Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Cytotoxicity of 13CRA on cholangiocarcinoma cells. (A) KKU-100 and (B) KKU-213B cells were treated with various concentrations of 13CRA for 12, 24 and 48 h. Cell viability was measured following treatment using sulforhodamine B assay, and the percentage of cytotoxicity was calculated. Data are presented as the mean ± SD from three independent experiments. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Sulforhodamine B Assay

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: 13CRA inhibits the self-renewal ability of cholangiocarcinoma cells. (A) KKU-100 and (B) KKU-213B cells were treated with various concentrations of 13CRA for 48 h. The viable-treated cells were harvested, and only 600 cells were cultured into each well for an additional 8 days to allow colony formation. The number of colonies was counted and calculated as the percentage of colonies formed compared to the untreated control. Data are presented as the mean ± SD from three independent experiments. * P<0.05 and ** P<0.01 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Cell Culture, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: 13CRA arrests the cell cycle at the G 2 /M phase in cholangiocarcinoma cells. (A) KKU-100 and (B) KKU-213B cells were treated with 1.25 or 2.5 µ M 13CRA for 48 h, and the cell cycle distribution was then examined using flow cytometry with propidium iodide DNA staining. Data are presented as the mean ± SD from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Flow Cytometry, Staining, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Effects of 13CRA on the expression of cell cycle regulatory genes in cholangiocarcinoma cells. KKU-100 and KKU-213B cells were treated with 1.25 or 2.5 µ M 13CRA for 48 h, and the expression levels of c-Myc , p21 and cyclin B1 genes were determined using reverse transcription-quantitative PCR. Quantification of the mRNA levels of (A) c-Myc , (B) cyclin B1 and (C) p21 in KKU-100 and (D) c-Myc , (E) cyclin B1 and (F) p21 in KKU-213B cells, which were normalized to those of β-actin , which acted as an internal control. Data are presented as the mean ± SD from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Effects of 13CRA on the expression of cell cycle-regulatory proteins in cholangiocarcinoma cells. (A) KKU-100 and KKU-213B cells were treated with 1.25 or 2.5 µ M 13CRA for 48 h, and the expression levels of p53, p21 and cyclin B1 proteins were assessed using western blot analysis. The intensity of the (B) p53, (C) p21 and (D) cyclin B1 protein bands in the KKU-100 and (E) p53, (F) p21 and (G) cyclin B1 in the KKU-213B cell lysates was quantified by normalization to β-actin. (H) The protein level of cyclin D1 and the intensity of the cyclin D1 bands in (I) KKU-100 and (J) KKU-213B cell lysates was quantified by normalization to β-actin. Data are presented as the mean ± SD from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Effects of 13CRA on the migration of cholangiocarcinoma cells. (A) KKU-100 and (B) KKU-213B cells were seeded into a Transwell plate, and treated with 1.25 and 2.5 µ M 13CRA for 24 h. The number of migrated cells was counted and calculated as a percentage of the migrated cells of the untreated control. Representative images of cell migration were captured under a light microscope (magnification, ×10). Data are presented as the mean ± SD from three independent experiments. *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Migration, Control, Light Microscopy

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Effects of 13CRA on the expression of epithelial-mesenchymal transition-related genes and proteins in cholangiocarcinoma cells. KKU-100 and KKU-213B cells were treated with 1.25 or 2.5 µ M 13CRA for 24 h, and expression levels of E-cad , Snail and vimentin genes were determined using reverse transcription-quantitative PCR. The mRNA levels of E-cad , Snail and vimentin in (A-C) KKU-100 and (D-F) KKU-213B cells were quantified by normalization to β-actin . (G) The expression levels of E-cad and vimentin proteins were assessed using western blot analysis and the intensity of the E-cad and vimentin bands was quantified by normalization to β-actin. Data are presented as the mean ± SD from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid; E-cad, E-cadherin.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: 13CRA inhibits the invasion and adhesion of cholangiocarcinoma cells. To examine cell invasion, the (A) KKU-100 and (B) KKU-213B cells were seeded into a Transwell pleated coated with Matrigel, and treated with 1.25 or 2.5 µ M 13CRA for 48 h. The numbers of invaded cells were counted and calculated as percentages of cell invasion vs. the untreated control. To examine cell adhesion, following treatment with 13CRA for 48 h, the (C) KKU-100 and (D) KKU-213B cells were harvested and seeded into a 96-well plate coated with fibronectin. Following 30 min of incubation, the cells were stained with crystal violet, their absorbance at 540 nm was read, and the percentage of cell adhesion was calculated. Representative images of cell adhesion and invasion were captured under a light microscope (magnification, ×10). Data are presented as the mean ± SD from three independent experiments. ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Control, Incubation, Staining, Light Microscopy

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: 13CRA downregulates the expression of adhesion- and invasion-related genes and proteins in cholangiocarcinoma cells. (A-D) KKU-100 and (E-H) KKU-213B cells were treated with 1.25 or 2.5 µ M 13CRA for 48 h. The expression levels of ICAM-1, COX-2 , MMP-2 and MMP-9 genes were determined using reverse transcription-quantitative PCR, and the quantification of the mRNA levels of ICAM-1 , COX-2 , MMP-2 and MMP-9 was performed via normalization to β-actin . (I) The expression levels of ICAM-1 and MMP9 proteins were assessed using western blot analysis and the intensity of the ICAM-1 and MMP9 bands was quantified by normalization to β-actin. Data are presented as the mean ± SD from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. the untreated control. 13CRA, 13- cis -retinoic acid; MMP , matrix metalloproteinase; ICAM-1 , intercellular adhesion molecule-1; COX-2 , cyclooxygenase-2.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: 13- cis -retinoic acid inhibits the self-renewal, migration, invasion and adhesion of cholangiocarcinoma cells

doi: 10.3892/ijmm.2023.5223

Figure Lengend Snippet: Inhibitory effects of 13CRA on the proliferation, migration, invasion and adhesion of CCA cells, and the possible underlying mechanisms. 13CRA, 13- cis -retinoic acid. MMP, metalloproteinase; ICAM-1, intercellular adhesion molecule-1; COX-2, cyclooxygenase-2; CCA, cholangiocarcinoma.

Article Snippet: The CCA cells were seeded in a six-well plate at a density of 2.5×10 5 cells/well overnight and then treated with 0.00, 1.25 and 2.50 µ M 13CRA in serum-free medium for 24 h. Following treatment, total RNA was isolated using TRIzol™ reagent (15596026, Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA synthesis was performed in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc.) using 5X iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. cDNA served as a template for qPCR amplification.

Techniques: Migration