Journal: PloS one

Article Title: NADPH-cytochrome P450 reductase is regulated by all-trans retinoic acid and by 1,25-dihydroxyvitamin D3 in human acute myeloid leukemia cells.

doi: 10.1371/journal.pone.0091752

Figure Lengend Snippet: Figure 3. Expression of POR gene and protein in transfected HL60 cells. HL60 cells were transfected with plasmids containing either control shRNA (CtrA) or POR shRNA and then (A) the expression of POR mRNA was tested in Real Time PCR. The bar charts show mean values (6SEM) of fold changes in mRNA levels relative to GAPDH mRNA levels. The control sample was calculated as 1. (B) The expression of POR protein in mitochondria of the cells exposed to 1,25D (10 nM) or ATRA (1 mM) for 96 h was detected using Western blot. Mitochondrial proteins were analyzed using anti-POR and anti-actin antibodies. Optical densities (OD) of POR bands divided by ODs of respective actin bands were calculated from 4 blots and mean values are given below the blots. doi:10.1371/journal.pone.0091752.g003

Article Snippet: Siliencing of POR gene by shRNA plasmids Electrotransfection by NeonH Transfection System (InvitrogenTM, Carlsbad, CA) was performed as follows: 16106 cells were transfected with 1 mg of control shRNA plasmid-A (sc-108060, Santa Cruz, CA) or POR shRNA plasmid (sc-35147-SH, Santa Cruz, CA).

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

Journal: PloS one

Article Title: NADPH-cytochrome P450 reductase is regulated by all-trans retinoic acid and by 1,25-dihydroxyvitamin D3 in human acute myeloid leukemia cells.

doi: 10.1371/journal.pone.0091752

Figure Lengend Snippet: Figure 4. Differentiation of transfected HL60 cells exposed to 1,25D or ATRA. HL60 cells were transfected with plasmids containing either control shRNA (CtrA) or POR shRNA and then were exposed to either 1,25D (10 nM) or ATRA (1 mM) for 96 h. The expression of differentiation markers CD11b (upper graph) and CD14 (bottom graph) was detected using flow cytometry. Mean values (6SEM) of percentages of positive cells are presented in Y-axis. Results that differ significantly from the result for respective control cells are marked with asterisks (*p,0.05; ***p,0.001), while the sample which differs from the appropriate CtrA sample is marked with hash (#p,0.05). doi:10.1371/journal.pone.0091752.g004

Article Snippet: Siliencing of POR gene by shRNA plasmids Electrotransfection by NeonH Transfection System (InvitrogenTM, Carlsbad, CA) was performed as follows: 16106 cells were transfected with 1 mg of control shRNA plasmid-A (sc-108060, Santa Cruz, CA) or POR shRNA plasmid (sc-35147-SH, Santa Cruz, CA).

Techniques: Transfection, Control, shRNA, Expressing, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

doi: 10.1074/jbc.RA120.016394

Figure Lengend Snippet: Expression level of Cygb in SMCs. Western blots of Cygb obtained from scr-RNA and Cygb siRNA-treated mouse aortic SMCs. The first three bands correspond to known amounts of purified Cygb. Bands 4 and 5 are from scr-RNA and Cygb siRNA-treated cells, respectively. The bar graph shows the mean value ±SD for n = 3 to 5. From the ratio of measured Cygb to total cellular protein concentration, the cellular concentration of Cygb was estimated to be 3.5 μM.

Article Snippet: SMCs were transfected with Cygb siRNA (Santa Cruz; sc-45548), B5R siRNA (Santa Cruz; sc-62174), B5 siRNA (Santa Cruz; sc-37378), or CPR siRNA (Santa Cruz; sc-35148) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations.

Techniques: Expressing, Western Blot, Purification, Protein Concentration, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

doi: 10.1074/jbc.RA120.016394

Figure Lengend Snippet: Effect of Cygb knock down on NO metabolism in SMCs. A , NO consumption by control, scr-RNA, or Cygb siRNA-treated mouse aortic SMCs. NO was injected as in Figure 1 . B , graph of the rate of NO decay ( V NO ) measured from control, scr-RNA, and siRNA-treated SMCs. A cell density of 3.5 × 10 6 SMCs•ml −1 was maintained in all groups. Values shown are means ± SD with n = 6. ∗∗Significant from scr-RNA at p < 0.001.

Article Snippet: SMCs were transfected with Cygb siRNA (Santa Cruz; sc-45548), B5R siRNA (Santa Cruz; sc-62174), B5 siRNA (Santa Cruz; sc-37378), or CPR siRNA (Santa Cruz; sc-35148) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations.

Techniques: Knockdown, Control, Injection

Journal: The Journal of Biological Chemistry

Article Title: Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

doi: 10.1074/jbc.RA120.016394

Figure Lengend Snippet: Expression levels of B5R, B5, or CPR in SMCs . Western blots of B5R ( A ), B5 ( B ), and CPR ( C ) obtained from scr-RNA and B5R, B5 and CPR siRNA-treated mouse aortic SMCs. The first three bands correspond to known amounts of purified B5R ( A ), B5 ( B ), or CPR ( C ). Bands 4 and 5 are from scr-RNA and B5R ( A ), B5 ( B ), or CPR ( C ) siRNA-treated cell homogenates, respectively. The bar graphs show the mean value ± SD for n = 3. From the ratio of each measured protein to total cellular protein concentration, the cellular concentrations of B5R, B5, and CPR are estimated at 0.88 μM, 0.38 μM, and 0.15 μM, respectively.

Article Snippet: SMCs were transfected with Cygb siRNA (Santa Cruz; sc-45548), B5R siRNA (Santa Cruz; sc-62174), B5 siRNA (Santa Cruz; sc-37378), or CPR siRNA (Santa Cruz; sc-35148) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations.

Techniques: Expressing, Western Blot, Purification, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

doi: 10.1074/jbc.RA120.016394

Figure Lengend Snippet: Effect of B5R, B5, and CPR on the rates of NO consumption by SMCs. A – C , NO consumption by scr-RNA versus B5R ( A ), B5 ( B ), and CPR ( C ) siRNA-treated (KD) mouse aortic SMCs. A cell density of 3.5 × 10 6 SMCs•ml −1 was used in all groups. NO was injected into the solution to achieve an initial concentration of 1.0 μM. Graph of the rates of NO metabolism ( V NO ) by scr-RNA versus B5R ( D ), B5 ( E ), and CPR ( F ) siRNA-treated (KD) SMCs. The bar graph shows the mean value ± SD for n = 6. ∗: Significant from scr-RNA at p < 0.05; ∗∗significant from scr-RNA at p < 0.001.

Article Snippet: SMCs were transfected with Cygb siRNA (Santa Cruz; sc-45548), B5R siRNA (Santa Cruz; sc-62174), B5 siRNA (Santa Cruz; sc-37378), or CPR siRNA (Santa Cruz; sc-35148) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations.

Techniques: Injection, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison

doi: 10.3390/ijms221910206

Figure Lengend Snippet: CYP2C9 WT and variants discussed in the current manuscript are shown with their respective amino acid changes.

Article Snippet: Human cytochrome P450 reductase was obtained from OriGene (Rockville, MD, USA).

Techniques:

Journal: The Journal of biological chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells.

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: Figure 15. Effects of POR and FDXR knockdown on pregnenolone synthesis in MGM-1 cells. siRNA knockdown of POR (A–D) or FDXR (E–H) in MGM- 1 cells. Knockdown efficiency was determined by qRT-PCR (A and E) and immunoblot (B, C, F, and G), with expression normalized to β-actin. B and F, representative immunoblots indicating successful knockdown. Twenty micrograms total protein were loaded into each lane. C and G quantification of protein levels in immunoblots in all experiments. D and H ELISA measurements of secreted pregnenolone in transfected MGM-1 cells. Data are presented as mean ± SD, N = 3. Statistics performed using unpaired t test. (* p < 0.05, ** p < 0.01, *** p < 0.001). qRT-PCR, quantitative RT-PCR.

Article Snippet: The cells were transfected with 5 nM scrambled negative control siRNA duplexes or gene-specific siRNA duplexes (Origene; #SR303637 for POR and #SR301560 for FDXR) using siTran 2.0 transfection reagents (Origene; #TT320001), according to manufacturer’s instructions.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection