p450-glo cyp3a4 (luciferin-pfbe) cell-based/biochemical assay kit  (Promega)

Journal: Stem cells (Dayton, Ohio)

Article Title: MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

doi: 10.1002/stem.2283

Figure Lengend Snippet: Upregulation of miR-194 upon hepatocytic differentiation of HepaRG liver progenitor cells. HepaRG cells were induced to differentiate to hepatocyte-like cells as described in Materials and Methods. (A): Pictures were taken at different phases of the culture: progenitor cells (day 2), intermediate (Day 14), and hepatocyte-like cells (day 28). h, hepatocyte-like areas; b, biliary-like areas. Bars, 50 μm. (B): Levels of HNF4A, ALDOB and CYP3A4 mRNAs during hepatocytic differentiation of HepaRG cells measured by quantitative PCR and shown as fold-changes and SEM, at day 14 and day 28 relative to day 2, in two independent experiments. (C): Mean bile canaliculi densities from three randomly selected fields in two independent experiments. (D): Graphs show quantification of secreted human albumin and CYP3A4 activity in HepaRG cells at day 2 and day 28 of the hepatocytic differentiation protocol. (E): Levels of miR-122 and miR-194 during hepatocytic differentiation of HepaRG cells measured by quantitative PCR and shown as fold-changes and SEM, at day 14 and day 28 relative to day 2, in two independent experiments.

Article Snippet: To check the induction of cytochrome P450 activities upon hepatocytic differentiation, we used the P450-GloTM CYP3A4 Assay (Luciferin-PFBE) Kit (Promega).

Techniques: Real-time Polymerase Chain Reaction, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

doi: 10.1002/stem.2283

Figure Lengend Snippet: Upregulation of miR-194 upon directed hepatocytic differentiation of hESCs. Differentiation of hESCs toward a hepatocyte fate was performed as described in Materials and Methods. (A): Images showing sequential morphological changes during differentiation from hESCs (pluripotent hESCs + ROCK inhibitor: day 0) to mature hepatocytes (day 20) through definitive endoderm (day 5), hepatic progenitor cells (day 10) and immature hepatocytes (day 15). Hepatocyte morphology including binucleated cells (black arrow). Bars, 50 μm. (B): Quantitative PCR was performed to measure the levels of hepatocyte markers (HNF4A, ALDOB, and CYP3A4) and miR-194 during the hepatocyte differentiation process of hESCs. The graphs show fold-changes and SEM relative to Day 0 in two independent experiments. (C): Western blotting was performed to measure the levels of OCT4 and ALB during the hepatocyte differentiation process of hESCs. Expression of α-tubulin was used as a protein loading control. (D): Immunofluorescence staining of SOX2 (green color, upper panel) and ALB (green color, lower panel) in hESCs at day 0 and day 20 of the hepatocytic differentiation protocol. Nuclei are stained with DAPI blue. Scale bar, 50 μm. (E): Graphs show quantification of secreted human albumin and CYP3A4 activity in hESCs at day 0 and day 20 of the hepatocytic differentiation protocol. (F): Level of miR-194 during hepatocytic differentiation of hESCs measured by quantitative PCR. The graphs show fold-changes and SEM relative to Day 0 in two independent experiments.

Article Snippet: To check the induction of cytochrome P450 activities upon hepatocytic differentiation, we used the P450-GloTM CYP3A4 Assay (Luciferin-PFBE) Kit (Promega).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, Immunofluorescence, Staining, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

doi: 10.1002/stem.2283

Figure Lengend Snippet: Overexpression of miR-194 accelerates hepatocytic differentiation of HepaRG cells. (A): Expression levels of miR-194 determined by quantitative PCR in HepaRG-empty and HepaRG-miR-194 cell lines. (B): Images of HepaRG-empty and HepaRG-miR-194 cell lines were taken at different phases of the culture: progenitor cells (day 2), intermediate (day 14), and hepatocyte-like cells (day 28). Bars, 50 μm. (C): Mean bile canaliculi densities assessed from three randomly selected fields in two independent experiments at day 14 of hepatocytic differentiation in HepaRG-empty and HepaRG-miR-194 cell lines. (D): Levels of HNF4A, ALDOB, ALB and miR-122 at day 14 of the differentiation process in HepaRG-miR-194 cell lines compared to HepaRG-empty cell lines. (E): Graphs show quantification of secreted human albumin and CYP3A4 activity from HepaRG-empty and HepaRG-miR-194 cell lines at day 28 of hepatocytic differentiation. The results were presented as means ± SEM from two independent experiments (P < 0.05).

Article Snippet: To check the induction of cytochrome P450 activities upon hepatocytic differentiation, we used the P450-GloTM CYP3A4 Assay (Luciferin-PFBE) Kit (Promega).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

doi: 10.1002/stem.2283

Figure Lengend Snippet: YAP1 inhibition induces hepatocytic differentiation in HepaRG cells. (A): YAP1 mRNA and protein expression levels determined respectively by quantitative PCR (left panel) and western blot analysis (right panel) in HepaRG-empty and HepaRG-YAP1 KD cell lines. α-tubulin was used to control protein loading. (B): Mean bile canaliculi densities of HepaRG-empty and HepaRG-YAP1 KD cell lines assessed from three randomly selected fields in two independent experiments. (C): Relative expression levels of HNF4A at day 14 and of HNF4A, ALDOB, CYP3A4 and ALB at day 28 of hepatocytic differentiation process in HepaRG-YAP1 KD compared to HepaRG-empty cell lines.

Article Snippet: To check the induction of cytochrome P450 activities upon hepatocytic differentiation, we used the P450-GloTM CYP3A4 Assay (Luciferin-PFBE) Kit (Promega).

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