Journal: Frontiers in Cell and Developmental Biology

Article Title: ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress

doi: 10.3389/fcell.2021.742049

Figure Lengend Snippet: Inhibition of MEK5/ERK5 pathway induces autophagy in serum starved and non-starved Cancer cells. (A,B) ERK5 inhibition induces sustained autophagy. Cells under serum starvation (0.5% FBS), (A) or cultured with 10% FBS ( B , Hela Cells) were treated with vehicle (DMSO) or 10 μM JWG-071 for the indicated times. Cells were lysed and levels of the autophagy-marker protein LC3 were visualized by immunoblotting. GAPDH levels are shown as a loading control. Blots are representative of three independent experiments. (C) ERK5 inhibition stimulates autophagy in a dose-dependent manner. Serum-starved cells were treated with DMSO or the indicated concentrations of JWG-071 for 24 h, and cell lysates were analysed by immunoblot as in (A,B) . Blots are representative of two separate experiments. (D) ERK5 or MEK5 inhibition impairs cellular ERK5 activity. Serum-starved cells were treated with the indicated inhibitors (5 μM), before incubation with 50 nM EGF for 30 min. Blots are representative of two separate experiments. (E) Inhibition of the MEK5/ERK5 pathway actives autophagy. Ishikawa cells were treated with either ERK5 inhibitors (5 μM JWG-071 or 10 μM XMD8-92) or MEK5 inhibitors (10 μM BIX 02188 or 10 μM BIX 02189) for 24 h. Controls were treated with vehicle. Autophagy was detected by immunoblotting as in (A,B) . GAPDH levels are shown as a loading control. (F) ERK5i AX15836 induces autophagy. MiaPaCa-2 cells were treated with 3 μM AX15836 for 12 or 24 h and autophagy was monitored as in (A,B) . Actin levels are shown as a loading control. (G) ERK5 silencing induces autophagy. MiaPaCa-2 cells were infected with lentiviral particles encoding for shRNA sequence to target ERK5. Levels of indicated proteins were detected by immunoblot analysis.

Article Snippet: ERK5 inhibitors JWG-071 (in-house synthesized), XMD8-92 (Tocris) and AX15836 (MedChemExpress), or MEK5 inhibitors BIX 02188 and BIX 02189 (Selleckchem) were diluted in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Inhibition, Cell Culture, Marker, Western Blot, Control, Activity Assay, Incubation, Infection, shRNA, Sequencing

Journal: Cell death & disease

Article Title: MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists.

doi: 10.1038/s41419-023-06229-6

Figure Lengend Snippet: Fig. 2 ERK5 kinase activity confers resistance to TRAIL-induced apoptosis in cancer cells. A Active ERK5 protects AN3CA EC cells to TRAIL- induced apoptosis. AN3CA EC cells were transfected with either ERK5 or a ERK5 kinase-dead mutant (D200A, ERK5-KD) together with a constitutively active form of MEK5 (MEK5-DD). Twenty-four hours after transfection cells were treated with 25 ng/ml TRAIL (8 h), and apoptosis was analyzed by flow cytometry (Annexin-V and PI staining). Left panel shows representative results of flow cytometry analysis. Right histograms show the quantification of apoptotic cells (early apoptotic Annexin-V + /PI- and late apoptotic Annexin-V + /PI+ cells). B, C ERK5 kinase activity impairs caspase-8 activation in response to TRAIL in AN3Ca (B) and Ishikawa (C) cells. Immunoblot analysis. Right histograms show the quantification of cleaved caspase-8 and −3 bands (mean ± SD, n = 3 independent biological experiments, one-way ANOVA followed by Bonferroni multiple comparison test). ***p < 0.001.

Article Snippet: ERK5 inhibitors JWG-071 (Merck), AX-15836 (MedChemExpress), and MEK5 inhibitors BIX02188 and BIX02189 (Selleckchem) were diluted in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Activity Assay, Transfection, Mutagenesis, Cytometry, Staining, Activation Assay, Western Blot, Comparison

Journal: Cell death & disease

Article Title: MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists.

doi: 10.1038/s41419-023-06229-6

Figure Lengend Snippet: Fig. 3 ERK5 or MEK5 genetic deletion sensitizes Ishikawa EC cells to DR agonists-induced apoptosis by favoring Caspase-8 activation. A Ishikawa wild type, MEK5-KO or ERK5-KO cells were treated for 24 h with 50 ng/ml TRAIL, 20 ng/ml TNFα or 100 ng/ml Anti-Fas activating antibody, and apoptotic cells were analyzed by flow cytometry (Annexin-V and PI staining). Right histograms show the quantification of apoptotic cells (mean ± SD, n = 3 independent biological experiments, one-way ANOVA). B, C MEK5 or ERK5 genetic deletion enhances caspase-8 activation in response to DR ligands. Ishikawa wild type, ERK5-KO (B) or MEK5-KO (C) cells were treated for 4 h with 50 ng/ml TRAIL, 20 ng/ml TNFα or 100 ng/ml Anti-Fas activating antibody. Caspase-8 and caspase-3 activation was monitored by immunoblot analysis. D MEK5 genetic deletion sensitizes 3D cultures of Ishikawa cells to DR ligands. 3D cultures were treated with 50 ng/ml TRAIL, 20 ng/ml TNFα or 100 ng/ml Anti-Fas activating antibody for 24 h, and cell viability was analyzed with LIVE/DEAD reagent (alive cells, green; dead cells, red). B, C similar results were obtained in three independent experiments.

Article Snippet: ERK5 inhibitors JWG-071 (Merck), AX-15836 (MedChemExpress), and MEK5 inhibitors BIX02188 and BIX02189 (Selleckchem) were diluted in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Activation Assay, Cytometry, Staining, Western Blot