Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 and NF- κ B signalling pathways are deregulated in human CC, with ERK5 expression correlating with increased NF- κ B activation. ( a ) Representative immunoblots of steady-state protein expression levels of MEK5, ERK5, NF- κ B, I κ B and β -actin in normal colon, colon adenomas and pMMR and dMMR colon carcinomas; ( b ) MEK5 and ERK5 steady-state protein levels; ( c ) NF- κ B, I κ B and NF- κ B/I κ B ratio; ( d ) correlations between ERK5 steady-state protein levels and NF- κ B, I κ B or NF- κ B/I κ B ratio; ( e ) representative immunoblots of steady-state levels of p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in normal colon, colon adenoma, and pMMR and dMMR colon carcinomas; and ( f ) representative immunohistochemistry for ERK5, p-NF- κ B and NF- κ B in human colon cancer samples. Immunoblot statistical significance was determined using the non-parametric statistical analysis Kruskal–Wallis test with Dunn's post test for selected comparisons and results are expressed as mean±S.E.M. for samples in each category; correlation statistical significance was determined using the non-parametric stastistical analysis Spearman test. pMMR, proficient mismatch repair system; dMMR, deficient mismatch repair system. Scale bar=100 μ m. * P <0.05, † P <0.01 and ‡ P <0.001 from normal colon

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: Correlation between MEK5, ERK5, NF- κ B and I κ B steady-state protein expression, NF- κ B activation and clinicopathological characteristics

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerates cell cycle progression in SW620 cells. ( a ) Representative immunoblot of ERK5 protein levels in SW620 cell lines with differential ERK5 activation, stable constitutive MEK5 activation ( CA-MEK5 ), dominant-negative MEK5 ( DN-MEK5 ), empty control ( Empty ) and wild-type SW620 cell line (WT). The developed cell model consistently showed that DN-MEK5 led to constitutive inhibition of ERK5 activation, CA-MEK5 led to constitutive ERK5 activation and Empty control cells displayed basal ERK5 activation. ( b ) FACS cell cycle analysis of CA-MEK5, DN-MEK5 and Empty SW620 cells, following release from dual-thymidine block and exposed to ( c ) XMD8-92 or ( d ) BAY11-7085. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot, Dominant Negative Mutation, Control, Inhibition, Cell Cycle Assay, Blocking Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerated cell cycle is associated with NF- κ B activation via reduction of I κ B steady-state levels. Immunoblot analysis of steady-state protein levels of ( a ) p-ERK5, ERK5 and p-ERK5/ERK5, and of ( b ) p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. ( c ) Representative immunoblots for p-ERK5, ERK5, p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in Empty, DN-MEK5 and CA-MEK5 SW620 cells when treated for 24 h with XMD8-92 and BAY11-7085, or untreated (no addition). Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05, † P <0.01 and ‡ P <0.001 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases cell migration in vitro . Cell migration was assessed by ( a ) wound-healing assay at 24 and 48 h after ‘wound' formation and ( b ) transwell migration assay, with cells allowed to migrate for 9 h after cell platting. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, In Vitro, Wound Healing Assay, Transwell Migration Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increased cell migration is associated with increased vimentin expression and NF- κ B activation. Immunoblot analysis of steady-state protein levels of ( a ) vimentin and ( b ) of p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. of at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases NF- κ B nuclear translocation and transcriptional activity via I κ B phosphorylation and degradation. Immunoblot analysis of steady-state protein levels of (a) p-I κ B, I κ B and p-I κ B/I κ B ratios, and ( b ) nuclear and cytosolic NF- κ B; ( c ) NF- κ B transcriptional activity, evaluated by dual luciferase assay with reporter plasmids. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P< 0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Translocation Assay, Activity Assay, Phospho-proteomics, Western Blot, Luciferase

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: In vivo , MEK5/ERK5 activation is associated with local invasion and regional lymphnode metastasis. As shown in the graphical abstract, ( a ) the injected tumours may grow locally in the caecum/colon infiltrating mucosa, sub-mucosa, muscularis propria and eventually sub-serosa. In addition, tumour growth may be focal (restricted to one foci, at the injection site) or multifocal (numerous foci spread throughout the caecum and colon, not restricted to the injection site). Furthermore, in addition to local invasion by tumour cells, the metastatic cascade encompasses intravasation into lymph vessels, extravasation out of the circulation, and survival and growth at secondary site. We injected 5 × 10 5 SW620 DN-MEK5 or CA-MEK5 cells into the wall of the caecum, in BALB/c scid mice, and mice were killed 30 or 60 days post injection. ( a ) Histopathological characteristics of DN-MEK5 and CA-MEK5 tumours regarding local invasion, extravasation and distant metastasis (to regional lymphnodes), and ( b) representative microphotographs of the multifocallity of CA-MEK5 tumours, compared with the focal lesions seen in DN-MEK5 tumours (white arrows, upper panel), of the lymph vessel invasion (black arrowhead, middle panel) and of the lymphnode metastasis (lower panel). *Tumour cells

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: In Vivo, Activation Assay, Injection

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: ( A ) Percentage of proliferation inhibition of A549, A427 and H460 cell lines treated with increasing doses of XMD8-92 or Seliciclib or in combination (top) 48 h after treatment and representative crystal violet-stained cells 72 h after drug treatment (bottom). The combination index (CI) showing the synergistic effect of combination of the 2 drugs is indicated; n = 3. ( B – D ) Relative quantification of Annexin V (AV) + Annexin V/PI (AV/PI)-positive cells by flow cytometry; n = 4 ( B ), colony forming capacity; n = 3 ( C ) and percentage of cell migration; n = 10 ( D ) of A549 cells treated with 10 µM XMD8-92 or Seliciclib or in combination, except for the colony formation assay in which cells were treated with 5 µM of each drug. ( E ) Immunoblot analysis of the indicated targets in A549 cells treated with the indicated doses of Seliciclib or XMD8-92 or in combination for 24 h. ( F ) Immunoblot analysis of the indicated targets in A549 cells treated with 10 µM XMD8-92 or 10 µM Seliciclib or in combination for 24 h. ( G ) Immunoblot analysis for the indicated targets in A549 cells transduced with shRNA control (pLKO.1 hygro + Tet-pLKO-puro) or a shRNA against CDK5 (Tet-pLKO-puro-shCDK5 + pLKO.1 hygro), or a shRNA against ERK5 (pLKO.1 hygro-shERK5 + Tet-pLKO-puro) or in combination (Tet-pLKO-puro-shCDK5 + pLKO.1 hygro-shERK5). After transduction and selection, cells were harvested for protein extraction 72 h after doxycycline (1 μg/mL) induction. ( H , I ) Relative cell number ( H ) and Annexin V (AV) + Annexin V/PI (AV/PI)-positive cell quantification by flow cytometry ( I ) in A549 cells treated as in ( G ) 72 h after doxycycline (1 μg/mL) induction; n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples. .

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Inhibition, Staining, Flow Cytometry, Migration, Colony Assay, Western Blot, Transduction, shRNA, Control, Selection, Protein Extraction

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: Relative quantification of cell death by flow cytometry analysis of Annexin V-Atto 633 (AV) + Annexin V/PI (AV/PI)-positive (left) and colony number (right) of A427 cells treated with XMD8-92 or Seliciclib (10 µM for apoptosis assay and 2.5 µM for colony formation each, respectively) alone or in combination; n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples.

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Flow Cytometry, Apoptosis Assay

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: ( A ) Representative scheme of the in vivo experiment workflow. ( B ) Representative hematoxylin & eosin (H&E) staining (left) and quantification of the average tumor size (right) of lung tissue from LSL-Kras G12D/WT ;p53 flox/flox mice 10 weeks after Cre induction and after 2 weeks of treatment with vehicle, XMD8-92 (50 mg/Kg), Seliciclib (50 mg/Kg) or combination. Scale bar: 1 mm; n mice/group: 6, 3, 4, 4. Graphical data are ± SEM. ( C ) Representative images of immunohistochemistry against Ki67 (left) and quantification of Ki67-positive cells (right) in lung tissue from LSL-Kras G12D/WT ;p53 flox/flox mice, treated as in ( B ). Scale bar: 100 μm; n mice/group: 3, 3, 4, 4. Graphical data are mean ± SD. ( D ) Tunel-positive cell quantification in lung tissue from LSL-Kras G12D/WT ;p53 flox/flox mice, treated as in ( B ); n mice/group: 4, 3, 4, 4. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples. .

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: In Vivo, Staining, Immunohistochemistry, TUNEL Assay

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: Body weight of Kras G12D/WT ;p53 flox/flox mice treated with vehicle or XMD8-92 or Seliciclib or combination of XMD8-92 and Seliciclib for 2 weeks.

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: ( A ) Hierarchical clustering (Pearson Correlation, average linkage) of genes with standard deviation at top 5% showing a clear separation between A549 cells treated with DMSO (Control) or XMD8-92 or Seliciclib or combination of the two drugs (10 µM each) for 12 h; n = 3. ( B ) Dotplot showing the results of KEGG pathway enrichment analysis of genes that are activated or suppressed in A549 treated with XMD8-92 and Seliciclib in combination (10 µM). ( C ) Immunoblot analysis of the indicated targets in A549 cells treated for 12 h with XMD8-92 and Seliciclib (10 µM for each drug) alone or in combination. ( D ) Quantification of DHR (ROS marker, green) (left) and representative flow cytometry histogram (right) of A549 cells treated as in ( C ); n = 3. ( E ) Quantification of DHR (ROS marker, green) in A549 cells treated with the combination of XMD8-92 and Seliciclib (10 µM) or VS-4718 (5 µM) in the presence or absence of the SOD mimetic, MnTMPyp (25 µM); n = 3. ( F ) Immunoblot analysis of the indicated targets in A549 cell line treated as in ( E ) except VS-4718: 2.5 µM. ( G ) Relative cell number (top) and representative crystal violet images of A549 cell line treated as in ( F ) for 96 h; n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples. .

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Standard Deviation, Control, Western Blot, Marker, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: ( A ) Quantification of DHR (ROS marker, green) in A549 cells treated with PF-562271 (10 µM) in the presence or absence of the SOD mimetic, MnTMPyp (25 µM); n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples. ( B ) Immunoblot analysis for the indicated targets in A549 cells line, treated with DMSO (control) or with a combination of XMD8-92 and Seliciclib (10 µM) or PF-562271 (5 µM) in the presence or absence of the SOD mimetic, MnTMPyp (25 µM). ( C ) Metascape-derived analysis of the functional categories associated to the 5 clusters from main Fig. . Enriched terms were filtered based on the enrichment score and accumulative hypergeometric P values ( P < 0.05). Remaining significant terms were then hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. Then 0.3 kappa score was applied as the threshold to cast the tree into term clusters.

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Marker, Western Blot, Control, Derivative Assay, Functional Assay

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: ( A ) Representative bright-field microscopy images of parental vehicle-treated A549 cells (left), VS-4718 tolerant cells (VS4718-T, middle) and VS4718-T upon withdrawal of the drug for 48 h (right). To obtain the VS-4718 tolerant (VS4718-T) cells, parental cells were treated with increasing doses of VS-4718 for 4 weeks and were after that maintained in 2.5 μM of VS-4718. Scale bars: 100 μm. ( B ) Percentage of proliferation inhibition of parental and VS4718-T A549 cells treated with increasing doses of VS-4718. Cell proliferation was determined 72 h post-treatment; n = 3. ( C ) Hierarchical clustering (Pearson Correlation, average linkage) of genes with standard deviation at top 5% showing a clear separation between A549 cells treated with VS-4718 (2.5 µM) for 12 h (acute) or rendered VS-4718 tolerant as described in ( A ); n = 3. ( D ) Analysis of the TRRUST module of Metascape showing that the genes of cluster 5 are identified as transcription factor targets (colored, left) and STRING database analysis showing the possible interaction between the different transcription factors (right). ( E ) Heatmap showing the expression profile of epithelial (KRT8,18) and mesenchymal markers of A549 cells treated as in ( C ). ( F ) Immunoblot for the indicated targets in A549 cells treated as in ( A ). The VS4718-T cells were maintained with 2.5 μM VS-4718. ( G ) Immunoblot for the indicated targets in A549 cells treated as in ( A ) and maintained at 2.5 μM. ( H ) Real-time PCR showing relative mRNA levels of ERK5 in A549 cells treated as in ( C ); n = 3. ( I ) Immunoblot for the indicated targets in A549 parental, VS4718-T and VS4718-T treated with XMD8-92 (10 μM). Heatmaps in ( C , E ) display a relative color scheme across samples that uses the minimum and maximum values in each row to convert the values into a scale ranging from 0 to 1. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n , number of biologically independent samples. .

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Microscopy, Inhibition, Standard Deviation, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: Body weight of Kras G12D/WT ;p53 flox/flox mice treated with vehicle or VS-4718 (FAKi) or a combination of VS-4718 and XMD8-92 (FAKi + ERK5i) for 2 weeks.

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer

doi: 10.1038/s44321-024-00138-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: For the experiment in Fig. , 10 weeks after Cre induction, mice were intraperitoneally injected once daily with vehicle (10% DMSO, 40% PEG 300; 5% Tween 80 and 45% Saline), XMD8-92 (HY-14443, MedChemExpress) and/or Seliciclib (MedChemExpress, HY-30237) at a dosage of 50 mg/kg (in 100 μl volume) for a period of 2 weeks.

Techniques: Recombinant, Plasmid Preparation, Sequencing, shRNA, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, In Vitro, In Vivo, TUNEL Assay, Software

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: ERK5 mediates pro-tumorigenic phenotype in non-small lung cancer cells induced by PGE2.

doi: 10.1016/j.bbamcr.2024.119810

Figure Lengend Snippet: Fig. 1. PGE2 and EP1 stimulation activates ERK5 in NSCLC cells. (A–C). Basal expression of ERK5 (115 kDa) in A549 cells transfected with lentiviral vectors carrying control shRNA encoding for a scrambled sequence (SC) or ERK5-specific shRNA (KD A or B) and in PC9 cells after 48 h of growth in 10 % FBS (A). ERK5 activation (115 kDa) in A549 SC (B) and PC9 (C) cells exposed to EGF (25 ng/ml, 15 min) or PGE2 (0.1 and 1 μM for 15 min). β-actin (45 kDa) was used as loading control. Blots are representatives of three independent experiments. Hyperphosphorylated ERK5 upshifted band is indicated by an arrow. (D). ERK5 phosphorylation (115 kDa) levels in A549 SC cells exposed to EGF (25 ng/ml), PGE2 (1 μM) with/without XMD8-92 (5 μM, 30 min pretreatment), or PGE2 receptor agonists (1 μM) for 15 min. (E). Quantification of blots reported in (D). CTR condition has assigned 1. *p < 0.05 and **p < 0.01 vs CTR. β-actin (45 kDa) was used as loading control. Blots are representatives of three independent experiments. (F). ERK5 phosphorylation (115 kDa) levels in PC9 cells exposed to EGF (25 ng/ml), PGE2 (1 μM), EP1 receptor agonist (17-phenyl trinor Prostaglandin E2 ethyl amide) (1 μM) or EGF (25 ng/ml) for 15 min. (G). Quantification of blots reported in (F). CTR condition has assigned 1. *p < 0.05 vs CTR. β-actin was used as loading control. Blots are representatives of three independent experiments. (H). Phosphorylation levels of ERK5 (T219/Y221) (115 kDa), p90RSK (T379) (90 kDa), and SGK (S78) (54 kDa) in A549 SC cells exposed to PGE2 (1 μM) or EP1 receptor agonist (1 μM) for 15 min. (I). Phosphorylation levels of ERK5 (T219/Y221) (115 kDa) and SGK (S78) (54 kDa) in PC9 cells exposed to PGE2 (1 μM) or EP1 receptor agonist (1 μM) for 15 min. (J). KLF2 expression (42 kDa) levels in A549 SC and KD exposed to PGE2 (1 μM) and EP1 (1 μM) for 60 min. Molecular weight markers on the left of blots.

Article Snippet: ERK5 inhibitor XMD8-92 was from Santa Cruz (Heidelberg, Germany) and dissolved in DMSO (10 mM).

Techniques: Expressing, Transfection, Control, shRNA, Sequencing, Activation Assay, Phospho-proteomics, Molecular Weight

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: ERK5 mediates pro-tumorigenic phenotype in non-small lung cancer cells induced by PGE2.

doi: 10.1016/j.bbamcr.2024.119810

Figure Lengend Snippet: Fig. 2. PGE2 induces NSCLC proliferation and cell cycle progression through ERK5 activation. A549 (SC, ERK5KD A and B) cell proliferation after 24 (A) and 48 (B) hours of treatment with PGE2 (1 μM) or EGF (25 ng/ml) in 1 % FBS. **p < 0.01 vs untreated cells (CTR condition) and ## p < 0.01 vs A549 SC treated with PGE2 or EGF; §§ p < 0.01 vs A549 SC treated with 10 % FBS. (C, D). Proliferation of A549 exposed to EGF (25 ng/ml) or PGE2 (1 μM) with or without XMD8-92 (5 μM, 30 min of pre-treatment) for 24 (C) and 48 (D) hours. *p < 0.05 and **p < 0.01 vs untreated cells (CTR condition); #p < 0.05 and ## p < 0.01 vs A549 SC treated with PGE2 or EGF alone. (E, F). Proliferation of PC9 exposed to EGF (25 ng/ml) or PGE2 (1 μM) with or without XMD8-92 (0.5 μM, 30 min of pre-treatment) for 24 (E) and 48 (F) hours. **p < 0.01 and ***p < 0.001 vs untreated cells (CTR condition); #p < 0.05 and ## p < 0.01 vs A549 treated with PGE or EGF alone. The percentage of cells at each stage of the cell cycle was analyzed by flow cytometry after DNA staining with propidium iodide. Quantification of cells residing in S phase (G) and G0/G1 (H) of cell cycle for A549 SC exposed to XMD8-92 (5 μM, 30 min of pre-treatment), PGE2 (1 μM) or their combination for 24 h. *p < 0.05 vs untreated cells (CTR condition). # p < 0.05 vs A549 SC treated with PGE2. (I). Quantification of cells residing in different phases of cell cycle G0 for A549 ERK5 KD exposed to PGE2 (1 μM) for 24 h. (J). c-Myc gene expression in A549 cells (SC, KD A and B) treated with PGE2 (0.1 μM and 1 μM) for 24 h. ***p < 0.001 vs untreated cells (CTR condition). ### p < 0.001 vs A549 SC treated with PGE2. (K). c-Myc (57 kDa) protein expression in A549 cells (SC, KD A) treated with PGE2 and EP1 receptor agonist (0 1 μM) for 48 h. (L). Quantification of blot reported in (K). CTR condition has assigned 1. *p < 0.05 vs CTR. β-actin (45 kDa) was used as loading control. Blots are representatives of three independent experiments. Molecular weight markers on the left of blots.

Article Snippet: ERK5 inhibitor XMD8-92 was from Santa Cruz (Heidelberg, Germany) and dissolved in DMSO (10 mM).

Techniques: Activation Assay, Flow Cytometry, Staining, Gene Expression, Expressing, Control, Molecular Weight

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: ERK5 mediates pro-tumorigenic phenotype in non-small lung cancer cells induced by PGE2.

doi: 10.1016/j.bbamcr.2024.119810

Figure Lengend Snippet: Fig. 3. PGE2 promotes NSCLC cell migration and invasion by activating EP1 and ERK5 signaling. (A, B). Scratch closure after 18 h of PGE2 treatment (1 μM) in A549 (SC, ERK5 KD A and B) cells. #p < 0.05 vs untreated cells (CTR condition). Scale bar, 100 μm. (C, D). PC9 cells scratch closure exposed to PGE2 (1 μM) for 18 h (1 % FBS) with or without XMD8-92 (0.5 μM, 30 min of pre-treatment). *p < 0.05 vs untreated cells (Ctr condition). (E). Tumor cell invasion evaluated by Boyden chamber assay in A549 SC exposed to EP receptors agonists (1 μM) for 8 h (1 % FBS) with or without XMD8-92 (5 μM, 30 min of pre-treatment). *p < 0.05 and **p < 0.01 vs untreated cells (Ctr condition); #p < 0.05 and ##p < 0.01 vs A549 treated with PGE2 or EP receptor agonist alone. (F). Invasion of PC9 cells. Cells were exposed to EP receptors agonists (1 μM) for 8 h (1 % FBS) with or without XMD8-92 (0.5 μM, 30 min of pre-treatment). *p < 0.05 and **p < 0.01 vs untreated cells (CTR condition); #p < 0.05 and ##p < 0.01 vs A549 treated with PGE2 or EP receptor agonist alone.

Article Snippet: ERK5 inhibitor XMD8-92 was from Santa Cruz (Heidelberg, Germany) and dissolved in DMSO (10 mM).

Techniques: Migration, Boyden Chamber Assay

Journal: Cardiovascular research

Article Title: Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

doi: 10.1093/cvr/cvu236

Figure Lengend Snippet: Figure 1 Erk5 inhibits endothelial migration via KLF2. (A–C) Primary HUVECs were transfected with the indicated siRNAs and after infection with an empty vector or a retrovirus encoding constitutively active MEK5 (MEK5D) either subjected to wound healing assays (A and B) or processed for BCAR1 mRNA analysis by qRT-PCR (C). (A and B) Knockdown of KLF2, but not KLF4, restores wound healing capacity of MEK5D-transduced ECs. (A) Represen- tativemicroscopicimages of n ¼ 6 experimentstaken18 h afterassayinitiation (72 h after siRNA transfection). Lower panels showtransparentoverlays of raw images and corresponding processed images used for quantification as detailed in Supplementary material online, Information. Scale bar represents 200 mm. (B) Quantification of percental gap closure +SD for the indicated combinations (n ¼ 6). (C) Effect of KLF2 and/or KLF4 depletion on MEK5D-induced BCAR1 mRNA expression. Data are derived from 4–5 individual experiments and represent mean fold values of GAPDH-normalized mRNA expression+ SD in relation to the vector + si-scrambled (Scr) control (arbitrarily set to 1). (D) Representative single cell tracking results of n ¼ 3 infection experiments in HUVECs, illustrating the inhibitoryeffect of MEK5D expression on individual cell trajectories(lines). XY diagrams showcell move- ments in x–y direction (in mm) with reference to their start position (0) over a period of 16 h. Each dot marks the endpoint of one of n ¼ 30 individually trackedcells. In (Band C), statisticallysignificant changesincomparisonwiththerespectiveexperimental control(Scr + MEK5D) orbetweenthe indicated groups(solidlines)weredeterminedbyone-wayANOVAfollowedbyHolm–Sidakmultiplicitycorrection.Statisticaldifferencestothenormalizedvector/ scr control in (C) were evaluated by one-sample t-test followed by Bonferroni–Holm multiplicity correction. Significant multiplicity-adjusted P-values are indicated by asterisks.

Article Snippet: The selective Erk5 inhibitors XMD8-92 (SC-361408) and BIX-02188 (S1530) were purchased from Santa Cruz and Selleck Chemicals.

Techniques: Migration, Transfection, Infection, Plasmid Preparation, Quantitative RT-PCR, Knockdown, Expressing, Derivative Assay, Control, Single Cell Tracking

Journal: Cardiovascular research

Article Title: Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

doi: 10.1093/cvr/cvu236

Figure Lengend Snippet: Figure 2 MEK5D expression or statin treatment resulted in decreased PAK1 expression in ECs. (A and B) HUVECs or HUAECs were infected with the indicated retroviruses and samples were taken 72 h post-infection. (A) qRT-PCR experiments showing average fold mRNA expression+SD of the indi- catedPAKisoformsorKLF2andKLF4inrelationtotheirexpressioninvector-infectedcells(arbitrarilysetto1).Datawerecalculatedfromn ¼ 4(HUVEC) or n ¼ 3 (HUAEC) experiments upon normalization to GAPDH expression. (B) Immunoblots showing isoform-specific repression of PAK1 by MEK5D. EfficientMEK5D expression wasmonitoredbyimmunoblots for MEK5and Erk5.Dataarerepresentativeofn ¼ 3independent experiments. (C)qRT-PCR experiments demonstrating dose-dependent reduction of PAK1 mRNA in HUVECs upon 24 h treatment with the indicated simvastatin concentrations. Data are derived from n ¼ 4 experiments. To allow comparison to the dose-dependency of simvastatin-induced Erk5 phosphorylation, a representative Erk5 immunoblot is additionally shown. The slower migrating band represents phosphorylated Erk5 (pErk5). (D) Western blot confirming simvastatin- dependent PAK1 repression and Erk5 phosphorylation in HUAECs. Tubulin immunoblots in (B–D) served as a loading control. Statistically significant changes of PAK1 expression in comparison with the respective experimental control [empty vector in (A) or diluent control in (C)] were determined by one-sample t-test. Asterisks indicate statistical significances of calculated uncorrected (A) or Bonferroni–Holm multiplicity-adjusted (C) P-values.

Article Snippet: The selective Erk5 inhibitors XMD8-92 (SC-361408) and BIX-02188 (S1530) were purchased from Santa Cruz and Selleck Chemicals.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Derivative Assay, Comparison, Phospho-proteomics, Control, Plasmid Preparation

Journal: Cardiovascular research

Article Title: Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

doi: 10.1093/cvr/cvu236

Figure Lengend Snippet: Figure 3 MEK5D-dependentPAK1repressionis mediatedbyErk5andKLF2. (A)Westernblot showingnormalization ofMEK5D-inducedPAK1protein expression upon incubation with the indicated concentrations of the specific Erk5 inhibitor XMD8-92 for 72 h. A blot for Erk5 is shown to confirm efficiency of Erk5 inhibition. Tubulin served as the loading control. Data are representative for two independent experiments. (B and C) Reversion of MEK5D-dependent PAK1 mRNA and protein repression by KLF2 siRNA. HUVECs were transfected with the indicated siRNAs and upon infection with the indicated retroviruses analysed for PAK1 mRNA (B) or protein expression (C) by qRT-PCR or immunoblot, respectively. Data in (B) are derived from n ¼ 4–5 experiments and represent mean fold values of GAPDH-normalized PAK1 mRNA expression+SD in relation to the vector + Scr control (set to 1). Differences relative to the Scr + MEK5D control or between indicated samples were statistically evaluated by one-way ANOVA or one-sample t-test followed by Holm–Sidak/Holm–Bonferroni multiplicity correction, respectively. Statistically different multiplicity-adjusted P-values are indicated by asterisks. Blots in (C) are representative of n ¼ 3 independent experiments.

Article Snippet: The selective Erk5 inhibitors XMD8-92 (SC-361408) and BIX-02188 (S1530) were purchased from Santa Cruz and Selleck Chemicals.

Techniques: Expressing, Incubation, Inhibition, Control, Transfection, Infection, Quantitative RT-PCR, Western Blot, Derivative Assay, Plasmid Preparation

Journal: Cardiovascular research

Article Title: Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

doi: 10.1093/cvr/cvu236

Figure Lengend Snippet: Figure 4 Flow-induced PAK1 repression depends on KLF2, but not on KLF4. (A–C) LSS represses PAK1. HUVECs (A and B) or HUAECs (C) were left untreated or exposed to LSS (20 dyn/cm2) for 72 h or the indicated periods of time. Subsequently, cells were harvested and analysed for Erk5 phosphorylation and PAK1 protein expression by immunoblot (A and C, right) or mRNA expression of KLF2, KLF4, and PAK1 by qRT-PCR (B and C, left), respectively. In (A), a lysate from MEK5D-transduced ECs was included as a positive control for comparison. Immunoblots for tubulin served as a loading control. (D) qRT-PCR analysis demonstrating reversion of LSS-induced PAK1 mRNA repression upon transfection with KLF2 siRNA. Immunoblots in (A and C) are representative of n ¼ 3 similar experiments. mRNA data in (B–D) are derived from n ¼ 3 experiments and represent GAPDH-normalized mean fold mRNA expression+ SD of the indi- cated genes in relation to the respective static or LSS-treated control (set to 1). Statistical evaluation was performed by one-way ANOVA followed by Holm–Sidak multiplicity correction (D, left, comparisons between the LSS/siRNA-co-treated groups and the LSS/scr control) orone-samplet-test (allothers).Statisticaldifferencesaccordingtoun- corrected or multiplicity-adjusted P-values are marked by asterisks.

Article Snippet: The selective Erk5 inhibitors XMD8-92 (SC-361408) and BIX-02188 (S1530) were purchased from Santa Cruz and Selleck Chemicals.

Techniques: Phospho-proteomics, Expressing, Western Blot, Quantitative RT-PCR, Positive Control, Comparison, Control, Transfection, Derivative Assay

Journal: Cardiovascular research

Article Title: Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

doi: 10.1093/cvr/cvu236

Figure Lengend Snippet: Figure 6 Model of Erk5-dependent migration control in ECs. (A) Stimulation of Erk5 activity by LSS leads to induction of KLF2 and sub- sequent loss of PAK1 expression, which limits migratory processes in healthy endothelium. (B) Flow dysfunction triggers loss of Erk5 activity and KLF2 expression, resulting in increased PAK1 levels and enhanced endothelial susceptibility for pro-migratory cues. Statin treatment can reactivate the Erk5/KLF2/PAK1 axis under such conditions restoring the anti-migratory phenotype of ECs (A). PAK1 regulation thus may serve as flow-sensitive switch to adjust endothelial susceptibility to pro-migratory cues, suggesting that interference with PAK1 signalling may be a promising strategy to suppress pathological migration in the context of vascular diseases such as atherosclerosis.

Article Snippet: The selective Erk5 inhibitors XMD8-92 (SC-361408) and BIX-02188 (S1530) were purchased from Santa Cruz and Selleck Chemicals.

Techniques: Migration, Control, Activity Assay, Expressing

Journal: Oncotarget

Article Title: Aberrant expression of homeobox gene SIX1 in Hodgkin lymphoma

doi:

Figure Lengend Snippet: A. The regulatory upstream region of SIX1 contains several potential binding sites for TFs, including MEF2C at −5593 bp (UCSC Genome Bioinformatics). Reporter gene assay analysis of this binding site in L-428 cells (insert) demonstrates an inhibitory impact of MEF2C on SIX1 transcription. B. RQ-PCR analysis after siRNA-mediated knockdown of MEF2C in L-428 cells resulted in increased SIX1 expression, indicating suppression of SIX1 by MEF2C. C. RQ-PCR analysis of SIX1 transcripts in HL cell lines after treatment with HDAC-inhibitor TSA shows decreased expression levels. D. RQ-PCR analysis of SIX1 transcripts after treatment of HL cell lines with MAPK7-inhibitor XMD8–92 shows decreased expression levels. E. RQ-PCR quantification of MEF2C transcripts in HL cell lines (left) and primary hematopoietic cells and tissues (right) demonstrates reduced expression of MEF2C in L-428 as compared to BM and B-cells. F. In silico expression analysis of MEF2C (left), HDAC9 (middle), and MAPK7 (right) in primary samples obtained from HL patients and from healthy donors (GSE12453). The data indicate significantly reduced expression levels of MEF2C and HDAC9 and enhanced levels of MAPK7 in HL patients as compared to normal B-cells.

Article Snippet: Treatments of cell lines were performed for 20 h with 10 μg/ml Trichostatin A (TSA) (Sigma, Taufkirchen, Germany), 10 μM XMD8–92 (Biozol, Eching, Germany), 10 μM IWR1 (R&D Systems, Wiesbaden, Germany), 20 ng/ml recombinant human Bone Morphogenic Protein 4 (BMP4) protein, 20 ng/ml Fibroblast Growth Factor 2 (FGF2) protein, 20 ng/ml Transforming Growth Factor beta (TGFb), or 20 ng/ml WNT5B protein (R&D Systems).

Techniques: Binding Assay, Reporter Gene Assay, Expressing, In Silico