Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A, Schematic diagram of high throughput screen of 2123 Selleckchem bioactive compound library at 100nM alone or in combination with the IC20 of TTM (dashed red line) in A375 or WM88. B,C, Normalized Percent Inhibition (NPI) of TTM + Compound versus NPI Compound graph for indicated cells treated with Selleckchem bioactive compound library alone or in combination with IC20 of TTM. Hits (blue circles) are defined as NPI TTM + Compound ≥ 20 (dashed red line). D,E, Observed effect versus expected effect graph for indicated cells of Hits from B and C. Hits (blue circles) are defined as Bliss Index ≥ 1.5 (dashed red line). F,G, Graphical representation of Hits defined as NPI TTM + Compound ≥ 20 and Bliss Index ≥ 1.5 in indicated cells. H, Venn diagram relationship between Compound Hits. I, NPI of 5 overlapping Hit Compounds from H alone or in combination with IC20 of TTM (dashed red line). J,K, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with vehicle or indicated concentrations of drugs. L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of drugs. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: High Throughput Screening Assay, Drug discovery, Inhibition

Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A,C, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells stably expressing two independent Dox-inducible shRNAs (#1 and #2) against indicated genes treated with indicated concentrations of TTM without or with Dox n=3. B,D, Scatter dot plot of TTM IC50 ± s.e.m in indicated cells cells stably expressing two independent Dox-inducible shRNAs against indicated genes treated without or with Dox. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3. E,G, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. F,H, Scatter dot plot of BH3 mimetic IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. I,J, Graphical representation of Bliss Index (observed effect versus expected effect) from A375 (E) and WM88 (G) at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: Stable Transfection, Expressing

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