erk5 inhibitor xmd8  (Santa Cruz Biotechnology)

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: KPNB1 inhibition reduces the amount of nuclear ERK5. (A, B) HEK293T overexpressing ERK5 and the constitutively active form of MEK5 (ERK5 + MEK5DD) (A) or A375 (B) cells transfected with two siRNA targeting KPNB1 (siKPNB1‐1 or siKPNB1‐2) or with non‐targeting control siRNA (siNT) were lysed, and western blot was performed on whole cell lysates or nuclear extracts with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). The graphs show average densitometric values ± SD of nuclear ERK5 protein levels normalized for fibrillarin content ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to siNT‐treated control cells. **, P < 0.01 and ***, P < 0.001. (C, D) HEK293T overexpressing ERK5 and the constitutively active form of MEK5 (ERK5 + MEK5DD) (C) or A375 (D) cells treated for 24 h with DMSO, used as vehicle, or increasing concentration of ivermectin (IVM) were lysed, and western blot was performed on whole cell lysates or nuclear extracts with the indicated antibodies. The graphs show average densitometric values ± SD of nuclear ERK5 protein levels normalized for fibrillarin content ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to vehicle‐treated control cells. *, P < 0.05 and **, P < 0.01.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Transfection, Control, Western Blot, Migration, Molecular Weight, Concentration Assay

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: Ivermectin prevents EGF‐induced nuclear translocation of ERK5. (A) 24‐h starved HeLa cells were treated with DMSO, used as vehicle, or 10 μ m ivermectin (IVM) for 24 h and then with 100 ng·mL −1 epidermal growth factor (EGF) for 30 min or the combination of the two. Cells were then lysed, and western blot was performed on whole cell lysates or nuclear extracts with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). The graph shows average densitometric values ± SD of nuclear ERK5 protein levels normalized for fibrillarin content ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to vehicle‐treated cells or between the indicated conditions. *, P < 0.05. (B) 24‐h serum‐starved HeLa cells were treated with DMSO, used as vehicle, or 5 μ m ivermectin (IVM) for 24 h and then with 100 ng·mL −1 epidermal growth factor (EGF) for 15 min or left untreated. Immunofluorescence analysis was performed staining ERK5 (red) and importin β1 (Imp β1, green). Confocal images were analysed to quantify ERK5 nuclear staining, represented in the graph as integrated optical density (IOD) ± SD ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to vehicle‐treated cells or between the indicated conditions. *, P < 0.05. Scale bar: 20 μm.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Translocation Assay, Western Blot, Migration, Molecular Weight, Immunofluorescence, Staining

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: Single molecule analysis confirms the efficacy of ivermectin in reducing ERK5 nuclear translocation. (A) Schematic representation of ERK5‐HaloTag‐JaneliaFluor646 system. Amino acid residues (a.a.); NLS: nuclear localization signal. (B) Confocal imaging of HeLa cells transfected with control empty vector, ERK5‐HaloTag or ERK5‐HaloTag and the constitutively active form of MEK5 (ERK5‐Halo + MEK5DD) and then labelled with JaneliaFluor646. Representative confocal images from three independent experiments are shown. Scale bar: 10 μm. (C) HeLa cells transfected with ERK5‐HaloTag and the constitutively active form of MEK5 (ERK5‐Halo + MEK5DD) were treated with 5 μ m ivermectin (IVM) or DMSO, used as vehicle, for 24 h and then lysed. Western blot was then performed on nuclear extracts with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). The graphs show the average densitometric values ± SD of nuclear ERK5 protein levels normalized for fibrillarin content ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to vehicle‐treated cells. *, P < 0.05. (D) Bright‐field images (BF, first row), maximum intensity projection images of super‐resolution videos (MIP, second row) and single frames of the latter overlapped with cytoplasmic and nuclear masks (third row), drawn based on bright‐field images (Fig. ), of HeLa cells transfected with an empty vector (first column), ERK5‐HaloTag (second column) or ERK5‐HaloTag and the constitutively active form of MEK5 (ERK5‐Halo + MEK5DD) (third column). Cells were left untreated (1–3 columns) or treated with 5 μ m ivermectin (IVM) for 24 h (forth column). All samples were then labelled with JaneliaFluor646. The graph represents nucleus/cytoplasmic density ratios (i.e. the number of ERK5 nuclear particles normalized for the nuclear area/the number of ERK5 cytoplasmic particles normalized for the cytoplasm area) ± SD of ERK5 single molecules detected in the super‐resolution videos in each sample condition. Number of measured cells/samples = 20 ( n = 3). P values calculated using Student's t ‐test refer to differences between the indicated conditions. *, P < 0.05. Scale bar: 10 μm.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Translocation Assay, Imaging, Transfection, Control, Plasmid Preparation, Western Blot, Migration, Molecular Weight

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: ERK5 interacts with importin β1. (A–D) HeLa cells overexpressing ERK5 and the constitutively active form of MEK5 (ERK5 + MEK5DD) (A, B) or A375 cells (C, D) were lysed, importin β1 (A, C) or ERK5 (B, D) was immunoprecipitated (IP) with specific antibodies as indicated, and western blot was then performed with the indicated antibodies, either reblotting the same membrane with different antibodies or running the same samples in parallel gels. IgG content (Heavy chain, Hc) was used as a loading control. Migration of molecular weight markers is indicated on the left (kDa). Impβ1, importin β1; l.e., low exposure; and h.e. high exposure. Images are representative of three independent experiments. (E) 24‐h serum‐starved HeLa cells were treated with 100 ng·mL −1 EGF for the indicated times or left untreated. Cells were then lysed, importin β1 was immunoprecipitated (IP), and western blot was performed with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). Impβ1, importin β1. Images are representative of three independent experiments. (F) 24‐h starved HeLa cells were treated with 100 ng·mL −1 EGF for 15 min or left untreated and then stained for ERK5 (yellow) and importin β1 (Impβ1, green). Confocal images were analysed to quantify the proximity ligation assay (PLA) (red) signal. The graph represents average values of PLA signal/cell ± SD. P values calculated using Student's t ‐test refer to differences with respect to untreated cells obtained from three independent experiments. P values calculated using Student's t ‐test refer to differences between the indicated conditions. *, P < 0.05. Scale bar: 20 μm.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Immunoprecipitation, Western Blot, Membrane, Control, Migration, Molecular Weight, Staining, Proximity Ligation Assay

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: AX15836 does not affect cancer cell proliferation. (A, B) A375 (A) and HeLa cells (B) were treated with increasing concentrations of AX15836, and MTT assays were performed after 72 h. The graphs report average data ± SD ( n = 3). (C) HeLa and A375 cells treated with DMSO, used as vehicle, or the indicated concentrations of AX15836 for 30 min were lysed, and western blot was performed on nuclear extracts with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). Images are representative of three independent experiments. (D) A375 cells were treated with DMSO, used as vehicle, or ivermectin (IVM) 10 μ m for 24 h and then with AX15836 5 μ m for 30 min or with the combination of the two drugs were lysed, and western blot was performed on nuclear or cytoplasmic extracts with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). The graphs show average densitometric values ± SD of ERK5 protein levels normalized for fibrillarin or tubulin content ( n = 3). P values calculated with ANOVA refer to differences with respect to vehicle‐treated cells or between indicated conditions. *, P < 0.05 and **, P < 0.01.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Migration, Molecular Weight

Journal: Molecular Oncology

Article Title: Importin subunit beta‐1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation

doi: 10.1002/1878-0261.13674

Figure Lengend Snippet: AX15836 inhibits cancer cell proliferation only in combination with ivermectin. (A, B) A375 (A) or HeLa (B) cells were treated with DMSO used as vehicle or increasing concentrations of AX15836 and of ivermectin (IVM) for 72 h, and MTT assay was performed. The graphs report average data ± SD ( n = 3). (C, D) Colony formation assay was performed with A375 (C) or HeLa (D) cells treated with DMSO used as vehicle (−/−), ivermectin (IVM) or AX15836 (AX) at the indicated concentrations or with their combination for 7 (A375) or 10 (HeLa) days. The graphs report average data ± SD ( n = 3). P values calculated using ANOVA refer to differences with respect to vehicle‐treated cells or between the indicated conditions. *, P < 0.05 and **, P < 0.01. § indicates synergistic effect (bliss test > 0) with respect to single treatments. (E) Parental and ERK5‐KO HUH‐7 cells were lysed, and western blot was performed with the indicated antibodies. Migration of molecular weight markers is indicated on the left (kDa). (F) Parental and ERK5‐KO HUH‐7 cells were treated with DMSO used as vehicle, AX15836 (AX) or ivermectin (IVM) at the indicated concentration for 72 h, and MTT assay was performed. The graphs report average data ± SD ( n = 3). P values calculated using Student's t ‐test refer to differences with respect to vehicle‐cells. *, P < 0.05; ns, not significant.

Article Snippet: Ivermectin (α/β1 importin inhibitor, IVM) [ ] and the ERK5 inhibitors XMD8‐92 [ ], JWG‐071 [ ] and AX15836 [ ] were from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: MTT Assay, Colony Assay, Western Blot, Migration, Molecular Weight, Concentration Assay

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: Frontiers in Cardiovascular Medicine

Article Title: Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition

doi: 10.3389/fcvm.2023.1187490

Figure Lengend Snippet: CHK1 regulates L-flow-induced SENP2 S344 phosphorylation, subsequently suppresses ERK5 and p53 SUMOylation: ( A ) HUVECs transduced with Ad-SENP2 WT or Ad-SENP2 S344A were exposed to L-flow for 0 and 30 min, and the levels of SENP2 (p-S344 and total), ERK5 (p-T-E-Y and total), and actin (loading control) were determined using Wes. An increase in p-ERK5-T-E-Y indicated successful generation of L-flow, in addition to elongated cell shape ( , ). ( B ) HUVECs were exposed to D-flow for 0, 20, 40, and 60 min, and the levels of SENP2 (p-S344 and total) were determined using immunoblotting. ( C ) HUVECs pretreated with 250 nM GDC 0575 were exposed to L-flow for 0, 20, 40, and 60 min, and the levels of SENP2 (p-S344 and total), CHK1 (p-S280, p-S345, and total), and actin were determined using Wes. ( D , upper panel) The graphs present quantified data from 3 independent experiments from ( C ) ( n = 3) (* p < 0.05, ** p < 0.01, two-way ANOVA). (D , lower panel ) The graphs present quantified data from 3 independent experiments from ( B ) ( n = 3). N.S. indicates no significance. (E–G) The graphs present quantified data from 3 independent experiments from ( C ) ( n = 3) (* p < 0.05, ** p < 0.01, two-way ANOVA). (H) WT mouse lung ECs (WT MLECs) and SENP2 S344A KI MLECs were exposed to L-flow for 0 and 30 min, and the levels of SENP2 (p-S344 and total) were determined by immunoblotting. (I ) WT MLECs and SENP2 S344A KI MLECs were exposed to L-flow for 0 and 30 min. Lysates were immuno-precipitated with the SUMO2/3 antibody (Signal-Seeker TM SUMOylation 2/3 Detection Kit, #BK162; Cytoskeleton, Inc.), and ERK5 or p53 antibody was used to detect SUMOylated ERK5 or p53 using Wes, respectively. The SUMO2/3 antibody was also used to confirm equal immunoprecipitation across the samples. Control beads were used as the negative control. ERK5, SENP2, and p53antibodies were used to detect their expression in total cell lysates using Wes. ( J ) The graphs present quantified data from 4 (upper panel) and 5 (lower panel) independent experiments (* p < 0.05, two-way ANOVA).

Article Snippet: CHK1 inhibitor (GDC 0575) and ERK5 inhibitor (XMD8-92, #S7525) were purchased from Selleck Chemicals LLC (Houston, TX, USA), dissolved in DMSO, and pretreated to ECs prior to flow exposure at the doses indicated in the figures.

Techniques: Phospho-proteomics, Transduction, Control, Western Blot, Immunoprecipitation, Negative Control, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition

doi: 10.3389/fcvm.2023.1187490

Figure Lengend Snippet: Radiation-induced EC activation through CHK1 downregulation: ( A )there was no significant difference in lipid-laden lesions between female WT → WT and WT → SENP2 S344A KI mice ( n = 3–4). ( B ) Expression levels of CHK1, SENP2 (p-S344 and total), VCAM1, ICAM1, and actin were evaluated in HUVECs exposed to 0, 2, 4, 6, 8 Gy of ionizing radiation (IR) for 24 h. ( C ) The graphs present quantified data from 3 independent experiments (** p < 0.01, two-way ANOVA). ( D , left panel) SUMOylated ERK5 was assessed in siRNA-treated HUVECs, and the lysates were immuno-precipitated as described in . ( D , right panel) The graphs present quantified data from 3 independent experiments (** p < 0.01, t -test). ( E , left panel) siRNA-treated HUVECs were exposed to D-flow for 24 h, and the expression of ICAM1, CHK1, and actin were determined using immunoblotting. ( E , right panel) The graphs present quantified data from 3 independent experiments (* p < 0.05, two-way ANOVA). ( F ) Expression levels of ICAM1, VCAM1, CHK1, and actin were evaluated in siRNA-treated HUVECs exposed to L-flow for 24 h using immunoblotting. ( G ) The graphs present quantified data from 3 or 4 independent experiments (* p < 0.05, ** p < 0.01, two-way ANOVA).

Article Snippet: CHK1 inhibitor (GDC 0575) and ERK5 inhibitor (XMD8-92, #S7525) were purchased from Selleck Chemicals LLC (Houston, TX, USA), dissolved in DMSO, and pretreated to ECs prior to flow exposure at the doses indicated in the figures.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition

doi: 10.3389/fcvm.2023.1187490

Figure Lengend Snippet: L-flow upregulates DDIAS expression via SENP2 S344 phosphorylation and ERK5 activation to suppress EC apoptosis: ( A ) the venn diagram illustrates the significant DEGs between the groups. ( B ) The heatmap displays the nine core genes, including DDIAS. ( C , left panel) Pretreatment with XMD8-92 (5 µM), an ERK5-specific inhibitor, suppresses L-flow-mediated upregulation of DDIAS expression in HUVECs after 24 h. ( C , right panel) The graphs present quantified data from 3 independent experiments (** p < 0.01, two-way ANOVA). ( D , left panel) HUVECs were overexpressed with DDIAS using the DDIAS plasmid or control plasmid, then exposed to IR for 24 h. The increased expression of cleaved caspase-3 induced by IR is reversed by DDIAS overexpression. ( D , right panel) The graphs present quantified data from 3 independent experiments (* p < 0.05, two-way ANOVA). “pCont” refers to the control plasmid, and “pDDIAS” refers to the DDIAS plasmid. ( E ) The scheme illustrates the mechanism of L-flow-induced SENP2 S344 phosphorylation in suppressing the EC pathophysiological stress response.

Article Snippet: CHK1 inhibitor (GDC 0575) and ERK5 inhibitor (XMD8-92, #S7525) were purchased from Selleck Chemicals LLC (Houston, TX, USA), dissolved in DMSO, and pretreated to ECs prior to flow exposure at the doses indicated in the figures.

Techniques: Expressing, Phospho-proteomics, Activation Assay, Plasmid Preparation, Control, Over Expression