Journal: Molecules

Article Title: Integrated UHPLC-Q-TOF/MS and Liver-on-a-Chip Evaluation of Chemical Composition Changes and Hepatotoxicity Differences in Yaomu Before and After Fermentation

doi: 10.3390/molecules31060994

Figure Lengend Snippet: Function verification and Oxidative stress of liver-on-a-chip. ( A ) Fluorescence imaging of CYP3A4 for THLE-2 cells cultured with liver-on-a-chip model. ( B ) Semi-quantitative results of CYP3A4 immunofluorescence. ( C ) After 12 h of dynamic perfusion in the blank group, perfusion administration of WFJ (7.8 mg/mL) and YM (7.8 mg/mL), and the released amount of ALB. ( D ) After 12 h of dynamic perfusion in the blank group, perfusion administration of WFJ (7.8 mg/mL) and YM (7.8 mg/mL), and the released amount of ALT. ( E ) After 12 h of dynamic perfusion in the blank group, perfusion administration of WFJ (7.8 mg/mL) and YM (7.8 mg/mL), and the released amount of AST. ( F ) Fluorescence imaging of ROS for THLE-2 cells cultured with liver-on-a-chip model. ( G ) Semi-quantitative results of ROS immunofluorescence. Significance levels indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The CYP3A4 rabbit polyclonal antibody (pAb) was purchased from Abclonal (Cat. No. A2544), the Cy3-conjugated goat anti-rabbit IgG secondary antibody was obtained from Absin (Cat. No. abs20024), and the cleaved caspase-3 polyclonal antibody was purchased from Proteintech (Cat. No. 25128-1-AP).

Techniques: Fluorescence, Imaging, Cell Culture, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO governs the activities of CYP2D6 and 3A4 in cells by modulating their heme content in a concentration-dependent bimodal fashion. GlyA-CHO cells underwent transfection to express FLAG- and MYC-tagged CYP3A4 or CYP2D6 in media enriched with 14 C Gly for 48 h, after which protein expression was ceased by Chx addition, and NOC18 was added at the designated concentrations. The cultures were maintained for 6 h before harvesting, followed by supernatant CYP activity assay and MYC Ab pulldown of the CYP to determine heme contents. A and B, CYP2D6 and CYP3A4 activities. C and D, CYP2D6 and CYP3A4 14 C heme counts. Data are the mean ± SD; n = 3 independent experiments. ∗∗∗ p < 0.001, one-way ANOVA. Ab, antibody; Chx, cycloheximide; CYP, cytochrome P450; 14 C-Gly, 14 C-glycine; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Concentration Assay, Transfection, Expressing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO governs the activities of CYP2D6 and 3A4 within HepG2 cells. HepG2 cells underwent transfection to express FLAG- and MYC-tagged CYP2D6 ( A ) or were induced by rifampicin to express CYP3A4 ( B ) in media enriched with 14 C Gly, after which protein expression was ceased by Chx addition and NOC18 was added to give the designated concentrations. After 6 h, cells were harvested, and the supernatants were subjected to a CYP activity assay. Data are the mean ± SD; n = 3 independent experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, one-way ANOVA. Chx, cycloheximide; CYP, cytochrome P450; HepG2, hepatocellular carcinoma G2 cell line; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples; UI, uninduced cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Expressing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO generated by immune-stimulated macrophage cells modulates the heme level and activity of CYP3A4 expressed in cocultured GlyA-CHO cells. A, outline of the experiment; RAW264.7 cells that had been activated or not for NO synthesis by culture with bacterial LPS were added in varying numbers to monolayers of GlyA-CHO cells expressing CYP3A4 incorporated with 14 C heme by previous culture with 14 C-Gly. Following 6 h of coulture, the nitrite concentration in the culture fluid was quantified ( B ) along with the CYP activity of the supernatant ( C ) and the 14 C heme counts in MYC Ab pulldowns of CYP3A4 from the supernatants ( D ). Data are the mean ± SD of three experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Generated, Activity Assay, Expressing, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: Kinetics of NO-induced alterations in CYP450 activities and heme contents. GlyA-CHO cells that were transfected to express CYP2D6 or 3A4 were incubated with 14 C-Gly to generate 14 C-labeled heme. Protein synthesis was then blocked with Chx, and NOC18 was added at concentrations of 0 ( red ), 5 (green), and 100 ( blue ) μM, followed by harvesting and analysis of the cell supernatants at designated time points. A and C, 14 C-heme counts in CYP2D6 and CYP3A4 MYC Ab pull-downs. B and D, CYP2D6 and CYP3A4 activities. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns = not significant, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; Chx, cycloheximide; CYP450, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Incubation, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO stimulates cell heme insertion into CYP3A4 by a GAPDH-dependent mechanism. GlyA-CHO cells that had been subjected to siRNA knockdown of GAPDH expression or treated with scrambled siRNA were administered 14 C-Gly and subsequently transfected to express CYP3A4 either alone or in conjunction with siRNA-resistant versions of wildtype HA-GAPDH or the heme-binding defective HA-GAPDH-H53A variant. Cells were subsequently treated with 0 or 5 μM NOC18, and after 6 h, the cell supernatants were prepared, and their CYP3A4 14 C-heme levels ( A ) and the activities ( B ) were assessed. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns, not significant, one-way ANOVA. 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; HA, hemagglutinin; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Knockdown, Expressing, Transfection, Binding Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: Effect of NOC18 exposures on Hsp90 association with CYP3A4. GlyA-CHO cells that had been transfected to express FLAG- and MYC-tagged CYP3A4 in media enriched with 14 C-Gly were given Chx and given NOC18 at the designated concentrations, cultured for 6 h, harvested, and the cell supernatants underwent MYC Ab pulldown and Western analysis to compare bound Hsp90 levels. A, representative Western blot indicating the relative levels of Hsp90 associated with CYP3A4. B, corresponding normalized band intensities. Data are the mean ± SD of three independent experiments, ∗∗ p < 0.01, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; Chx, cycloheximide; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; Hsp90, heat shock protein 90; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO-driven heme insertion in CYP3A4 requires cell Hsp90 activity. GlyA-CHO cells underwent transfection to express CYP3A4 in the presence of 14 C-Gly. Cells then received 10 μM radicicol (an Hsp90 inhibitor) 1 h prior to the addition of 0 or 5 μM NOC18. The cells were harvested at the indicated times, and supernatants were analyzed to determine CYP3A4 14 C-heme content ( A ) and activity ( B ) at 6 h. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns, not significant, one-way ANOVA. 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; Hsp90, heat shock protein 90; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Activity Assay, Transfection

Journal: Endocrinology

Article Title: TGF-β Induces Endometriotic Progression via a Noncanonical, KLF11-Mediated Mechanism.

doi: 10.1210/en.2016-1194

Figure Lengend Snippet: Figure 3. Differential CYP3A4 expression levels affected cell proliferation in Ishikawa cells: (A) Ishikawa cells were transfected with 2.5 g pcDNA3/His-CYP3A4 or pcDNA3/His (EV) control. The cells were photographed by live-imaging, counted and analyzed using the IncuCyte Real Time Live Imaging System every 3 hours and absolute cell numbers as well as percentage of 100% well confluence determined. CYP3A4 transfected Ishikawa cells replicated faster, which was reflected in a significantly increased growth trajectory starting after 24 hours. The differential growth trajectory between CYP3A4 and EV transfected cells diverged significantly through the next 48 hours. Results represent mean and SD obtained from 3 biological replicates. B, CYP3A4 expression levels were increased in CYP3A4 compared to EV transfectants at 48 hours post-transfection; GAPDH was used as loading control. C, Ishikawa cells were transfected with increasing doses of CYP3A4siRNA or scrambled control (50 nM, 100 nM and 200 nM). Cells transfected with CYP3A4siRNA demonstrated a dose dependent decrease in proliferation. The growth trajectory significantly diverged from Scr control (200 nM shown) after 24 hours and was significantly lower at 72 hours. The response to CYP3A4siRNA was dose dependent. D, CYP3A4 expression levels were decreased in a dose dependent manner in Ishikawa cells transfected by CYP3A4siRNA compared to Scr control (200 nM shown) in transfectants at 48 hours post-transfection; GAPDH was used as loading control. E, Total DNA content, a marker of cell number was increased in Ishikawa cells transfected with CYP3A4 compared to EV transfected controls at 72 hours. Results represent mean SD from 3 independent replicates; * P .05 compared to corresponding control (F) In Ishikawa cells transfected with CYP3A4 siRNA (50, 100 or 200 nM), cell numbers were decreased in a dose dependent manner compared to Scr control (200 nM shown) at 72 hours. Results represent mean SD from 3 independent replicates; * P .05 compared to corresponding control

Article Snippet: Cell Proliferation Assay Ishikawa cells were transfected with either pcDNA3/Cyp3A4 or CYP3A4siRNA (Santa Cruz Biotech.) or scrambled control (50 nM, 100 nM or 200 nM) for 24 hours as described above.

Techniques: Expressing, Transfection, Control, Imaging, Marker

Journal: Clinical and Translational Science

Article Title: A Long‐Term Human Liver Spheroid Model for Assessing Silencing and Durability of GalNAc ‐Conjugated siRNAs

doi: 10.1111/cts.70536

Figure Lengend Snippet: Characterization and long‐term culture of liver spheroids. (A) Representative brightfield images of primary human hepatocytes aggregating into a spheroid over 7 days. (B) Fluorescent images of primary human hepatocytes at different stages of spheroid formation and culture, stained for ASGR1 (green) and nuclei (blue). (C) Analysis of mRNA expression of ASGR1, AHSA1, Albumin and CYP3A4 in spheroids at different times over 14 days. ( n = 4–5 independent experiments). ALB, albumin; ASGR1, asiologlycoprotein receptor 1; AHSA1, activator of 90 kDa heat shock protein ATPase homolog 1; CYP3A4, cytochrome P450 3A4.

Article Snippet: CYP3A4 , Hs00604506_m1 , Thermo Fisher.

Techniques: Staining, Expressing

Journal: Clinical and Translational Science

Article Title: A Long‐Term Human Liver Spheroid Model for Assessing Silencing and Durability of GalNAc ‐Conjugated siRNAs

doi: 10.1111/cts.70536

Figure Lengend Snippet: siRNA uptake and distribution. (A) Illustrative immunofluorescent images of primary human hepatocytes following 4‐h incubation with GalNAc‐conjugated fluorescent siRNA (fluo‐siRNA) (200 nM) on poly‐l‐lysine coated slides (left) and liver spheroid treated for 4 h with fluo‐siRNA (200 nM) cryosectioned and stained with phalloidin (green) (right). (B) Illustrative immunofluorescent images of cryosection liver spheroids 7 days post treatment with fluo‐siRNA (200 nM) (red) in comparison to ASGR1 expression (green). (C) Representative immunofluorescent image of liver spheroids treated with fluo‐siRNA (red) and stained for CYP3A4 (green). The mean fluorescent signal of the fluo‐siRNA was measured in the CYP3A4+ and CYP3A4− area of the spheroid ( n = 4). ** p < 0.01.

Article Snippet: CYP3A4 , Hs00604506_m1 , Thermo Fisher.

Techniques: Incubation, Staining, Comparison, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 CDS-luciferase activities are reduced by individual or combined shRNAs in CHL cells. (A) CHL cells co-transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different shRNA expression plasmids showed 20%–30% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. (B) CHL cells transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different combinations of shRNA expression plasmids showed 40%–50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. Firefly luciferase activity was normalized to corresponding Renilla luciferase activity, and the control group was set as 100%. * P <0.05; *** P <0.001 compared to the corresponding control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: The cells were incubated with primary antibody against CYP3A4 (AVIVA Systems Biology, California, USA) diluted in 1% BSA-PBS at 4 °C overnight and washed three times with PBS.

Techniques: Luciferase, Transfection, shRNA, Expressing, Plasmid Preparation, Activity Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Combined shRNAs are effective in targeting CYP3A4 CDS in HEK293 cells. (A) HEK293 cells transfected with CYP3A4 CDS-luciferase reporter and three shRNA expression plasmids showed about 50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. *** P <0.001 compared to the control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment). (B) Fluorescent microscopic pictures of CYP3A4 expression in HEK293 cells at 48 h after transient transfection with CYP3A4 CDS-luciferase reporter plasmid alone (CYP3A4), along with control plasmid (pS-NC) or three shRNA expression plasmids (S1+S2+S3).

Article Snippet: The cells were incubated with primary antibody against CYP3A4 (AVIVA Systems Biology, California, USA) diluted in 1% BSA-PBS at 4 °C overnight and washed three times with PBS.

Techniques: Transfection, Luciferase, shRNA, Expressing, Plasmid Preparation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: The mixture of three shRNA plasmids inhibits endogenous CYP3A4 mRNA expression in HepG2 cells. (A) CYP3A4 mRNA expression was elevated 2-fold in HepG2 cells by rifampicin (50 μmol/L for 48 h), as determined by RT-PCR analysis. (B) and (C) The shRNA expression plasmid mixture suppressed CYP3A4 mRNA levels in HepG2 cells. (D) The shRNA expression plasmid mixture did not change CYP3A5 mRNA expression in the HepG2 cells. Relative CYP3A4/5 mRNA expression level was normalized to corresponding GAPDH mRNA level, and the control group was set as 100%. *** P <0.001, compared to the vehicle control. n =3 in each group, which refers to the number of independent transfection samples in a representative experiment.

Article Snippet: The cells were incubated with primary antibody against CYP3A4 (AVIVA Systems Biology, California, USA) diluted in 1% BSA-PBS at 4 °C overnight and washed three times with PBS.

Techniques: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Expression of endogenous CYP3A4 protein is suppressed by combined shRNAs in HepG2 cells. CYP3A4 protein expression (A) in HepG2 cells (treated with rifampicin) was suppressed by about 50% (B) after the transfection with the shRNA expression plasmid mixture, compared to pS-NC control plasmid. CYP3A4 protein expression level was normalized to corresponding GAPDH level in HepG2 cells, and control group was set as 100%. *** P <0.001, compared to the negative control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: The cells were incubated with primary antibody against CYP3A4 (AVIVA Systems Biology, California, USA) diluted in 1% BSA-PBS at 4 °C overnight and washed three times with PBS.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Control, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 shRNAs alter the chemosensitivity of cells. Transfection of HepG2 cells with CYP3A4 shRNAs reduces the sensitivity to GA (15:1) or GA (17:1), compared to the cells transfected with control plasmid. Cytotoxicity was determined by the MTT assay. The inhibition rate relative to the control (0%) was calculated for each concentration of drug, and the IC 50 value was estimated by fitting the data to a Hill equation .

Article Snippet: The cells were incubated with primary antibody against CYP3A4 (AVIVA Systems Biology, California, USA) diluted in 1% BSA-PBS at 4 °C overnight and washed three times with PBS.

Techniques: Transfection, Control, Plasmid Preparation, MTT Assay, Inhibition, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 CDS-luciferase activities are reduced by individual or combined shRNAs in CHL cells. (A) CHL cells co-transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different shRNA expression plasmids showed 20%–30% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. (B) CHL cells transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different combinations of shRNA expression plasmids showed 40%–50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. Firefly luciferase activity was normalized to corresponding Renilla luciferase activity, and the control group was set as 100%. * P <0.05; *** P <0.001 compared to the corresponding control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: The membranes were blotted with antibodies against antibodies of rabbit anti-human CYP3A4 (AVIVA Systems Biology, California, USA).

Techniques: Luciferase, Transfection, shRNA, Expressing, Plasmid Preparation, Activity Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Combined shRNAs are effective in targeting CYP3A4 CDS in HEK293 cells. (A) HEK293 cells transfected with CYP3A4 CDS-luciferase reporter and three shRNA expression plasmids showed about 50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. *** P <0.001 compared to the control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment). (B) Fluorescent microscopic pictures of CYP3A4 expression in HEK293 cells at 48 h after transient transfection with CYP3A4 CDS-luciferase reporter plasmid alone (CYP3A4), along with control plasmid (pS-NC) or three shRNA expression plasmids (S1+S2+S3).

Article Snippet: The membranes were blotted with antibodies against antibodies of rabbit anti-human CYP3A4 (AVIVA Systems Biology, California, USA).

Techniques: Transfection, Luciferase, shRNA, Expressing, Plasmid Preparation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: The mixture of three shRNA plasmids inhibits endogenous CYP3A4 mRNA expression in HepG2 cells. (A) CYP3A4 mRNA expression was elevated 2-fold in HepG2 cells by rifampicin (50 μmol/L for 48 h), as determined by RT-PCR analysis. (B) and (C) The shRNA expression plasmid mixture suppressed CYP3A4 mRNA levels in HepG2 cells. (D) The shRNA expression plasmid mixture did not change CYP3A5 mRNA expression in the HepG2 cells. Relative CYP3A4/5 mRNA expression level was normalized to corresponding GAPDH mRNA level, and the control group was set as 100%. *** P <0.001, compared to the vehicle control. n =3 in each group, which refers to the number of independent transfection samples in a representative experiment.

Article Snippet: The membranes were blotted with antibodies against antibodies of rabbit anti-human CYP3A4 (AVIVA Systems Biology, California, USA).

Techniques: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Expression of endogenous CYP3A4 protein is suppressed by combined shRNAs in HepG2 cells. CYP3A4 protein expression (A) in HepG2 cells (treated with rifampicin) was suppressed by about 50% (B) after the transfection with the shRNA expression plasmid mixture, compared to pS-NC control plasmid. CYP3A4 protein expression level was normalized to corresponding GAPDH level in HepG2 cells, and control group was set as 100%. *** P <0.001, compared to the negative control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: The membranes were blotted with antibodies against antibodies of rabbit anti-human CYP3A4 (AVIVA Systems Biology, California, USA).

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Control, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 shRNAs alter the chemosensitivity of cells. Transfection of HepG2 cells with CYP3A4 shRNAs reduces the sensitivity to GA (15:1) or GA (17:1), compared to the cells transfected with control plasmid. Cytotoxicity was determined by the MTT assay. The inhibition rate relative to the control (0%) was calculated for each concentration of drug, and the IC 50 value was estimated by fitting the data to a Hill equation .

Article Snippet: The membranes were blotted with antibodies against antibodies of rabbit anti-human CYP3A4 (AVIVA Systems Biology, California, USA).

Techniques: Transfection, Control, Plasmid Preparation, MTT Assay, Inhibition, Concentration Assay