Journal: Scientific Reports

Article Title: Profiling of CYP4Z1 and CYP1B1 expression in bladder cancers

doi: 10.1038/s41598-021-85188-4

Figure Lengend Snippet: Association between CYP1B1 expression and clinicopathologic features.

Article Snippet: For CYP1B1 immunodetection, sections were covered with rabbit polyclonal antibody specific for CYP1B1 (NBP2-24722) (Novus Biological, USA) at a concentration of 10 μg/ml for one hour at room temperature.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Profiling of CYP4Z1 and CYP1B1 expression in bladder cancers

doi: 10.1038/s41598-021-85188-4

Figure Lengend Snippet: CYP1B1 expression in different types of bladder cancers. Tumours were classified on the bases of histological type. ( A ) Normal bladder tissue, ( B ) Urothelial carcinoma, ( C ) Squamous cell carcinoma, ( D ) Papillary adenocarcinoma, and ( E ) Mucinous adenocarcinoma.

Article Snippet: For CYP1B1 immunodetection, sections were covered with rabbit polyclonal antibody specific for CYP1B1 (NBP2-24722) (Novus Biological, USA) at a concentration of 10 μg/ml for one hour at room temperature.

Techniques: Expressing

Journal: Journal of Oral Biology and Craniofacial Research

Article Title: Evaluation of CYP1B1, oxidative stress and phase II detoxification enzyme status in oral cancer progression model

doi: 10.1016/j.jobcr.2024.02.001

Figure Lengend Snippet: (a) Negative CYP1B1 expression in apparently normal mucosa (CWOT) (40x) (b) CYP1B1 expression in apparently healthy mucosa with tobacco exposure (CWT) showing intense cytoplasmic positivity (40x) (c) CYP1B1 expression in OED showing moderate cytoplasmic positivity (40x) (d) CYP1B1 expression in OSCC showing moderate cytoplasmic intensity (40x).

Article Snippet: The primary antibody used was CYP1B1 rabbit polyclonal (50 μl, Sino Biological) and the procedure followed was as per the instructions of the manufacturer.

Techniques: Expressing

Journal: Advanced Science

Article Title: Burn‐Induced Gut Microbiota Dysbiosis Aggravates Skeletal Muscle Atrophy by Tryptophan‐Kynurenine Mediated AHR Pathway Activation

doi: 10.1002/advs.202409296

Figure Lengend Snippet: Kyn induces L6 myotube proteolysis through the activation of the AHR pathway. A) L6 myotubes were treated with Kyn (50, 100, or 150 µmol) or vehicle for 24 h. Myotube morphology was examined via H&E staining (scale bar, 50 µm). B) Effects of Kyn on the diameter and length of L6 myotubes ( n = 3 per group). C) Changes in SLC38A2 and AHR protein expression in L6 myotubes treated with Kyn (50, 100, or 150 µmol) or vehicle for 24 h ( n = 3 per group). D) L6 myotubes pre‐treated with SLC38A2 siRNA (100 nmol) and incubated with Kyn (100 µmol). H&E staining was used to examine the myotube morphology and effects of different treatments on the diameter and length of L6 myotubes (scale bar, 50 µm. n = 3 per group). E) Bar graph showing RT‐qPCR quantification of AHR gene expression levels in L6 myotubes transfected with SCL38A2 siRNAs or vehicle ( n = 3 per group). F) L6 myotubes were pretreated with DMSO or Kyn supplementation. The expression of AHR, HSP90, Cul4B, and GAPDH was determined by western blot analysis (input). Immunoprecipitation was performed with AHR or IgG antibody. HSP90, Cul4B, HSP90‐bound AHR, and Cul4B‐bound AHR were determined by western blot analysis (IP; n = 3). G) L6 myotubes pre‐treated with AHR siRNA (100 nmol) and incubated with Kyn (100 µmol). H&E staining was used to examine the myotube morphology and effects of different treatments on the diameter and length of L6 myotubes (scale bar, 50 µm. n = 3 per group). H,I) Changes in CYP1A1, CYP1A2, CYP1B1, AHRR, MAFbx, and MuRF‐1 protein expression in L6 myotubes transfected with AHR siRNAs or vehicle ( n = 3 per group). All quantitative data were analyzed using one‐way ANOVA and are shown as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; and *** p ≤ 0.001.

Article Snippet: The membranes were incubated overnight with primary antibodies: rat IDO‐1 (Proteintech, 13268‐1‐AP), β‐actin (Servicebio, GB12001), MAFbx (Proteintech, 67172‐1‐Ig), MuRF‐1 (Proteintech, 55456‐1‐AP), HSP90 (Proteintech, 13171‐1‐AP), Cul4B (Proteintech, 12916‐1‐AP), AHR (Proteintech, 28727‐1‐AP), CYP1A1 (Proteintech, 13241‐1‐AP), CYP1A2 (Proteintech, 19936‐1‐AP), CYP1B1 (Proteintech, 18505‐1‐AP), AHRR (Servicebio, GB115154), and GAPDH (Proteintech, 10494‐1‐AP).

Techniques: Activation Assay, Staining, Expressing, Incubation, Quantitative RT-PCR, Gene Expression, Transfection, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: qRT-PCR analysis of CYP1B1 and CYP1A1 in 20 matched normal and tumor pairs derived from bladder tissue. Each bar represents an average of triplicate reactions. The numbers in the X axis correspond to patient numbers. Ta/T1 and T2/T3 represent the different stages of tumors according to TNM classification. ns not statistically significant, * statistically different p< 0.05.

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques: Quantitative RT-PCR, Derivative Assay

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: qRT-PCR analysis of CYP1B1 and CYP1A1 in 20 matched normal and tumor pairs derived from colon tissue. Each bar represents an average of triplicate reactions. The numbers in the X axis correspond to patient numbers. Ta/T1 and T2/T3 represent the different stages of tumors according to TNM classification. ns not statistically significant, * statistically different p< 0.05.

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques: Quantitative RT-PCR, Derivative Assay

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: Box plots indicate mean ± STDEV for (A) bladder and (B) colon tumor and normal samples. Statistical analysis was conducted using paired T test and Wilcoxon ranks test. Statistical differences were obtained for bladder (n=20) and colon tumors (n=20) vs normals (p< 0.05 for CYP1A1 and CYP1B1).

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques:

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: Diosmetin (10 μM) was incubated with recombinant CYP1A1 (1mg/ml) and CYP1B1 (1mg/ml) at various time points in the presence of NADPH (5mM) and MgCl2 (0.5mM) as described in Materials and Methods. (A) LC-MS analysis of diosmetin metabolism by CYP1A1. Mass spectrometric trace indicated two positively charged ions (287, 301) and one negatively charged ion (315) that correspond to diosmetin, 3´,4´,5,7 tetrahydroxy flavone (luteolin) and hydroxy - 4´ methoxy tetrahydroxy flavone (hydroxy diosmetin) respectively. (B) Enzyme profile of diosmetin metabolism by CYP1A1. Diosmetin was incubated for 20 min with increasing enzyme concentrations corresponding to 0.5, 0.8, 1.2 and 3,5 mg/ml. Green line to orange line: increasing concentrations of CYP1A1 recombinant enzyme. (C) Enzyme profile of diosmetin metabolism by CYP1B1. Diosmetin was incubated for 20 min with increasing enzyme concentrations corresponding to 0.5, 0.8, 1.2 and 3,5 mg/ml. Orange line to black line: increasing concentrations of CYP1B1 recombinant enzyme. (D) Time dependence analysis of diosmetin (10 μM) metabolism by CYP1A1 at 1, 5, 10, 15, 30, 60 min. Black to blue line: 1-60 min. (E) Time dependence analysis of diosmetin metabolism by CYP1B1 (1mg/ml) at 1, 5, 10, 15, 30, 60 min time intervals. Black to blue line: 1-60 min. (F) Michaelis-Menten kinetics of luteolin production by CYP1A1 and CYP1B1-mediated metabolism of 4´ methoxy 3´,5,7 trihydroxy flavone indicating maximum activity (Vmax).

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques: Incubation, Recombinant, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: Group pairs were compared using Mann-U-Whitney test. Bars depict the mean values. Scatterplot depicting mRNA levels of CYP1A1 and CYP1B1 genes in tumor samples of different TNM status and normal samples of (A) bladder and (B) colorectal origin. Statistical significance was set at p < 0.05. Arrows and horizontal lines indicate groups compared with statistical tests.

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques:

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: Correlation of CYP1A1 and CYP1B1 mRNA T/N expression ratio with CYP1 activity T/N expression ratio in (A) bladder and (B) colon tumors using linear regression analysis.

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques: Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

doi: 10.1371/journal.pone.0082487

Figure Lengend Snippet: Reduction of CYP1 activity in colon (n=3) and bladder tumors (n=4) by (A) CYP1B1 antibody (1:500) (B) CYP1A1 antibody (1:300).

Article Snippet: For CYP1 enzyme inhibition experiments, the assay was carried out with microsomal protein and diosmetin as described above in the presence of human CYP1B1 antibody (Santa Cruz, Heidelberg, Germany) at 1:500 dilution in PBS 1% FBS.

Techniques: Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-019-1337-2

Figure Lengend Snippet: AHR and GPER are involved in CYP1B1 induction by 3MC. a 1 μM 3MC induces the mRNA expression of CYP1B1 in SkBr3 breast cancer cells and CAFs, as indicated. Data obtained by real-time PCR in three independent experiments performed in triplicate were normalized to the expression of 18S and shown as fold changes of CYP1B1 expression upon treatment with 3MC with respect to cells treated with vehicle (−). Evaluation of CYP1B1 protein levels in SkBr3 cells ( b ) and CAFs ( e ) upon treatment for 6 h with vehicle (−), 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15. The up-regulation of CYP1B1 protein levels induced by a 6 h treatment with 1 μM 3MC is abrogated in SkBr3 cells ( c ) and CAFs ( f ) transfected for 24 h with shGPER. d , g Efficacy of GPER silencing. β-actin serves as a loading control. Results shown are representative of three independent experiments. h Luciferase activities of CYP1B1 promoter constructs in SkBr3 cells and CAFs treated for 18 h with vehicle (−) and 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15, as indicated. CYP1B1 protein levels in SkBr3 cells ( i ) and CAFs ( j ) transfected for 18 h with a vector or DN/c-Fos construct and then treated for 6 h with vehicle (−) and 1 μM 3MC, as indicated. β-actin serves as a loading control. Results shown are representative of three independent experiments. k Luciferase activities of CYP1B1 promoter plasmids in SkBr3 cells and CAFs transfected for 8 h with CYP1B1 constructs, a vector or DN/c-Fos construct and then treated for 18 h with vehicle (−) and 1 μM 3MC. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by treatments were calculated. Each column represents the mean ± SD of three independent experiments, each performed in triplicate. (■) indicates P < 0.05 for cells receiving treatments versus vehicle (−)

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Aldrich, Milan, Italy), probed overnight at 4 °C with antibodies against CYP1B1 (TA339934) and cyclin D1 (TA801655) (purchased from OriGene Technologies, DBA, Milan, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy), AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), c-Fos (E8), pEGFR Tyr 1173 (sc-12,351), EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14) and β-actin (C-2) (purchased from Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Construct, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-019-1337-2

Figure Lengend Snippet: AHR and GPER are involved in the up-regulation of cyclin D1 by 3MC. a Cyclin D1 mRNA expression in SkBr3 cells and CAFs treated with vehicle (−) and 1 μM 3MC, as indicated. mRNA expression of cyclin D1 in SkBr3 cells ( b ) and CAFs ( d ) upon treatments for 18 h with vehicle (−) and 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15, as indicated. Data obtained by real-time PCR in three independent experiments performed each in triplicate were normalized to 18S expression and shown as fold changes of Cyclin D1 expression induced by treatments with respect to cells treated with vehicle (−). Cyclin D1 protein levels in SkBr3 cells ( c ) and CAFs ( e ) upon treatments for 18 h with vehicle (−) and 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15, as indicated. Cyclin D1 protein levels in SkBr3 cells ( f ) and CAFs ( j ) upon treatments for 18 h with vehicle (−) and 1 μM 3MC alone or in combination with 5 μM CYP1B1 activity inhibitor TMS. Cyclin D1 protein levels in SkBr3 cells ( g ) and CAFs ( k ) transiently transfected with shRNA or shCYP1B1 for 24 h and then treated for 18 h with vehicle (−) and 1 μM 3MC. h, l Efficacy of CYP1B1 silencing. Cyclin D1 protein levels in SkBr3 cells ( i ) and CAFs ( m ) treated for 18 h with vehicle (−) and 1 μM 3MC alone or in combination with 100 nM SP1 inhibitor Mithramycin A (MTM A). β-actin serves as a loading control. Results shown are representative of three independent experiments. (■) P < 0.05 for cells receiving treatments versus vehicle (−)

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Aldrich, Milan, Italy), probed overnight at 4 °C with antibodies against CYP1B1 (TA339934) and cyclin D1 (TA801655) (purchased from OriGene Technologies, DBA, Milan, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy), AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), c-Fos (E8), pEGFR Tyr 1173 (sc-12,351), EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14) and β-actin (C-2) (purchased from Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Transfection, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-019-1337-2

Figure Lengend Snippet: Transduction pathways involved in the proliferative effects triggered by 3MC. The proliferation of SkBr3 cells ( a ) and CAFs ( e ) induced by 1 μM 3MC is prevented by 1 μM AHR inhibitor CH223191, 100 nM GPER antagonist G15, 5 μM CYP1B1 inhibitor TMS and 100 nM SP1 antagonist mithramycin A (MTM A). The growth effects induced by 1 μM 3MC in SkBr3 cells ( b ) and CAFs ( f ) are prevented silencing either CYP1B1 or GPER expression. Cells were transfected every 2 days with shRNA, shCYP1B1 or shGPER, treated every day with ligands and then counted on day 5. Efficacy of the silencing of CYP1B1 ( c , g ) and GPER ( d , h ). β-actin serves as a loading control. Proliferation of cells treated with vehicle (−) was set as 100% upon which cell growth induced by treatments was calculated. Each data point is the mean ± SD of three independent experiments performed in triplicate. i Representative images of SkBr3 spheroids (a single spheroid per well) grown on agar-coated plates after 20 days treatment with vehicle (−) or 1 μM 3MC alone or in combination with 1 μM AHR inhibitor CH223191, 100 nM GPER antagonist G15, 5 μM CYP1B1 inhibitor TMS and 100 nM SP1 antagonist MTM A, as indicated. j Evaluation of SkBr3 cell growth upon treatments, as indicated, vehicle (−) was set as 100% upon which the results induced by treatments was calculated. Each column represents the mean ± SD of two independent experiments, each performed in triplicate. (■) indicates P < 0.05 for cells receiving treatments versus vehicle (−)

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Aldrich, Milan, Italy), probed overnight at 4 °C with antibodies against CYP1B1 (TA339934) and cyclin D1 (TA801655) (purchased from OriGene Technologies, DBA, Milan, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy), AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), c-Fos (E8), pEGFR Tyr 1173 (sc-12,351), EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14) and β-actin (C-2) (purchased from Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Transduction, Expressing, Transfection, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-019-1337-2

Figure Lengend Snippet: Schematic representation of CYP1B1 regulation by both AHR and GPER signaling

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Aldrich, Milan, Italy), probed overnight at 4 °C with antibodies against CYP1B1 (TA339934) and cyclin D1 (TA801655) (purchased from OriGene Technologies, DBA, Milan, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy), AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), c-Fos (E8), pEGFR Tyr 1173 (sc-12,351), EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14) and β-actin (C-2) (purchased from Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: