apoptosis in the 100 μg/ml BBR group was 52.45%, followed by the 50 μg/ml BBR group (35.37%) and the 25 μg/ml BBR group (25.18%), which were higher than those of the CM (3.61%) or DMSO group (3.46%) (P < 0.05). A 0.5 mg/ml concentration of VX-702, a p38 MAPK inhibitor, significantly decreased the rate of apoptosis (3.04%) compared to the BBR group (P < 0.05). c Detection of apoptosis-associated proteins Bax, Bcl-2, p38 MAPK and p-p38 MAPK through western blotting. d The histograms of expression for Bax, Bcl-2, p38 MAPK and p-p38 MAPK. The 50 and 100 μg/ml concentrations of BBR significantly increased the expression of Bax and decreased the expression of Bcl-2 compared to the CM, DMSO or VX-702 groups (P < 0.05). Although only 100 μg/ml BBR upregulates the expression of p38 MAPK, p-p38 MAPK expression is increased by BBR in a dose-dependent manner compared to the CM, DMSO or VX-702 groups (P < 0.05). The error bar represents the SD (n = 3). *P < 0.05 versus CM group; **P < 0.05 versus VX-702 group

Article Snippet: The p38 MAPK inhibitor VX-702 was also employed (SD5960, Beyotime, China).

Techniques: Staining, Concentration Assay, Western Blot, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-β signalling

doi: 10.1007/s12079-019-00525-7

Figure Lengend Snippet: Correlation analysis between effects of berberine (BBR) on TGF-β receptors and p38 phosphorylation by Pearson

Article Snippet: The p38 MAPK inhibitor VX-702 was also employed (SD5960, Beyotime, China).

Techniques: Phospho-proteomics

Journal: Journal of Cell Communication and Signaling

Article Title: Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-β signalling

doi: 10.1007/s12079-019-00525-7

Figure Lengend Snippet: Schematic diagram for relevant mechanisms of CCD-18Co-induced EMT and berberine treatment. TGF-β, one of the cytokines produced by CCD-18Co myofibroblasts, binds TGF-β receptors (TβRII and TβRI) and induces EMT transformation of colon epithelial HCoEpiC cells through Smads signaling pathway (Green lines. Arrows represent activation or promotion). Berberine can inhibit the TGF-β/Smads signaling to suppress the CCD-18Co-stimulated EMT. Berberine also promotes apoptosis of HCoEpiCs through activation of p38 MAPK pathway (Yellow lines, Arrows represent activation or promotion, flat lines represent inhibition or down-regulation)

Article Snippet: The p38 MAPK inhibitor VX-702 was also employed (SD5960, Beyotime, China).

Techniques: Produced, Transformation Assay, Activation Assay, Inhibition