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Figure 4. MYLK-AS1 activates <t>EGFR/HER2-ERK</t> signaling pathway in HCC. (A) BEL-7402 cells were transfected with MYLK-AS1 siRNAs (100 nM) or control siRNA (100 nM). The MYLK-AS1 knockdown effect was detected by RT-qPCR. Western blot was performed to determine the expression of EGFR/HER2-ERK signaling pathway-related genes as indicated. β-actin was used as a loading control. (B) MYLK-AS1 siRNAs (50 nM, 100 nM and 200 nM) or control siRNA (200 nM) were transfected into MHCC97-H cells. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). (C) HepG2 cells were transfected with MYLK-AS1 (5 μg) or empty vector. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). All experiments were conducted three times independently and representative immunoblot results were shown. Data were presented as the mean ± SD (*P < 0.05, **P < 0.01).
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Figure 4. MYLK-AS1 activates EGFR/HER2-ERK signaling pathway in HCC. (A) BEL-7402 cells were transfected with MYLK-AS1 siRNAs (100 nM) or control siRNA (100 nM). The MYLK-AS1 knockdown effect was detected by RT-qPCR. Western blot was performed to determine the expression of EGFR/HER2-ERK signaling pathway-related genes as indicated. β-actin was used as a loading control. (B) MYLK-AS1 siRNAs (50 nM, 100 nM and 200 nM) or control siRNA (200 nM) were transfected into MHCC97-H cells. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). (C) HepG2 cells were transfected with MYLK-AS1 (5 μg) or empty vector. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). All experiments were conducted three times independently and representative immunoblot results were shown. Data were presented as the mean ± SD (*P < 0.05, **P < 0.01).

Journal: International journal of biological sciences

Article Title: Long noncoding RNA MYLK-AS1 promotes growth and invasion of hepatocellular carcinoma through the EGFR/HER2-ERK1/2 signaling pathway.

doi: 10.7150/ijbs.43062

Figure Lengend Snippet: Figure 4. MYLK-AS1 activates EGFR/HER2-ERK signaling pathway in HCC. (A) BEL-7402 cells were transfected with MYLK-AS1 siRNAs (100 nM) or control siRNA (100 nM). The MYLK-AS1 knockdown effect was detected by RT-qPCR. Western blot was performed to determine the expression of EGFR/HER2-ERK signaling pathway-related genes as indicated. β-actin was used as a loading control. (B) MYLK-AS1 siRNAs (50 nM, 100 nM and 200 nM) or control siRNA (200 nM) were transfected into MHCC97-H cells. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). (C) HepG2 cells were transfected with MYLK-AS1 (5 μg) or empty vector. The MYLK-AS1 overexpression effect was measured by RT-qPCR. Western blot was performed as in (A). All experiments were conducted three times independently and representative immunoblot results were shown. Data were presented as the mean ± SD (*P < 0.05, **P < 0.01).

Article Snippet: After blocking, the membranes were then incubated with primary antibodies against EGFR (Santa Cruz Biotechnology), phosph-EGFR (Santa Cruz Biotechnology), HER2 (Santa Cruz Biotechnology), RAS (Santa Cruz Biotechnology), RAF1 (Santa Cruz Biotechnology), MEK1/2 (Cell Signaling), phosph-MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling), phosph-ERK1/2 (Cell Signaling), and β-actin (Santa Cruz Int.

Techniques: Transfection, Control, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Plasmid Preparation

Figure 5. MYLK-AS1 regulates proliferation and invasion of HCC cells through the EGFR/HER2-ERK signaling pathway. (A) HepG2 cells were transfected with pcDNA3.0-MYLK-AS1 (MYLK-AS1) vector or empty vector (EV). Cells were treated with PD98059 or GW583340 as indicated, with DMSO as control. Cell proliferation was then determined by CCK-8 assay. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results (** P < 0.01 at day 4). (B) HepG2 cells were transfected as in (A). The cell invasion changes were detected by transwell assay. All experiments were conducted three times independently, and the data were presented as the mean ± SD (**P < 0.01). (C) Representative immunoblot of HepG2 cells transfected as in (A) with the indicated antibodies. The experiments have been repeated 3 times with similar results. (D and E) HepG2 cells were transfected with ERK siRNA or EGFR siRNA as indicated, with control siRNA as control. Twenty four hours later, the cells were transfected with pcDNA3.0-MYLK-AS1 (MYLK-AS1) vector or empty vector (EV) as indicated. After 24 h, the transfected cells were collected and replated into new wells as indicated. Cell proliferation was then determined by CCK-8 assay for the indicated times. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results (** P < 0.01 at day 4).

Journal: International journal of biological sciences

Article Title: Long noncoding RNA MYLK-AS1 promotes growth and invasion of hepatocellular carcinoma through the EGFR/HER2-ERK1/2 signaling pathway.

doi: 10.7150/ijbs.43062

Figure Lengend Snippet: Figure 5. MYLK-AS1 regulates proliferation and invasion of HCC cells through the EGFR/HER2-ERK signaling pathway. (A) HepG2 cells were transfected with pcDNA3.0-MYLK-AS1 (MYLK-AS1) vector or empty vector (EV). Cells were treated with PD98059 or GW583340 as indicated, with DMSO as control. Cell proliferation was then determined by CCK-8 assay. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results (** P < 0.01 at day 4). (B) HepG2 cells were transfected as in (A). The cell invasion changes were detected by transwell assay. All experiments were conducted three times independently, and the data were presented as the mean ± SD (**P < 0.01). (C) Representative immunoblot of HepG2 cells transfected as in (A) with the indicated antibodies. The experiments have been repeated 3 times with similar results. (D and E) HepG2 cells were transfected with ERK siRNA or EGFR siRNA as indicated, with control siRNA as control. Twenty four hours later, the cells were transfected with pcDNA3.0-MYLK-AS1 (MYLK-AS1) vector or empty vector (EV) as indicated. After 24 h, the transfected cells were collected and replated into new wells as indicated. Cell proliferation was then determined by CCK-8 assay for the indicated times. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results (** P < 0.01 at day 4).

Article Snippet: After blocking, the membranes were then incubated with primary antibodies against EGFR (Santa Cruz Biotechnology), phosph-EGFR (Santa Cruz Biotechnology), HER2 (Santa Cruz Biotechnology), RAS (Santa Cruz Biotechnology), RAF1 (Santa Cruz Biotechnology), MEK1/2 (Cell Signaling), phosph-MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling), phosph-ERK1/2 (Cell Signaling), and β-actin (Santa Cruz Int.

Techniques: Transfection, Plasmid Preparation, Control, CCK-8 Assay, Transwell Assay, Western Blot

Figure 6. Knockdown of MYLK-AS1 inhibits tumor growth in vivo. (A) Nude mice were seeded with MYLK-AS1 shRNA-expressing MHCC97-H cells and control shRNA-expressing MHCC97-H cells. The volume of the tumors was calculated every week after transplantation (mean ± SD; n = 10). The mice were killed 21 d after implantation and the weight of the tumors was measured. **P < 0.01 at day 21. (B) Representative immunoblot of the tumors from (A) with the indicated antibodies. (C) A proposed model underlying the role of MYLK-AS1 in hepatoma cell proliferation, migration and invasion via regulation of the EGFR/HER2-ERK1/2 pathway.

Journal: International journal of biological sciences

Article Title: Long noncoding RNA MYLK-AS1 promotes growth and invasion of hepatocellular carcinoma through the EGFR/HER2-ERK1/2 signaling pathway.

doi: 10.7150/ijbs.43062

Figure Lengend Snippet: Figure 6. Knockdown of MYLK-AS1 inhibits tumor growth in vivo. (A) Nude mice were seeded with MYLK-AS1 shRNA-expressing MHCC97-H cells and control shRNA-expressing MHCC97-H cells. The volume of the tumors was calculated every week after transplantation (mean ± SD; n = 10). The mice were killed 21 d after implantation and the weight of the tumors was measured. **P < 0.01 at day 21. (B) Representative immunoblot of the tumors from (A) with the indicated antibodies. (C) A proposed model underlying the role of MYLK-AS1 in hepatoma cell proliferation, migration and invasion via regulation of the EGFR/HER2-ERK1/2 pathway.

Article Snippet: After blocking, the membranes were then incubated with primary antibodies against EGFR (Santa Cruz Biotechnology), phosph-EGFR (Santa Cruz Biotechnology), HER2 (Santa Cruz Biotechnology), RAS (Santa Cruz Biotechnology), RAF1 (Santa Cruz Biotechnology), MEK1/2 (Cell Signaling), phosph-MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling), phosph-ERK1/2 (Cell Signaling), and β-actin (Santa Cruz Int.

Techniques: Knockdown, In Vivo, shRNA, Expressing, Control, Transplantation Assay, Western Blot, Migration

Western blot to evaluate cell cycle related protein expressions (A) and protein abundance of Wnt/β-catenin pathway (B) . Analysis of GEO database shows correlation between DACH1 and β-catenin downstream targets Nr-CAM (C) , DKK1 (D) , PTTG1 (E) and SOX9 (F) by one-way ANOVA test.

Journal: Oncotarget

Article Title: DACH1 is a novel predictive and prognostic biomarker in hepatocellular carcinoma as a negative regulator of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: Western blot to evaluate cell cycle related protein expressions (A) and protein abundance of Wnt/β-catenin pathway (B) . Analysis of GEO database shows correlation between DACH1 and β-catenin downstream targets Nr-CAM (C) , DKK1 (D) , PTTG1 (E) and SOX9 (F) by one-way ANOVA test.

Article Snippet: The primary antibodies used were rabbit polyclone DACH1 (ProteinTech group), β-catenin (ProteinTech group), phosph-β-catenin (Santa Cruz), GSK3β (ProteinTech group), phosph-GSK3β (Santa Cruz), cyclin D1 (Cell Signaling Technology), β-actin (Cell Signaling Technology), CDK4 (Santa Cruz), cyclin B1 (Santa Cruz), cyclin E1 (Santa Cruz), PCNA (Santa Cruz) and α-vinculin (Sigma).

Techniques: Western Blot, Quantitative Proteomics