Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 5. mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. (A) Knockdown of Mnk1 does not abrogate rapamycin-induced eIF4E phosphorylation. H157 cells that were not transfected (NT) or were transfected with control (Ctrl) or Mnk1 siRNA for 48 h were treated with 10 nM rapamycin (Rap). After 3 h, the cells were subjected to preparation of whole-cell protein lysates. (B) Modulation of eIF4E phosphorylation by mTOR inhibitors in WT, 1-KO, 2-KO, and Mnk1/Mnk2 DKO cells. The indicated MEF lines were treated with 10 nM rapamycin (R) or RAD001 (R1) for 3 h and then subjected to preparation of whole-cell protein lysates. The indicated proteins in these experiments were detected by Western blot analysis.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Knockdown, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 6. Inhibition of Mnk-dependent eIF4E phosphorylation by a Mnk1 inhibitor (C) augments rapamycin-mediated growth inhibition (A and B) in human lung cancer cells. (A) The individual cell lines, as indicated, were seeded in 96-well plates. On the next day, they were treated with the indicated concentrations of rapamycin (Rap) alone, 2.5 M CGP57380 (CGP) alone, and their combination. After 3 days, the plates were subjected to determination of cell numbers using the sulforhodamine B assay. The bars are means of four replicate determinations plus standard deviations. (B) H460 cells at a density of 250 cells per well were seeded in 12-well plates. On the second day, the cells were treated with the indicated concentrations of rapamycin (Rap) alone, CGP57380 (CGP) alone, and their combination. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with crystal violet. The colonies in each well were counted. The bars are means of three replicate determinations plus standard deviations. (C) H157 cells were pretreated with the indicated concentrations of CGP57380 (CGP) for 30 min and then cotreated with 10 nM rapamycin (Rap) for 1 h or 24 h. The cells were then subjected to preparation of whole-cell protein lysates for detection of the indicated proteins using Western blotting.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Inhibition, Phospho-proteomics, Sulforhodamine B Assay, Staining, Western Blot

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 3. Fractionation of human peripheral blood mononuclear cells (PBMCs) from lean normal donors suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressed approximately the same level of p27 as unfractionated PBMCs. Panels a to d show the results of the western immunoblot analysis of p27, total 4E-BP1 and total MNK1 in unfractionated PBMCs obtained from lean normal African Americans (AAs) (n = 3) and lean normal Caucasian Americans (CAs) (n = 3). Panels a to d also include the results of the western immunoblot analysis of p27, total 4E-BP1 and total MNK1 in T (CD3+) cells (n = 3) and monocytes (CD14+) (n = 3) isolated from unfractionated PBMCs us- ing Miltenyi’s AutoMAC Simmunomagnetic cell sorting protocols (ZenBio Inc., Research Triangle Park, North Carolina, USA and Miltenyi Biotech Inc., Auburn, California, USA). The unfractionated PBMCs used to isolate T (CD3+) cells and monocytes (CD14+) were obtained from the mixtures of lean normal African and Caucasian Americans (AAs and CAs). decreased—by about 40%—relative to the unfractionated PBMCs. Surprisingly, this extent of the decrease in the expression of MNK1 in the T (CD3+) cells did not interfere with the expression of the p27 itself in the same T (CD3+) cells. In summary, the results presented above suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressed the same level of p27 as unfractionated PBMCs in either lean normal African or Caucasian Americans.

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Fractionation, Western Blot, Isolation, FACS, Expressing

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 4. Regarding the results presented in Figure 3, panels a and b present the hypothetical upstream molecular signaling pathways-inclu- ding 4E-BP1 and MNK1-of the expression of p27 in human PBMCs ob- tained from lean normal donors as compared to those in (1) the livers from genetically homozygous obese diabetic rats or mice [2] and (2) hu- man breast cancer cell lines in vitro [12]-[14]. press decreased—by about 40%—relative to the unfractionated PBMCs. Surprisingly, this extent of the decrease in the expression of MNK1 in the T (CD3+) cells did not interfere with the expression of the p27 itself in the same T (CD3+) cells. In summary, the results presented above suggested that all sub-populations of PBMCs, including T (CD3+) cells and monocytes (CD14+), probably expressedthe same level of p27 as unfractionated PBMCs in either lean normal African or Caucasian Americans.

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Protein-Protein interactions, Expressing, In Vitro

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 5. Severe depression—by over 95%—of the expression of p27 in the peripheral blood mononuclear cells (PBMCs) obtained from obese type 2 diabetic African Americans (AAs) was probably caused primarily by 1) the severe depression of the MNK1 upstream molecular signaling pathways of the expression of p27 and secondarily by 2) the moderate increase in the phosphorylation of 4E-BP1 upstream molecular signaling pathways of the expression of p27. Panels a to h present the results of the western immunoblot analysis of (Panels a and b) p27 (n = 3), (Panels a and c) total 4E-BP1 (n = 3), (Panels a and d) 4E-BP1 phosphorylated at Thr 37/46 (n = 3), (Panels a and e) total MNK1 (n = 3), (Panels a and f) MNK1 phosphorylated at Thr 197/202 (n = 3), (Panels a and g) total eIF4E (n = 3), and (Panels a and h) eIF4E phosphorylated at Ser 209 (n = 3). lean normal African and Caucasian Americans (AAs and CAs) and Panel b of Figure 6 for primarily obese type 2 diabetic African Americans (AAs). Stated briefly, the severe depression of the expression of p27 in the PBMCs from obese type 2 diabetic African Americans was caused primarily by 1) the severe depression of the MNK1 upstream molecular signaling pathways and secondarily by 2) the moderate increase in the phosphoryla-

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Expressing, Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: American Journal of Molecular Biology

Article Title: Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

doi: 10.4236/ajmb.2014.43013

Figure Lengend Snippet: Figure 6. Regarding the results presented in Figure 5, panels a and b present the hypothetical MNK1 and 4E-BP1 upstream mo- lecular signaling pathways of the expression of p27 in the PBMCs obtained from lean normal African and Caucasian Americans (AAs nd CAs) (Panel (a)) and primarily obese type 2 diabetic Af- rican Americans (AAs) (Panel (b)). tion of the 4E-BP1 upstream molecular signaling pathways of the expression of p27. Panels a, e and f of Figure 5 show that the expressions of total MNK1 and MNK1 phosphorylated at Thr 197/202 in the PBMCs from obese type 2 diabetic African Americans (AAs) were severely depressed-by ap- proximately 82.2% and 83.3%, respectively-relative to the lean normal African Americans (AAs). The results presented above in the previous section indicated that the depression of MNK1 by approximately 40% did not significantly decrease the expression of p27. The results presented here in the Panels a and b of Figure 5 sug- gested that the depression of total MNK1 and MNK1 phosphorylated at Thr 197/202 by over 80% was associ- ated with the severe depression of the expression of p27—by over 95%—in the PBMCs from obese type 2 dia- betic African Americans (AAs) relative to the lean normal African Americans (AAs). It could have been that the depression of the expression of MNK1 by 40% was not enough to severely depress the expression of p27. Lastly, the results presented in the Panels a, g and h of Figure 5 indicated that the expression of the total eIF4E was significantly increased but the expression of the eIF4E phosphorylated at Ser 209 was severely de- pressed in the PBMCs from obese type 2 diabetic African Americans (AAs). This observation suggested that the eIF4E unphosphorylated at Ser 209 might protect the 5’-N7-methylguanosine cap (5’-m7G cap)in the 5’-untran- slated region (5’-UTR) of the p27 mRNA, thereby severely depressing the inherent cap-independent translation initiation of p27 mRNA into p27 protein in obese type 2 diabetic African Americans [13] [15] [16].

Article Snippet: The following primary antibodies were purchased from the Cell Signaling Technology (Danvers, Massachusetts, USA): 1) 4E-BP1 (total) and phospho-4E-BP1 (Thr37/46); 2) phospho-MNK1 (Thr197/202); 3) eIF4E (total) and phospho-eIF4E (Ser209); and 4) GAPDH; Additionally, the following primary antibodies were obtained from the Santa Cruz Biotechnology (Dallas, Texas, USA): 1) p27Kip1; 2) 4E-BP1; 3) MNK1; 4) eIF4E; and 5) GAPDH.

Techniques: Protein-Protein interactions, Expressing

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, PDAC cells (AsPC1, CD18 and Panc1) were grown on tissue culture plastic or in three-dimensional type I collagen (2mg/ml) for 24 hours. The cell lysates were analyzed for eIF4E phosphorylation on Ser209 (p-eIF4E) by Western blotting. B, PDAC cells were grown for 24 hours on tissue culture plastic or in three-dimensional type I collagen (2mg/ml). The cell lysates were analyzed for MNK1 phosphorylation on Thr197/202 by Western blotting. C, PDAC cells growing in three-dimensional type I collagen were treated with DMSO or CGP57380 (2.5 μM) for 24 hours and the effect on eIF4E phosphorylation was determined by Western blotting. D, PDAC cells were transfected with control siRNA (siCtrl) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on MNK1 and MNK2 mRNA expression was determined by qRT-PCR. Effect on MNK1 protein levels and eIF4E phosphorylation was determined by Western blotting. The results are representative of at least three independent experiments. E, Human pancreatic TMAs containing 27 pancreatic tumor specimens were immunostained with anti-p-eIF4E antibody and assessed for fibrosis using trichrome staining. Samples were obtained on an IRB-approved protocol. The relative immunostaining and the extent of fibrosis were graded as low (0 or 1+) or high (2+ or 3+). The relationship between p-eIF4E expression in the tissue samples and the extent of fibrosis was assessed by Fisher’s exact test.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Western Blot, Transfection, Control, Expressing, Quantitative RT-PCR, Staining, Immunostaining

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: Chemoresistant CD18 (CD18-CR) cells were generated as detailed in Materials and Methods. A, CD18 and CD8-CR cells growing on tissue culture plastic were examined by phase contrast microscopy. Lysates from CD18 and CD18-CR cells were analyzed for E-cadherin, vimentin and ZEB1 protein and mRNA expression by Western blotting and qRT-PCR. B, CD18 and CD18-CR cells were plated onto thin-layer type I collagen matrix overlaid with colloidal gold, allowed to migrate for 24 hours, and the tracks were photographed and quantified. *, p<0.05. C, CD18 and CD18-CR cells were grown on tissue culture plastic or in three-dimensional type I collagen (2mg/ml) for 24 hours. The cell lysates were analyzed for eIF4E phosphorylation on Ser209 by Western blotting. D, CD18-CR cells growing in three-dimensional type I collagen were treated with DMSO or CGP57380 (2.5 μM) for 24 hours and the effect on eIF4E phosphorylation was determined by Western blotting. E, CD18-CR cells were transfected with control siRNA (siCtrl) or a combination of MNK1 and MNK2 specific siRNAs for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on MNK1 and MNK2 mRNA expression was determined by qRT-PCR. Effect on MNK1 protein levels and eIF4E phosphorylation was determined by Western blotting. The results are representative of at least three independent experiments. See also Supplemental Figure S1.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Generated, Microscopy, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, CD18-CR cells were treated with CGP57380 (2.5 μM) for 72 hours and the effect on E-cadherin and vimentin protein was determined by Western blotting (left). The effect on E-cadherin and vimentin mRNA expression was determined by qRT-PCR (right). B, CD18-CR previously treated with DMSO or CGP57380 (2.5 μM) for 48 hours were plated onto thin-layer type I collagen matrix overlaid with colloidal gold, allowed to migrate for 24 hours in the presence of DMSO or CGP57380 (2.5 μM), and the tracks were photographed and quantified. *, p<0.05. C and D, CD18-CR cells were transfected with control siRNA (siCtrl), MNK1 siRNA (siMNK1), MNK2 siRNA (siMNK2) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on p-eIF4E, E-cadherin and vimentin expression was determined by Western blotting (C). The effect on E-cadherin and vimentin mRNA expression was determined by qRT-PCR (D). The results are representative of at least three independent experiments.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A and B, CD18-CR cells were transfected with control siRNA (siCtrl) or ZEB1-specific siRNA (siZEB1) for 72 hours, and the effect on E-cadherin and vimentin protein expression was determined by Western blotting (A, left). CD18-CR cells growing on glass coverslips were transfected with siCtrl or siZEB1 for 72 hours. The cells were then stained for E-cadherin, actin using phalloidin and DAPI to counterstain the nuclei (A, right). The transfected cells were plated onto thin-layer type I collagen matrix overlaid with colloidal gold, allowed to migrate for 24 hours, and the tracks were photographed and quantified (B). **, p<0.01. C, CD18-CR and AsPC1 cells in 3D collagen were treated with CGP57380 (2.5 μM) for 72 hours and the effect on ZEB1 mRNA was determined by qRT-PCR. The effect on ZEB1 protein was determined by Western blotting. D, CD18-CR cells were transfected with control siRNA (siCtrl), MNK1 siRNA (siMNK1), MNK2 siRNA (siMNK2) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were then processed for MNK1, MNK2 and ZEB1 mRNA and protein expression by Western blotting and qRT-PCR. E, CD18-CR cells on tissue culture plastic were treated with CGP57380 (5 μM) for 48 hours. The cells were subjected to hypotonic lysis followed by separation of equal amounts of supernatant, as measured by OD at 260 nm, on 10%–50% sucrose gradients and the OD at 254 nm was recorded. The OD254 is shown as a function of gradient depth. The levels of ZEB1 and GAPDH mRNA in the whole cell lysates and in the polysomal fractions were determined by qRT-PCR. The relative amounts of ZEB1 mRNA in the polysomal fractions were compared to the relative amounts of ZEB1 mRNA in the whole cell lysates. The results are representative of three independent experiments.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Transfection, Control, Expressing, Western Blot, Staining, Quantitative RT-PCR, Lysis

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, CD18-CR cells were transfected with control siRNA (siCtrl) or eIF4E-specific siRNA (sieIF4E) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for eIF4E, ZEB1, E-cadherin and vimentin mRNA expression by qRT-PCR and the lysates were analyzed for eIF4E, ZEB1, E-cadherin and vimentin protein expression by Western blotting. CD18-CR cells growing on glass coverslips were transfected with siCtrl, sieIF4E or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 72 hours. The cells were then stained for actin using phalloidin and DAPI to counterstain the nuclei. B, CD18-CR cells were transfected with siCtrl or sieIF4E for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for miR-200b, miR-200a, miR200c and miR-141 microRNAs and normalized using RNU48 as internal control. C, CD18-CR cells were transfected with siCtrl or siMNK1/2 for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were then processed for miR-200b, miR-200a, miR200c and miR-141 microRNAs and normalized using RNU48 as internal control. D, CD18-CR cells were transfected with control siRNA (siCtrl) or hnRNPA1-specific siRNA (sihnRNPA1) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for hnRNPA1, ZEB1, E-cadherin and vimentin mRNA expression by qRT-PCR and the lysates were analyzed for hnRNPA1, ZEB1, E-cadherin and vimentin protein expression by Western blotting. The results are representative of three independent experiments. See also Supplemental Figures S2–5.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, CD18-CR cells were grown in three-dimensional type I collagen (2mg/ml) and fresh serum-containing medium supplemented with DMSO or CGP57380 (2.5 μM) was added every other day for 5 days. The effect on colony size in three-dimensional type I collagen was examined by phase-contrast microscopy and size of the individual colonies was measured. **, p<0.01. B, CD18-CR cells were transfected with control siRNA (siCtrl) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 72 hours. The effect on colony size in three-dimensional collagen was examined by phase-contrast microscopy and size of the individual colonies was measured. *, p<0.05. C, CD18 and CD18-CR cells were processed for Aldefluor activity in the presence or absence of aldehyde dehydrogenase inhibitor DEAB by FACS analysis. E, CD18-CR cells were treated with DMSO or CGP57380 (2.5 μM) for 48 hours and then processed for Aldefluor activity by FACS analysis. The results are representative of three independent experiments.

Article Snippet: {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}} CGP57380 was obtained from Santa Cruz. siRNAs against MNK1 and MNK2 were purchased from Dharmacon, ZEB1 siRNA was obtained from Life Technologies, while eIF4E and hnRNPA1 siRNAs were from Santa Cruz.

Techniques: Microscopy, Transfection, Control, Activity Assay

Journal: Neuropharmacology

Article Title: Ciliary neurotrophic factor signaling in the rat orbitofrontal cortex ameliorates stress-induced deficits in reversal learning.

doi: 10.1016/j.neuropharm.2019.107791

Figure Lengend Snippet: Fig. 4. CNTF elicits a p38-dependent protein translation pathway. Rats were either left undisturbed in their home cages (No Stress) or were subjected to CIC stress (CIC), and injected with saline (VEH) or 50 nM CNTF (CNTF) 30 min before collection of OFC tissue. Male rats: CNTF increased pJAK2, pT-Akt, pS9-GSK3β and peIF4E in both non-stressed and stressed animals; CNTF increased pS6K only in non-stressed rats (A, B, C, D, E, *p < 0.05 and **p < 0.01 vs. respective vehicles, n = 4–6). Female rats: CNTF increased pJAK2, pT-Akt, pS9-GSK3β and peIF4E in both non-stressed and stressed animals; CNTF increased pS6K only in non-stressed rats (G, H, I, J, K, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. respective vehicles, n = 4–6). Panels F and L show images of representative western blots. CNTF induces p38 (M) and Mnk1 (P) and this effect is not sensitive to the PI3K inhibitor LY294002 (*p < 0.05, and ***p < 0.001 vs. VEH, n = 4–5). CNTF increases p- p38 and pMnk1 in both non-stressed and stressed male (N, Q) and female rats (O, R, *p < 0.05, **p < 0.01 vs. respective vehicles, n = 4–6). S. Proposed model of CNTF actions. CNTF (50 nM) binding to gp130/LIFR/CNTFRα activates JAK2, which in turns activates PI3K/Akt. Akt phosphorylates and inhibits GSK3β. Akt also inhibits TSC, an inhibitor of mTOR, thereby activating this pathway. mTOR phosphorylates S6K, a regulator of global protein synthesis. This branch is induced by CNTF in non-stressed rats, but it does not seem to be activated in stressed rats. In parallel to regulation of Akt, CNTF also induces the p38/Mnk1/eIF4E cascade that controls specialized mRNA translation. Finally, at 50 nM, CNTF does not seem to activate ERK or STAT3 in the OFC (dotted arrows).

Article Snippet: Subsequently, membranes were stripped with Restore Plus (Fisher Scientific) and re-probed with antibodies against un-phosphorylated STAT3 (mouse mAb 1:2,000, Santa Cruz Biotechnology, sc-8019), JAK2 (rabbit mAb, 1:2,000, CST 3230), Akt (mouse mAb, 1:1,000, CST 2920), GSK3β (mouse mAb, 1:1,000, CST 9832), S6K (rabbit pAb, 1:1,000, CST 9202), eIF4E (rabbit pAb, 1:1,000, CST 9742), ERK (rabbit pAb, 1:5,000, Santa Cruz Biotechnology, sc-94), p38 MAPK (rabbit mAb, 1:2,000, CST 8690), and Mnk1 (mouse mAb, 1:1,000, Novus Biologicals NBP211526), or GAPDH (CST; 1:10,000).

Techniques: Injection, Saline, Western Blot, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

doi: 10.1158/1541-7786.MCR-17-0397

Figure Lengend Snippet: MNK1 is required for ATO-induced eIF4E phosphorylation in GBM and arsenic binds to MNK1 and activates kinase activity. A and B, U87 (A) and LN229 (B) cells were transfected with control siRNA or siRNA targeting MNK1, MNK2, or combination. Cells were treated with or without ATO (2 μmol/L) for 90 minutes. Whole cell lysates were subjected to immunoblotting with antibodies against MNK1, phospho-eIF4E (Ser209), or eIF4E. C and D, Cells from A and B were collected and RNA was extracted. Expression of MKNK1 and MKNK2 genes was determined by qRT-PCR normalizing to GAPDH. Data represent means ± SEM of two independent experiments. E, LN229 (MNK1 expressing) or LN18 (MNK1 undetectable) cells were treated with or without ATO (5 μmol/L) for 90 minutes. Whole cell lysates were subjected to SDS-PAGE followed by transfer to PVDF membranes. Membranes were incubated with antibodies against phospho-eIF4E (Ser209), eIF4E, or MNK1. F, LN229, U87, or recombinant MNK1-GST protein were treated with DMSO or the biotinylated arsenic compound, As-Biotin (AsB), for 2 hours. Whole cell lysates or recombinant protein were incubated with streptavidin beads overnight. Streptavidin pull-down and input control were subjected to immunoblotting with an antibody against MNK1. G, Recombinant MNK1-GST protein was treated with increasing concentrations of ATO for 30 minutes in the presence of the MNK1 peptide substrate, EIF2S. Kinase activity was assessed using the ADP-Glo Kinase Assay. Data represent means ± SEM of three independent experiments. Unpaired, two-tailed t test, **, P ≤ 0.01.

Article Snippet: In vitro kinase assay Recombinant, active MNK1 protein (SignalChem) was used for the in vitro kinase assay.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, SDS Page, Incubation, Recombinant, Kinase Assay, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

doi: 10.1158/1541-7786.MCR-17-0397

Figure Lengend Snippet: Expression of MES GSC markers correlate with MKNK1 and MKNK1 expression predicts poor survival in GBM patients. A and B, Multiple correlation analysis of MES and PN GSC markers in TCGA patients. Pearson’s correlation analysis,****, P ≤ 0.0001. C, Heatmap showing expression of MKNK1, MES GSC markers (ALDH1A3, CD44) and PN GSC markers (OLIG2, SOX2) in different tumor regions: microvascular proliferation (MP), infiltrating tumor (IT), leading edge (LE), and cellular tumor (CT). Bar graphs depict summarized data from heatmap. Comparisons between MKNK1 and SOX2 means in different tumor regions is shown with Tukey’s honest significant difference test,*, P ≤ 0.05; **, P ≤ 0.01;***, P ≤ 0.001. D, Expression of MKNK1 in GBM and low-grade gliomas: oligodendroglioma (OG), oligoastrocytoma (OA), and astrocytoma (AS). One-way ANOVA,*, P ≤ 0.05;****, ≤ 0.0001. E and F, Overall survival (OS) analysis of low-grade glioma (E), all GBM (F), and MGMT promoter unmethylated; non-G-CIMP GBM (G) patients with high and low expression of MKNK1 (MNK1). Log-rank (Mantel-Cox) test P-values are shown. Expression data downloaded from GlioVis (http://gliovis.bioinfo.cnio.es; ref. 57). Heatmap generated using the IVY Glioblastoma Project. ©2015 Allen Institute for Brain Science. Ivy Glioblastoma Atlas Project. Available from: glioblastoma.alleninstitute.org.

Article Snippet: In vitro kinase assay Recombinant, active MNK1 protein (SignalChem) was used for the in vitro kinase assay.

Techniques: Expressing, Generated

Journal: Molecular Therapy. Nucleic Acids

Article Title: Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells

doi: 10.1038/mtna.2015.50

Figure Lengend Snippet: MNK1b aptamers recognize a region between amino acids common to both isoforms and the specific MNK1b region, MNK1bSR . ( a ) Analysis of the binding of aptamers with MNK1b was performed by ELONA. GST-MNK1b was plated at 0.2 µg/well (3 pmol/well) and then incubated with Bio-apMNK2F and Bio-apMNK3R at 0–40 nmol/l concentrations. The bars represent the mean ± SEM of 4–6 independent experiments. ( b ) Proteins MNK1a, MNK1b, MNK1aΔ77, and MNK1ΔCt were plated at 3 pmol/well and then incubated with aptamers Bio-apMNK2F, Bio-apMNK3R, and Bio-38x(AG) at 20 nmol/l. Bars represent the mean ± SEM of 4–6 independent experiments (** P < 0.01 compared to apMNK2F; a P < 0.001 compared to blank). ( c ) A scheme displaying MNK1a, MNK1b, MNK1aΔ77, and MNK1ΔCt possible sites of interaction with the aptamers and the extent of binding for aptamers to the different forms of the kinases.

Article Snippet: In vitro MNK1a activity was assayed as follows: 0.25 μg recombinant activated MNK1a (GST-MNK1a T385D) (BPS Bioscience, EEUU) was preincubated with 0.2 μmol/l of each aptamers (6 pmol/tube) or the same concentration of the MNK1 inhibitor (CGP57380) for 5 minutes at 30 °C, and the kinase reaction was performed using as substrate 200 μmol/l of the peptide substrate (RRRLSSLRA) in 20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl 2 , 100 μmol/l ATP, and 1 μCi [γ - 32 P] ATP (Hartmann Analytic, Germany) for 30 minutes at 30 °C.

Techniques: Binding Assay, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells

doi: 10.1038/mtna.2015.50

Figure Lengend Snippet: Aptamers detect MNK1a/b in cells . Aptacytochemistry in MDA-MB-231 cells stably overexpressing ( a ) MNK1a, ( b ) MNK1b, or ( c ) transiently overexpressing MNK1b. Experiments were performed using Alexa-488-conjugated aptamers apMNK2F and apMNK3R (2 pmol/coverslip) and ( a ) MNK1 (C-20), ( b ) MNK1 (M-20), or ( c ) anti-Flag antibodies as described in the Materials and Methods section. Confocal microscopy images corresponding to the staining of nuclei with Hoechst (blue), antibodies (red), and aptamers (green). The overlay images (merge) are also shown. Bar = 20 µm.

Article Snippet: In vitro MNK1a activity was assayed as follows: 0.25 μg recombinant activated MNK1a (GST-MNK1a T385D) (BPS Bioscience, EEUU) was preincubated with 0.2 μmol/l of each aptamers (6 pmol/tube) or the same concentration of the MNK1 inhibitor (CGP57380) for 5 minutes at 30 °C, and the kinase reaction was performed using as substrate 200 μmol/l of the peptide substrate (RRRLSSLRA) in 20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl 2 , 100 μmol/l ATP, and 1 μCi [γ - 32 P] ATP (Hartmann Analytic, Germany) for 30 minutes at 30 °C.

Techniques: Stable Transfection, Confocal Microscopy, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells

doi: 10.1038/mtna.2015.50

Figure Lengend Snippet: Effects of aptamers on kinase activity and translation in vitro . ( a ) The kinase activity assay was performed using the peptide RRRLSSLRA as a substrate and the recombinant activated MNK1a (GST-MNK1a T385D) as a kinase in the absence (control) or the presence of 6 pmol for each aptamer. In parallel, the same concentration of CGP57380 was used as a positive control. Percentages were calculated relative to the control. Bars represent the mean ± SEM of 2–4 independent experiments (* P < 0.05 compared to the control). ( b ) In vitro translation assay performed with a Rabbit Reticulocyte Lysate System in the absence (control) or presence of 4 µmol/l aptamers. Luciferase activity was measured after different incubation times. The data are expressed as arbitrary units (A.U.) and represent the mean ± SEM of 2–4 independent experiments.

Article Snippet: In vitro MNK1a activity was assayed as follows: 0.25 μg recombinant activated MNK1a (GST-MNK1a T385D) (BPS Bioscience, EEUU) was preincubated with 0.2 μmol/l of each aptamers (6 pmol/tube) or the same concentration of the MNK1 inhibitor (CGP57380) for 5 minutes at 30 °C, and the kinase reaction was performed using as substrate 200 μmol/l of the peptide substrate (RRRLSSLRA) in 20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl 2 , 100 μmol/l ATP, and 1 μCi [γ - 32 P] ATP (Hartmann Analytic, Germany) for 30 minutes at 30 °C.

Techniques: Activity Assay, In Vitro, Kinase Assay, Recombinant, Concentration Assay, Positive Control, Luciferase, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells

doi: 10.1038/mtna.2015.50

Figure Lengend Snippet: MNK1b aptamers inhibit proliferation, migration, and colony formation in vitro . ( a ) MDA-MB-231 cells were plated at a density of 6 × 10 3 cells/well in p96 plates. After 16–24 hours, aptamers were transfected at 5–50 nmol/l concentrations for 72 hours, and MTT activity assays were performed. The graph represents the mean ± SEM of 2–3 independent experiments. (* P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control; a P < 0.001, b P < 0.01, c P < 0.05 compared to 38x(AG)). ( b ) Effects of aptamers on MDA-MB-231 cell migration with a wound-healing assay for 24 hours. Transfected cells were allowed to form a confluent monolayer and were treated with mitomycin C. Representative photomicrographs of initial and final wounds are shown. Bar = 1,000 µm (upper and middle panels). The lower panel shows a higher magnification (bar = 200 µm) of the wounds after 24 hours. Cells transfected in the absence of aptamers were used as a control. ( c ) Effect of the aptamers on colony formation. Transfected MDA-MB-231 cells were seed at 1 × 10 3 cells/well in six-well plates. After 8–9 days, the colonies were fixed, stained, and counted. Bars represent the mean ± SEM of four independent experiments. * P < 0.05. Cells transfected in the absence of aptamers were used as a control. ( d ) MDA-MB-231 cells stably transfected with Myc-pcDNA3 (control), Myc-MNK1a (MNK1a), and Myc-MNK1b (MNK1b) were plated at a density of 6 × 10 3 cells/well in p96 plates. After 16–24 hours, aptamers were transfected at 12.5 nmol/l concentration for 72 hours and MTT activity assays were performed. The graph represents the mean ± SEM of four independent experiments. b P < 0.01, c P < 0.05 compared to the control.

Article Snippet: In vitro MNK1a activity was assayed as follows: 0.25 μg recombinant activated MNK1a (GST-MNK1a T385D) (BPS Bioscience, EEUU) was preincubated with 0.2 μmol/l of each aptamers (6 pmol/tube) or the same concentration of the MNK1 inhibitor (CGP57380) for 5 minutes at 30 °C, and the kinase reaction was performed using as substrate 200 μmol/l of the peptide substrate (RRRLSSLRA) in 20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl 2 , 100 μmol/l ATP, and 1 μCi [γ - 32 P] ATP (Hartmann Analytic, Germany) for 30 minutes at 30 °C.

Techniques: Migration, In Vitro, Transfection, Activity Assay, Wound Healing Assay, Staining, Stable Transfection, Concentration Assay