Journal: International Journal of Molecular Sciences

Article Title: Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication

doi: 10.3390/ijms22147420

Figure Lengend Snippet: Toll-like receptor (TLR) signaling pathway-related genes significantly regulated by zinc chloride in Huh7 cells.

Article Snippet: SB202190, a potent and selective p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication

doi: 10.3390/ijms22147420

Figure Lengend Snippet: Toll-like receptor (TLR) signaling pathway-related genes significantly regulated by zinc chloride in PLC/PRF/5 cells.

Article Snippet: SB202190, a potent and selective p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication

doi: 10.3390/ijms22147420

Figure Lengend Snippet: Knockdown of mitogen-activated protein kinase kinase 3 (MAP2K3) could lead to a decrease in HAV subgenomic and genomic RNA replication: ( a ) effect of 5 μM zinc chloride (ZnCl 2 ) for 24 h on MAP2K3 and GAPDH expression at the protein level. A Western blot test demonstrated the MAP2K3 and GAPDH expression, using a specific antibody for MAP2K3 and for GAPDH, respectively; ( b ) the MAP2K3/GAPDH ratios from three independent experiments were measured using Adobe Photoshop CS5 (Adobe Inc., San Jose, CA, USA); ( c ) effect of the knockdown of MAP2K3 by specific siRNA compared with the control siRNA (si-C) on MAP2K3 and β-tubulin expression. A Western blot test demonstrated the MAP2K3 and β-tubulin expression using a specific antibody for MAP2K3 and for β-tubulin, respectively. ( d ) The MAP2K3/β-tubulin ratios from three independent experiments were measured; ( e ) the effect of the knockdown of MAP2K3 on the HAV genotype IB subgenomic replicon replication in HuhT7; ( f ) the effect of the knockdown of MAP2K3 on HAV genotype IIIA HA11-1299 replication in Huh7. The data are expressed as the means ± standard deviations of triplicate determinations from one experiment which was representative of three independent experiments. The original images for the blots ( a , c ) are shown in the .

Article Snippet: SB202190, a potent and selective p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Knockdown, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication

doi: 10.3390/ijms22147420

Figure Lengend Snippet: Knockdown of mitogen-activated protein kinase kinase 3 (MAP2K3) could enhance the interferon-β promoter activities stimulated by polyinosinic–polycytidylic acid (poly(I:C)). ( a ) IHH cells * p < 0.05 vs. other groups. ** p < 0.05 vs. the control siRNA (si-C) without poly(I:C) group; ( b ) Huh7 cells.

Article Snippet: SB202190, a potent and selective p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Knockdown, Control

Journal: International Journal of Molecular Sciences

Article Title: Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication

doi: 10.3390/ijms22147420

Figure Lengend Snippet: SB202190, a selective inhibitor of the MAP2K3–p38 MAPK signaling pathway, enhanced the interferon-β promoter activity stimulated by polyinosinic–polycytidylic acid (poly(I:C)). ( a ) IHH cells; ( b ) Huh7 cells. SB202190 was used at a concentration of 10 nM for 16 h.

Article Snippet: SB202190, a potent and selective p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activity Assay, Concentration Assay

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Danu modulates the phosphorylation of p38 MAPK, Erk1/2, Akt, mTOR and the expression of beclin 1, LC3-I, and LC3-II in MCF10A, MCF7, and MDA-MB-231 cells. Notes: MCF10A, MCF7, and MDA-MB-231 cells were treated with Danu at 0.01, 0.1, and 0.5 μM for 24 hours, and the protein samples were subjected to Western blotting assay. ( A ) Representative bolts of p-p38 MAPK, p38 MAPK, p-Erk1/2, Erk1/2, p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II, and ( B ) bar graphs showing the ratio of p-p38 MAPK/p38 MAPK, p-Erk1/2/Erk1/2, p-Akt/Akt, and p-mTOR/mTOR, and the relative levels of beclin 1 and LC3-II. β-actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; conc, concentration; SD, standard deviation; ANOVA, analysis of variance; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; Akt, protein kinase B; mTOR, mammalian target of rapamycin; LC3, microtubule-associated protein 1A/1B-light chain 3.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Effects of Danu treatment over 72 hours on phosphorylation levels of p38 MAPK, Erk1/2, Akt, and mTOR, and the levels of beclin 1, LC3-I, and LC3-II in MCF10A, MCF7, and MDA-MB-231 cells. Notes: Cells were treated with Danu for 4, 8, 12, 24, 48, and 72 hours, and β-actin was used as the internal control. ( A ) Representative blots of p-p38 MAPK, p38 MAPK, p-Erk1/2, Erk1/2, p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in MCF10A cells, ( B ) MCF7 cells, and ( C ) MDA-MB-231 cells. Abbreviations: Danu, danusertib; conc, concentration; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; Akt, protein kinase B; mTOR, mammalian target of rapamycin; LC3, microtubule-associated protein 1A/1B-light chain 3

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Concentration Assay

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Bar graphs showing the ratio of p-p38 MAPK/p38 MAPK, p-Erk1/2/Erk1/2, p-Akt/Akt, and p-mTOR/mTOR, and the relative levels of beclin 1 and LC3-II in ( A ) MCF10A cells, ( B ) MCF7 cells, and ( C ) MDA-MB-231 cells. Notes: Cells were treated with Danu for 4, 8, 12, 24, 48, and 72 hours, and β-actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; conc, concentration; SD, standard deviation; ANOVA, analysis of variance; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; Akt, protein kinase B; mTOR, mammalian target of rapamycin; LC3, microtubule-associated protein 1A/1B-light chain 3; hr, hour.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Concentration Assay, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Effects of various chemical modulators on the expression levels of beclin 1 and LC3-II in MCF7 cells and MDA-MB-231 cells. Notes: Cells were pretreated with WM (10 μM), bafilomycin A1 (100 nM), SB202190 (10 μM), or U0126 (10 μM) for 30 minutes, 0.5 μM Danu was added and incubated for a further 24 hours, cells were harvested, and protein samples were subjected to Western blotting assay. ( A ) Representative blots of beclin 1 and LC3-II in MCF7 cells and MDA-MB-231 cells, and ( B ) bar graphs showing the relative levels of beclin 1 and LC3-II. β-actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; conc, concentration; SD, standard deviation; ANOVA, analysis of variance; LC3, microtubule-associated protein 1A/1B-light chain 3; WM, wortmannin; DMSO, dimethyl sulfoxide; Baf A1, bafilomycin A1.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Concentration Assay, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Effects of two autophagy inhibitors on the activation of p38 MAPK and Erk1/2 in MCF7 and MDA-MB-231 cells. Notes: MCF7 and MDA-MB-231 cells were pretreated with WM (10 μM) or bafilomycin A1 (100 nM) for 30 minutes before adding 0.5 μM Danu for further incubation of 24 hours. Cells were then harvested and protein samples were subjected to Western blotting assay. ( A ) Representative blots of p-p38 MAPK, p38 MAPK, p-Erk1/2, and Erk1/2 in MCF7 and MDA-MB-231 cells, and ( B ) bar graphs showing the ratio of p-p38 MAPK/p38 MAPK and p-Erk1/2/Erk1/2 in MCF7 and MDA-MB-231 cells. β-actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; SD, standard deviation; ANOVA, analysis of variance; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; WM, wortmannin; DMSO, dimethyl sulfoxide; Baf A1, bafilomycin A1.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Effects of specific p38 MAPK and Erk1/2 inhibitors on Danu-induced autophagy in MCF7 cells and MDA-MB-231 cells. Notes: Cells were pretreated with SB202190 (10 μM) or U0126 (10 μM) for 30 minutes, 0.5 μM Danu was added for further incubation of 24 hours, and cells were harvested. The percentage of autophagic MCF7 and MDA-MB-231 cells was examined by flow cytometry with Cyto-ID ® as the green stain for autophagic vacuoles. ( A ) Flow cytometric dot plots showing the autophagic MCF7 and MDA-MB-23 cells, and ( B ) bar graphs showing the percentage of autophagic MCF7 and MDA-MB-231 cells. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; SD, standard deviation; ANOVA, analysis of variance; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; DMSO, dimethyl sulfoxide.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Effects of p38 MAPK and Erk1/2 inhibitors on the activation of p38 MAPK, Erk1/2, and Akt, and the expression of beclin 1 and LC3-I and -II in MCF7 and MDA-MB-231 cells. Notes: MCF7 and MDA-MB-231 cells were pretreated with SB20290 (10 μM) or U0126 (0 μM) for 30 minutes before adding 0.5 μM Danu for further incubation of 24 hours. Cells were then harvested and protein samples were subjected to Western blotting assay. ( A ) Representative blots of p-p38 MAPK, p38 MAPK, p-Erk1/2, Erk1/2, p-Akt, Akt, beclin 1, and LC3-I and II in MCF7 and MDA-MB-231 cells, and ( B ) bar graphs showing the ratio of p-p38 MAPK/p38 MAPK, p-Erk1/2/Erk1/2, and p-Akt/Akt in MCF7 and MDA-MB-231 cells. β-actin was used as the internal control. Data are expressed as the mean ± SD of three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; SD, standard deviation; ANOVA, analysis of variance; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk/2, extracellular signal-regulated kinases 1 and 2; Akt, protein kinase B; LC3, microtubule-associated protein 1A/1 B-light chain 3; DMSO, dimethyl sulfoxide.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

doi: 10.2147/DDDT.S74412

Figure Lengend Snippet: Knockdown of the p38 MAPK and Erk1/2 blocks Danu-induced autophagy. Notes: MCF7 cells were transfected with control siRNA (100 nM), p38 MAPK-specific siRNA (100 nM), or Erk1/2-specific siRNA (100 nM) and then treated with Danu (0.5 μM) or DMSO for 24 hours. The protein samples were subjected to Western blotting assay. ( A ) Representative blots of p-p38 MAPK, p38 MAPK, p-Erk1/2, Erk1/2, p-Akt, Akt, beclin 1, and LC3-I and II in MCF7 and MDA-MB-231 cells and ( B ) bar graphs showing the ratio of p-p38 MAPK/p38 MAPK, p-Erk1/2/Erk1/2, and p-Akt/Akt and the relative levels of beclin 1 and LC3-II in MCF7 cells. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. ** P <0.01 and *** P <0.001 by one-way ANOVA. Abbreviations: Danu, danusertib; SD, standard deviation; ANOVA, analysis of variance; p, phosphorylated; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated kinases 1 and 2; Akt, protein kinase B; LC3, microtubule-associated protein 1A/1B-light chain 3; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA.

Article Snippet: Bafilomycin A1 (a known inhibitor of the late phase of autophagy, which prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes), wortmannin (WM, an irreversible and selective phosphatidylinositol 3-kinase [PI3K] inhibitor and a blocker of autophagosome formation), and SB202190 (an autophagy inducer and specific p38 mitogen-activated protein kinase [MAPK] inhibitor) were purchased from Invivogen Inc. (San Diego, CA, USA).

Techniques: Transfection, Western Blot, Standard Deviation, Small Interfering RNA