Article Figures & Data
Figures
- Fig. 1.
Changes of gene expression by knockdown of G9a. (A) Efficiency of knockdown of G9a at mRNA (left) and protein (right) levels in HepaRG cells at day 4 after transfection with siG9a in comparison with siNC. CYP3A4 protein was also determined. ACTB was used as a housekeeping protein for a loading control. (B) Changes of mRNA expression of P450 genes at day 4 after siRNA treatment. RT-PCR data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. (C) Enzyme activity of CYP3A4 at day 4 after siRNA treatment. Enzyme activity of CYP3A4 was determined by using the P450-Glo CYP3A4 assay. Fold changes of siG9a treated cells were calculated by comparing with the siNC control. The sample size for each treatment group is n = 3 to 4. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
- Fig. 2.
Changes of gene expression by overexpression of G9a. (A) Efficiency of G9a overexpression at mRNA (left) and protein (right) levels in HepaRG cells at day 4 after transduction with AdV-G9a vs. AdV-GFP. CYP3A4 protein was also determined. ACTB was used as a housekeeping protein for a loading control. (B) Changes of expression of P450 genes at day 4 after adenovirus treatment. RT-PCR data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. (C) Enzyme activity of CYP3A4 at day 4 after adenovirus treatment. Enzyme activity of CYP3A4 was determined by using the P450-Glo CYP3A4 assay. Fold changes of AdV-G9a treated cells were calculated by comparing with the AdV-GFP control. The sample size for each treatment group is n = 3. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
- Fig. 3.
Changes of mRNA expression of nuclear receptor genes by knockdown (A) and overexpression (B) of G9a. Expression of nuclear receptor genes at day 4 after siRNAs or adenovirus treatment was quantified by RT-PCR. The sample size for each treatment group is n = 3. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
- Fig. 4.
(A) Accumulation of fatty acids in HepaRG cells visualized using Oil Red O staining 24 hours after treatment. (B) Cellular viability measured using quantification of ATP by CellTiter-GLO assay after 24 and 48 hours post-treatment. (C) Cellular damage caused by fatty acid treatment measured using lactate dehydrogenase release assay after 24 and 48 hours post-treatment. (D) Intracellular accumulation of triglycerides upon fatty acid treatment quantified using fluorescence assay at 24 hours post-treatment. (E) mRNA expression of CYP3A4 at 24 hours after fatty acid treatment measured using RT-PCR. (F) Protein expression of CYP3A4 using Western blot. Data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. The sample size for each treatment group is n = 3 to 4. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; **P < 0.01 compared with the DMSO treated controls using two-tailed unpaired Student’s t test.
- Fig. 5.
Changes of mRNA expression of P450 genes after knockdown (A) and overexpression (B) of G9a followed by fatty acid treatment at 24 hours.RT-PCR data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. Fold changes of siNC OA/PA treated cells were calculated by comparing with the siNC DMSO control, siG9a OA/PA treated cells were calculated by comparing with siG9a DMSO control, AdV-GFP OA/PA treated cells were calculated by comparing with AdV-GFP DMSO control, and AdV-G9a OA/PA treated cells were calculated by comparing with AdV-G9a DMSO control. The sample size for each treatment group is n = 3. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
- Fig. 6.
Changes of mRNA expression of nuclear receptors after knockdown (A) and overexpression (B) of G9a followed by fatty acid treatment at 24 hours. RT-PCR data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. Fold changes of siNC OA/PA treated cells were calculated by comparing with the siNC DMSO control, siG9a OA/PA treated cells were calculated by comparing with siG9a DMSO control, AdV-GFP OA/PA treated cells were calculated by comparing with AdV-GFP DMSO control, and AdV-G9a OA/PA treated cells were calculated by comparing with AdV-G9a DMSO control. The sample size for each treatment group is n = 3. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
- Fig. 7.
Changes of mRNA expression of FGF21 gene by knockdown (A) and overexpression (B) of G9a at day 4. Changes of mRNA expression of nuclear receptors after knockdown (C) and overexpression (D) of G9a followed by fatty acid treatment at 24 hours. RT-PCR data analysis was performed using 2−ΔΔCT method. Mean cycle threshold values of TBP and GAPDH were used for ΔCT calculations. The fold change of siG9a was calculated by comparing with siNC, AdV-G9a with AdV-GFP, siG9a OA/PA with siG9a DMSO, and AdV-G9a OA/PA with AdV-G9a DMSO. The sample size for each treatment group is n = 3. Data are presented as means ± S.D. from a single experiment. The experiment was repeated once to confirm the findings. **P < 0.01; ***P < 0.001 compared with the controls using two-tailed unpaired Student’s t test.
