xmd8 92  (MedChemExpress)

Journal: bioRxiv

Article Title: VCAN is essential for ERK5-driven tumorigenesis in soft tissue sarcoma

doi: 10.1101/2025.05.28.654281

Figure Lengend Snippet: ERK5 regulates VCAN promoter activity in SK-LMS-1 cells. (A) Assessment of MAPK7 interference in SK-LMS-1 cells by lentiviral infection carrying the PLKO.1-shScramble (shSCR) or shRNA for MAPK7 (PLKO.1-shRNA ERK5-1/2) vectors. Relative mRNA levels were evaluated by RT-qPCR (left panel) and protein levels were detected by western blot using Vinculin as a loading control (right panel). (B) VCAN relative mRNA levels in SK-LMS-1 cells infected with shSCR and shERK5-1/2 measured by RT-qPCR. (C) VCAN (left panel) and CDKN1A ( right panel) relative mRNA levels measured by RT-qPCR in SK-LMS-1 cells treated for 18 hours with XMD8-92 (5 µM) and JWG-071 (5 µM) inhibitors. (D) VCAN (left panel) and CDKN1A (right panel) relative mRNA levels measured by RT-qPCR in HEK-293T cells treated as in C. (E) Luciferase activity assay in HEK-293T cells transiently transfected with different plasmids: luciferase control plasmid without promoter sequences (Control-L), reporter of the VCAN promoter’s activity (VCAN-L), hyperactive form of MEK5 (MEK5DD) plus a WT ERK5 (ERK5) or an inactive ERK5 (ERK5-KD) (left panel). Protein expression was measured by western blot, using HA antibody for ERK5 levels and Vinculin as loading control (right panel). (F) Luciferase activity assay in SK-LMS-1 cells transiently transfected with the same plasmids as in panel E. (G) SK-LMS-1 cells were transduced with lentiviral vector pBabe Control (pBABE Cont.) or expressing the hyperactive form of MEK5 (MEK5DD), and selected cells were analyzed by western blot against the indicated antibodies (left panel). VCAN relative mRNA levels were evaluated by RT-qPCR in these cells (right panel). Graphics represent the mean +/-SD of 3 independent experiments. The unpaired Student’s t-test was used to assess statistical significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: XMD8-92 and JWG-071 were obtained from Selleckchem (Deltaclon, Madrid, Spain).

Techniques: Activity Assay, Infection, shRNA, Quantitative RT-PCR, Western Blot, Control, Luciferase, Transfection, Plasmid Preparation, Expressing, Transduction

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: 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 vitro , Tocris #4132 , Cat# 4132.

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