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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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: XMD8-92 for in vivo (50 mg/Kg) , MedChemExpress , Cat# HY-14443.

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

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

Journal: Oncotarget

Article Title: Changes in metabolism affect expression of ABC transporters through ERK5 and depending on p53 status

doi: 10.18632/oncotarget.23305

Figure Lengend Snippet: (A) HuH7 cells were transfected with control siRNA or siERK5. Thirty-six hours later mRNA expression was analyzed by qPCR. (B) Primary human hepatocytes were transfected with control siRNA or siERK5 at day 1 and 3 post seeding. 96 h later mRNA levels were assessed. (C) Jurkat cells were transfected with a small hairpin RNA for ERK5 (shERK5) or ERK5 expression vector. 36h later mRNA expression was analyzed by qPCR. (D) Jurkat cells were treated with 10 μM XMD8-92 or 10 μM BIX01289 for 24 h before analyzing expression of ABCB1 by FACs. The data represent means ± SD n=3; * p < 0.05, ** p < 0.01, *** p < 0.001 Student's t-test compared to empty vector transfected cells (control).

Article Snippet: The ERK5 inhibitor XMD8-92 and the MEK5 inhibitor BIX02189 were from Selleck.

Techniques: Transfection, Control, Expressing, Plasmid Preparation