1. BAY , a novel MNK1 inhibitor, targets oncogenic protein expression and shows potent anti-tumor activity 
Susann Santag, Franziska Siegel, Antje M. Wengner, Claudia Lange, Ulf Bömer, Knut Eis, Florian Pühler, Philip Lienau, Linda Bergemann, Martin Michels, Franz von Nussbaum, Dominik Mumberg, Kirstin Petersen 
Cancer Letters 
Volume 390, Pages (April 2017)
DOI: /j.canlet
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2. Fig. 1
BAY mediated effects on eIF4E phosphorylation. Western blot analysis of total and phosphorylated eIF4E in A549 and NCI-H460 cells treated with BAY  at the indicated concentrations or with vehicle control (DMSO) for 2 h.
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3. Fig. 2
BAY mediated regulation of cell cycle and cell cycle-associated proteins. (A–C) NSCLC cell lines (A, B) and the AML cell line MOLM-13 (C) treated with BAY (10 μM) for 24 h were analyzed by Western blot for the indicated proteins. Cyclin A2 was not detected in MOLM-13 cells. (D) NSCLC cell lines A549 and NCI-H460 and the AML cell line MOLM-13 treated with BAY (10 μM) for 24 h were analyzed by flow cytometry-based propidium iodide staining. Graphs are representatives of 3 independent experiments. Statistical significances between BAY and DMSO in each cell line are presented in the Supplementary Fig. S2A (n = 3).
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4. Fig. 3
BAY mediated effects on apoptosis. (A–B) Cells treated with BAY (10 μM) or vehicle control (DMSO) for 1–2 h or 24 h were analyzed by Western blot for the indicated proteins. (C) MOLM-13 AML cells treated with BAY (10 μM) for 24 h were analyzed by flow cytometry-based annexin/propidium iodide staining. The ratio of Annexin positive cells in % is shown (mean +/− standard deviation). Statistical significance was calculated by two-tailed Student's t-test. (D) A549 cells treated with BAY (10 μM) or DMSO and indicated concentrations of docetaxel for 24 h were analyzed by Western blot for the indicated proteins.
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5. Fig. 4
BAY mediated reduction of cell migration and expression of proteins involved in EMT. (A) A549, NCI-H460 and NCI-H2030 cells treated with BAY (10 μM) or vehicle control (DMSO) for 24 h were analyzed by Western blot for the indicated proteins. (B) A549 cells pre-treated with TGF-β1 (5 ng/ml) for 10 h were treated with BAY  (10 μM) or vehicle control (DMSO) for 24 h before cells were analyzed by Western blot for the indicated proteins. (C) Scratch wound healing assay was performed using NCI-H2030 cells treated with BAY (10 μM) or vehicle control (DMSO). Pictures of scratch wound closure were captured at the indicated time points. Edges of scratches were highlighted manually afterwards only to illustrate cell migration. Representative pictures of 3 independent wells are shown. (D and E) Unmodified pictures were analyzed by ImageJ and percentage change of wound area (i.e. wound closure) after the incubation time was calculated for 12 independent wells in parallel. Experiment shown here is a representative of 3 independent experiments and graphs represent the mean with standard deviation. Statistical significance was calculated by two-tailed Student's t-test.
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6. Fig. 5
BAY mediated effects on the release of pro-inflammatory cytokines in human whole blood. Human whole blood diluted in medium was incubated with BAY  in the indicated concentrations or with vehicle control (DMSO). After 30 min preincubation, E. coli LPS (5 ng/ml) was added and samples were incubated for 24 h followed by harvesting. (A) Release of pro-inflammatory cytokines was analyzed by cytokine assay from the supernatant. Graphs are representatives of 3 independent experiments and show the mean with standard deviation. (B) Pelleted cells were analyzed by Western blot for the indicated proteins. No stimulus: control samples without LPS and with DMSO treatment.
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7. Fig. 6
Anti-tumor activity of BAY in lung cancer xenograft models. (A) Response of NCI-H460 xenograft in nude rats (10 animals/group) to treatment with BAY monotherapy. Rats were treated orally with BAY or vehicle control. (B) Response of A549 xenograft in nude mice (12 animals/group) to treatment with BAY in monotherapy and in combination therapy with docetaxel. Mice were treated with the indicated doses BAY p.o., docetaxel (15/10 mg/kg once weekly i.v.; dose reduction from 15 to 10 mg/kg on day 32) or vehicle control. For combination therapy, the indicated doses of BAY were given in combination with the fixed dose of docetaxel applied in monotherapy. (C) Ex vivo mode of action analysis of A549 tumors harvested from mice 1 h after the final monotherapy treatment with vehicle or the indicated dose BAY Tumor samples were lysed and analyzed by Western blot for the indicated proteins. (D) Quantification of p-eIF4E and CDC25C protein levels from (C), normalized to total protein or loading control, is represented as a percentage of the vehicle group. (E) Ex vivo mode of action analysis of mouse whole blood from an independent A549 efficacy study performed under comparable conditions as the previous A549 model. Samples were harvested after final monotherapy treatment with vehicle or BAY (200 mg/kg p.o.). Samples were stimulated with E. coli LPS for 24 h before plasma samples were analyzed for the release of the indicated cytokines by sandwich immunoassays. (F) Response of patient-derived NSCLC xenograft model Lu7462 in nude mice (12 animals/group) to treatment with BAY in monotherapy or combination therapy with pemetrexed. Mice were treated with the indicated doses of BAY p.o. or pemetrexed (50 mg/kg QDx7 i.p.). For all in vivo studies, tumor growth is depicted by tumor volume (mean +/− SEM) and statistical significance was calculated by two-way analysis of variation, applying Bonferroni post-testing for multiple comparisons. For ex vivo cytokine release assays, statistical significance was calculated by two-tailed Student's t-test with Welsh correction because of unequal variances. p.o.; per oral application; i.v., intravenous application; QD, once daily; BID, twice daily; i.p., intraperitoneal application.
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8. Supplementary Fig. S3
Extended mode of action analysis of BAY (A) Analysis of mRNA expression of BAY regulated downstream targets in A549 lung cancer cells. Cells treated for 24 h with BAY (10 μM) or vehicle control (DMSO) were harvested, and mRNA expression was analyzed by qPCR for the indicated probes. Group differences were analyzed by Student's t-test (CCNB1) or by Student's t-test with Welsh correction, because of unequal variances (MYC). (B) Analysis of block of proteasomal degradation on BAY mediated effects. COLO 205 cells treated for 24 h with BAY (10 μM), proteasomal inhibitor MG-132 (2 μM, Sigma Aldrich) or vehicle control (DMSO) were analyzed by Western blot for the indicated proteins. n.s., non-significant.
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9. Supplementary Fig. S4
Expression of EMT markers in NSCLC cell lines. A549, NCI-H460, NCI-H2030 and were analyzed by Western blot for the indicated proteins. While expression of E-cadherin is associated with the epithelial type, expression of Vimentin, N-cadherin and ZEB-1 are associated with the mesenchymal type.
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10. Supplementary Fig. S5
Analysis of body weight change in NSCLC xenograft models (A) NCI-H460, (B) A549 and (C) Lu7462. Body weight was measured 2–3 times/week for all animals until studies were terminated. Body weight change illustrated as mean of percent change from initial body weight at the time of study initiation.
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