mek3 6  (Santa Cruz Biotechnology)

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: MKK3 and MKK6 gene expression levels in ERMS and NSM samples. Using the R2-Genomics Analysis and Visualization Platform, MKK3 or MKK6 gene expression was assessed in ERMS primary tumours or NSM across different datasets: (A) Barr and Assmann datasets, (B) Davicioni and Hofman datasets, (C) Schafer Welle dataset. Statistical analysis was performed using one-way ANOVA. (A-C) Right panels illustrate the relative expression of MKK3 and MKK6 in ERMS samples. ERMS, embryonal rhabdomyosarcoma; NSM, normal skeletal muscle.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Gene Expression, Expressing

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: Role of MKK3 and MKK6 in the control of proliferation and differentiation of RD cells. (A) MKK3 and MKK6 western blots are shown as the control of the transfection with dnMKK3, caMKK3 or caMKK6 vectors; western blot analysis of Myc, ERK-PO4 and ERK, cyclin D1 and p21 in RD cells transfected with caMKK6, dnMKK3 or caMKK3. GAPDH was used as a loading control. (B) Western blot analysis of myogenic differentiation markers, MHC, myogenin and p38-PO4 using the same samples as in (A). Tubulin was used as a loading control. Phospho-kinases were also normalised for unphosphorylated isoforms. The numbers on the left of the blots indicate the protein size (kDa). Experiments were performed three times. (A and B) Right panels illustrate the quantitative evaluations of the different western blots performed, expressed as the mean ± SD. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test: ***P<0.001; **P<0.01 vs. CMV. MHC, myosin heavy chain.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Control, Western Blot, Transfection

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: Morphological and functional changes induced by MKK6 or MKK3 overexpression in RD cells. (A) Western blots showing MKK3 and MKK6 expression as the control of the transfection with the specific vectors. Tubulin was used as a loading control. The numbers on the left of the blots indicate the protein size (kDa). (B) Differences in viable RD cell number in cells transfected with empty vector (CMV), caMKK3 or caMKK6 assessed using the trypan blue exclusion assay. Histograms represent the mean value ± SD of three independent experiments. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test: **P<0.01; *P<0.05 vs. CMV. (C) Phase contrast images of RD cells transfected with CMV, caMKK3 or caMKK6. MKK6 induces typical elongated myogenic morphology not present in caMKK3 transfected RD cells. (D) CMV-, caMKK3- or caMKK6-transfected cells after immunofluorescence staining with Myc or MHC antibodies. DAPI was used for nuclear staining. Scale bars, 50 µm. Experiments were performed twice. MHC, myosin heavy chain.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Functional Assay, Over Expression, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Trypan Blue Exclusion Assay, Immunofluorescence, Staining

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: Anti-oncogenic and pro-myogenic signals are mediated by p38 activation in RD cells. (A) Proliferation of RD cells overexpressing MKK6 treated with or without SB203580 (5 µM) assessed using trypan blue exclusion assay. Histograms represent the mean value ± SD of two independent experiments. Statistical analyses were performed using two-way ANOVA with Tukey's post hoc test: **P<0.01; *P<0.05 vs. CMV; # P<0.05 vs. caMKK6. (B) Morphological evaluation of RD cells transfected with caMKK6 treated with or without SB203580 (5 µM). Scale bars, 50 µm. Experiments were performed twice. (C) RD cells transfected with empty vector (CMV) or caMKK6 and treated with or without the SB203580 (5 µM) p38 inhibitor were analysed for phospho-active p38 expression level. GAPDH was used for protein quantification. Phospho-p38 was also normalised for total unphosphorylated isoform. MKK6 expression is shown as a transfection control. (D) Western blots of MHC, myogenin and Myc in RD cells transfected with CMV or caMKK6 and treated with or without SB203580 (5 µM) p38 inhibitor. (C and D) The numbers on the left of the blots indicate the protein size (kDa). Lower panels represent quantitative evaluations of the western blots expressed as the mean ± SD. Statistical analyses were performed using two-way ANOVA with Tukey's post hoc test: ***P<0.001; **P<0.01; *P<0.05 vs. CMV; ### P<0.001; ## P<0.01 vs. caMKK6. Experiments were performed three times. MHC, myosin heavy chain.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Activation Assay, Trypan Blue Exclusion Assay, Transfection, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: TE cell differentiation is dependent on MKK6/p38 pathway activation. TE cells transfected with CMV or caMKK6 were treated with 5 µM SB203580 or left untreated; western blots of MKK6, Myc, ERK-PO4, p38-PO4, myogenin and MHC were normalised to tubulin. Phospho-kinases were also normalised for unphosphorylated isoforms. The numbers on the left of the blots indicate the protein size (kDa). Lower panel represent histograms of the quantitative evaluations of the western blots expressed as the mean ± SD. Statistical analyses were performed using two-way ANOVA with Tukey's post hoc test: ***P<0.001; *P<0.05 vs. CMV; ### P<0.001; ## P<0.01 vs. caMKK6. Experiments were performed three times. TE cells, TE671 cells; MHC, myosin heavy chain.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Cell Differentiation, Activation Assay, Transfection, Western Blot

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: MKK6 is induced by MEK/ERK inhibitors in RD cells. RD cells were treated with 10 µM U0126 or 10 nM trametinib and the expression levels of MKK6-PO4, p38-PO4, MHC and myogenin were examined using western blot analysis. GAPDH and unphosphorylated kinases were used to normalise MKK6 and p38 (3 h and O/N panel); tubulin and GAPDH were used to normalise MHC and myogenin, respectively (3 days panel). The numbers on the left of the blots indicate the protein size (kDa). Lower panels represent histograms of the quantitative evaluations of the western blots, expressed as the mean ± SD. Statistical analyses were performed by using one-way ANOVA with Dunnett's post hoc test: ***P<0.001; **P<0.01; *P<0.05 vs. negative control. Experiments were performed three times. MHC, myosin heavy chain; O/N, overnight; C, negative control.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Expressing, Western Blot, Negative Control

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: AKT is induced by MEK/ERK inhibition. RD and TE cells were treated with 10 µM U0126 or left untreated and lysates were analysed for AKT-PO4 Ser473 and AKT-PO4 Thr308 expression. Both phosphorylation levels were increased after O/N, 1 day and 3 days of treatments. The numbers on the left of the blots indicate the protein size (kDa). In the lower panels, quantitative evaluations of the western blots are shown as the mean ± SD. Statistical analyses were performed using a Student's t-test: ***P<0.001; **P<0.01; *P<0.05 vs. negative control (control). Experiments were performed twice. TE cells, TE671 cells; O/N, overnight.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques: Inhibition, Expressing, Phospho-proteomics, Western Blot, Negative Control, Control

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: AKT activation is part of myogenic differentiation in RD cells. (A) Cells transfected with empty vector (CMV), caMKK3 or caMKK6 were analysed for AKT-PO4 Ser473, AKT-PO4 Thr308 and p38-PO4 expression. Both AKT phosphorylation sites and p38 were activated by caMKK6 transfection, whereas they were absent in CMV and caMKK3 transfected cells; MKK3 and MKK6 overexpression is shown as a transfection control. GAPDH was used for protein quantification. Phospho-kinases were also normalised for unphosphorylated isoforms. The numbers on the left of the blots indicate the protein size (kDa). Right panel represents histograms of the quantitative evaluations of the western blots, expressed as the mean ± SD. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test: ***P<0.001 vs. CMV. (B) Western blots showing the reduced AKT phosphorylation in caMKK6-transfected RD cells treated with 5 µM SB203580. (C) Both AKT phosphorylation levels were not activated by U0126 in p38-silenced RD cells, whilst they were present in scramble control-transfected (SCR) cells treated with U0126. GAPDH was used as loading control. (B and C) The numbers on the left of the blots indicate the protein size (kDa). Lower panels represent histograms of the quantitative evaluations of the western blots, expressed as the mean ± SD. Statistical analyses were performed using two-way ANOVA with Tukey's post hoc test: ***P<0.001; **P<0.01; *P<0.05 vs. CMV or SCR; ### P<0.001 vs. caMKK6; §§§ P<0.001 vs. SCR-U0126. Experiments were performed twice.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

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

Journal: Oncology Reports

Article Title: Anti-oncogenic and pro-myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma

doi: 10.3892/or.2022.8363

Figure Lengend Snippet: MKK3 and MKK6 kinases in ERMS cells. Summary diagram describing the differential role played by MKK3 and MKK6 in controlling growth arrest and myogenic differentiation in ERMS cells. MHC, myosin heavy chain.

Article Snippet: Filters were blocked with 5% non-fat dry milk or 3% BSA for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-Myc (cat. no. sc-40; 1:300), anti-ERK-PO4 E-4 (cat. no. sc-7383; 1:500), anti-ERK1/2 C-9 (cat. no. sc-514302; 1:500), anti-p38-PO4 (cat. no. sc-166182; 1:1,000), anti-p38 (cat. no. sc-535; 1:500), anti-MKK3 (cat. no. sc-961; 1:500), anti-cyclin D1 (cat. no. sc-20044; 1:1,000), anti-p21 (cat. no. sc-6246; 1:200), anti-GAPDH (cat. no. sc-47724; 1:500) and anti-tubulin (cat. no. sc-5286; 1:500) (all from Santa Cruz Biotechnology, Inc.); anti MKK6-PO4 D8E9 (MEK3/6) (cat. no. 12280; 1:1,000), anti-MKK6 D31D1 (cat. no. 8550; 1:1,000), anti-AKT-PO4 Thr308 (cat. no. 4056; 1:1,000), anti-AKT-PO4 Ser473 (cat. no. 9271; 1:1,000) and AKT (cat. no. 9272; 1:1,000) (all from Cell Signalling Technology, Inc.); anti-myosin heavy chain (MHC; cat. no. MF20; 1:300) and anti-myogenin (cat. no. F5D; 1:300) were monoclonal from hybridoma supernatant (all from Developmental Studies Hybridoma Bank).

Techniques:

Journal: Cell Death & Disease

Article Title: MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis

doi: 10.1038/s41419-021-03729-1

Figure Lengend Snippet: (A , B) Cells were transfected with shRNA against MTA2 (shMTA2) and siRNA against MEK3 (si-MEK3) and then subjected to ( A ) migration and invasion assay or ( B ) immunodetection of MTA2, MEK3, p38, and MMP12. ( C , D) Cells were transfected with shRNA against MTA2 (shMTA2) and siRNA against ASK1 (si-ASK1) and then subjected to ( C ) migration and invasion assay or ( D ) immunodetection of MTA2, ASK1, MEK3, p38, and MMP12. β-actin signal was used as internal control. ** and #, P < 0.01 and P < 0.05 compared with shLuc and shMTA2 cells alone, respectively.

Article Snippet: The antibodies source and dilution factor: MTA2 (sc-55566; 1:1000), ERK (sc-514302; 1:1000), p-p38 mitogen activated protein kinase (p-p38) (sc-166182; 1:1000), p38 (sc-7972; 1:1000), p-JNK (sc-6254; 1:1000), JNK (sc-571; 1:1000), ASK1 (sc-5294; 1:1000), p-MEK3 (sc-8407; 1:1000), MEK3 (sc-960; 1:1000), c-Jun (sc-74543; 1:1000), c-Fos (sc-8047; 1:1000), lamin B (sc-374015; 1:2000), YB1 (sc-18057; 1:1000), β-actin (sc-69879; 1:2000), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG (1:10000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, shRNA, Migration, Invasion Assay, Immunodetection, Control

Journal: Cell Death & Disease

Article Title: MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis

doi: 10.1038/s41419-021-03729-1

Figure Lengend Snippet: SiHa cells were transfected with shRNA against MTA2 (shMTA2) and then subjected to ( A ) nucleus fractionation and immunodetection of nuclear MTA2, phosphylated YB1 (p-YB1), and YB1 or ( B ) immunofluorescent detection of MTA2, p-YB1, and MMP12 by confocal microscopy. ( C – E) Cells were transfected with shMTA2 and siRNA against YB1 (si-YB1) and then subjected to ( C ) migration and invasion assay, ( D ) AP1 reporter assay, or ( E ) MMP12 mRNA assessment via qRT-PCR. F Cells were transfected with shMTA2 and then subjected to immunoprecipitation by using anti-p-YB1 antibody and immunodetection of the indicated poteins. ( G , H) Cells were transfected with shMTA2 combined with siRNA against ASK1 (si-ASK1), MEK3 (si-MEK3), or p38 (si-p38) and then subjected to ( G ) immunodetection of the indicated proteins or ( H ) MMP12 mRNA expression assessment via qRT-PCR. ** and #, P < 0.01 and P < 0.05 compared with shLuc and shMTA2 cells alone, respectively.

Article Snippet: The antibodies source and dilution factor: MTA2 (sc-55566; 1:1000), ERK (sc-514302; 1:1000), p-p38 mitogen activated protein kinase (p-p38) (sc-166182; 1:1000), p38 (sc-7972; 1:1000), p-JNK (sc-6254; 1:1000), JNK (sc-571; 1:1000), ASK1 (sc-5294; 1:1000), p-MEK3 (sc-8407; 1:1000), MEK3 (sc-960; 1:1000), c-Jun (sc-74543; 1:1000), c-Fos (sc-8047; 1:1000), lamin B (sc-374015; 1:2000), YB1 (sc-18057; 1:1000), β-actin (sc-69879; 1:2000), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG (1:10000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, shRNA, Fractionation, Immunodetection, Confocal Microscopy, Migration, Invasion Assay, Reporter Assay, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: Cell Death & Disease

Article Title: MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis

doi: 10.1038/s41419-021-03729-1

Figure Lengend Snippet: Our findings indicate that MTA2 knockdown induces activation of the ASK1/MEK3/p38 cascade, which subsequently leads to YB1 phosphorylation and the binding of p-YB1 and AP1, inhibiting AP1 transcriptional activity and MMP12 expression, thereby reducing cervical cancer metastasis.

Article Snippet: The antibodies source and dilution factor: MTA2 (sc-55566; 1:1000), ERK (sc-514302; 1:1000), p-p38 mitogen activated protein kinase (p-p38) (sc-166182; 1:1000), p38 (sc-7972; 1:1000), p-JNK (sc-6254; 1:1000), JNK (sc-571; 1:1000), ASK1 (sc-5294; 1:1000), p-MEK3 (sc-8407; 1:1000), MEK3 (sc-960; 1:1000), c-Jun (sc-74543; 1:1000), c-Fos (sc-8047; 1:1000), lamin B (sc-374015; 1:2000), YB1 (sc-18057; 1:1000), β-actin (sc-69879; 1:2000), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG (1:10000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Activation Assay, Phospho-proteomics, Binding Assay, Activity Assay, Expressing