Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Primers used for quantitative PCR analyses.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Narrowing down of the domain involved in the induction of COL1A1 expression in LX-2 cells. (A) Overexpression of both full-length fibrinogen domain of TNX [TNX-FG] and ITGA11 failed to induce the expression of fibrosis marker genes, including ACTA2, COL1A1 and TGFB1 . LX-2 cells were transfected with expression vectors for hTNX-FG (lane 2), ITGA11 (lane 3) and hTNX-FG and ITGA11 (lane 4) in DMEM/0.5% FBS. (B) Induction of COL1A1 expression by overexpression of both hTNX-FGF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGF (lane 2), hTNX-FGL (lane 3), ITGA11 (lane 4), hTNX-FGF and ITGA11 (lane 5) and hTNX-FGL and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Induction of COL1A1 expression by overexpression of both hTNX-FGFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFF (lane 2), hTNX-FGFL (lane 3), ITGA11 (lane 4), hTNX-FGFF and ITGA11 (lane 5) and hTNX-FGFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (D) Induction of COL1A1 expression by overexpression of both hTNX-FGFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFF (lane 2), hTNX-FGFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFF and ITGA11 (lane 5) and hTNX-FGFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (A-D) As a control, RNA from the cells without transfection (lane 1) was used. The cell lysate was prepared 48 h after transfection and then RNA was purified. Subsequently, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. The expression level of each gene in the control was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FG, fibrinogen-related domain of human tenascin-X; hTNX-FGF, first half of hTNX-FG; hTNX-FGL, latter half of hTNX-FG; hTNX-FGFF, first half of hTNX-FGF; hTNX-FGFL, latter half of hTNX-FGF; hTNX-FGFFF, first half of hTNX-FGFF; hTNX-FGFFL, latter half of hTNX-FGFF.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Marker, Transfection, Control, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Identification of the minimal sequence responsible for induction of COL1A1 expression in LX-2 cells. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (B) Overexpression of both hTNX-FGpeptide2-5 and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGpeptide2-5 (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGpeptide2-5 and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Overexpression of both hTNX-FGFFFM and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFM (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFM and ITGA11 (lane 6) in DMEM/0.5% FBS. After transfection followed by cell culture, cell lysate extraction and RNA purification, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. (A-C) The expression level of each gene in the control (lane 1, RNA from cells without transfection) was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG); hTNX-FGFFFM, PRDCGEEMQNGAGAS peptide from hTNX-FG; hTNX-FGFFFL, QNGAGASRTSTIFL peptide from hTNX-FG; hTNX-FGpeptide2-5, GGLRIPF peptide from hTNX-FG.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Sequencing, Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 in addition to inhibitors. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a TGFBRI inhibitor (SB525334). DMSO (lanes 1, 2 and 3) and SB525334 (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). (B) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a YAP inhibitor (verteporfin). DMSO (lanes 1, 2 and 3) and vertepofin (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). Subsequently, the cells were cultured followed by cell lysate extraction, RNA purification and reverse transcription-quantitative PCR. (A and B) The expression level of each gene [ ACTA2, COL1A1 and TGFB1 for (A) and ACTA2, COL1A1 and YAP1 for (B)] in the control (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (lane 1) is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1); ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP, Yes-associated protein; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Effect of YAP1 knockdown on induction of COL1A1 expression. YAP1 was knocked down with YAP1 siRNA prior to overexpression of both hTNX-FGFFFF and ITGA11, and then the expression levels of ACTA2 and COL1A1 were analyzed by reverse transcription-quantitative PCR in LX-2 cells. RNA from cells treated with transfection of control siRNA only (lane 1), transfection of control siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 2) and transfection of YAP1 siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 3) was used. The expression level of each gene ( ACTA2, COL1A1 and YAP1 ) in the control (control siRNA only) (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (control siRNA) is shown (n=3). Data are presented as the mean ± SD. **P<0.01 vs. control (lane 1); # P<0.05, ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. At the bottom of the figure, the relative expression levels of YAP1, hTNX-FGFFFF and ITGA11 are also shown, setting lane 2 to 1.0, since hTNX-FGFFFF expression was not detected in lane 1. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP1, Yes-associated protein 1; siRNA, small interfering RNA; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Knockdown, Expressing, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Cotransfection, Small Interfering RNA