Fig 1: Microarray analysis and construction of PPI networks(A) Top ten KEGG pathways annotation. KEGG pathway enrichment. The X-axis represents the adjusted P-value, Y-axis is the KEGG pathway. (B) Heatmap of Cd-induced nephrotoxicity and the expression of related genes involved in chemical carcinogenesis pathways. Green: relatively low expression; red: relatively high expression; scale, normalized expression value. (C) PPI diagram of differentially expressed genes related to Cd-induced renal damage and chemical carcinogenesis pathway identified by microarray in the present study. (D) PPI diagram of genes related to the Cd and chemical carcinogenesis pathway in CTD. (E) Venn diagram showing the overlap of differentially expressed genes related to Cd-induced renal damage and chemical carcinogenesis pathway identified by microarray in the present study and the genes related to Cd-related chemical carcinogenesis pathway in the CTD database. (F) Merging of the PPI networks. Yellow nodes indicate the two overlapping genes. (G) CYP1B1 and UGT2B mRNA expression by quantitative reverse transcription polymerase chain reaction (RT-qPCR). (H) CYP1B1 and UGT2B expression by Western blot assay. PPI diagram, the size of a node is determined by the degree of freedom: the greater the degree of freedom, the larger the node. The color of the node is set according to the degree of freedom, going from blue (low degree of freedom) to yellow (high degree of freedom). The thickness of the edge represents the betweenness: the larger the betweenness, the thicker the edge.
Fig 2: Overview of hippocampal anatomy and locations of image acquisition. Stitched image (a) of ipsilateral hippocampus of female rat subjected to penetrating traumatic brain injury and a survival time of 3 days. Acquired at 20× magnification using a Nikon Eclipse Ni-E microscope. Acquired at Bregma: -3.84 mm. RNAscope in-situ hybridization marked CYP1B1 mRNA in brown. Black boxes (b–f) show location and scope of image captured as well as CYP1B1 mRNA markings. Box (b) depicts CA1, (c) CA2, (d) CA3, (e) CA4 and (f) DG. Abbreviations: CA: Cornu Ammonis, CYP1B1: Cytochrome P450 1B1, DG: Dentate Gyrus.
Fig 3: Representative images from ipsilateral subregion CA2 using RNAscope and ViewRNA. Images captured at 20× magnification from the same female rat subjected to penetrating traumatic brain injury and a survival time of 3 days. Captured at Bregma: -3.84 mm. Image (a) shows CYP1B1 mRNA marked in brown (RNAscope) and a cresyl violet counterstaining. Image (b) shows CYP1B1 mRNA marked in red (ViewRNA) and a methyl green counterstain. Expression was greatest in CA2 irrespective of sex, intervention and survival time using both RNAscope (a) and ViewRNA (b). Abbreviations: CA2: Cornu Ammonis 2.
Fig 4: Properties of CYP1B1 promoter polymorphisms. A, CYP1B1 expression in clinical samples. Representative immunostaining of CYP1B1 by polymorphism status in benign prostatic hyperplasia (BPH) specimens. (a) None to weak staining, (b) moderate staining, and (c) stronger staining. (d) Staining score categorized by polymorphism status for rs2551188, rs2567206 and rs10175368. Score was significantly higher for minor genotype at rs2551188 (G/A+A/A), rs2567206 (C/T+T/T) and rs10175368 (G/A+A/A) compared to respective major genotypes. Number of specimens for each polymorphic status is indicated in parentheses. Data expressed as mean ± SD. *P < .05, determined by two-tailed Mann-Whitney U test. B, Effect of CYP1B1 polymorphisms on luciferase activity in prostate cancer cells. PC-3 and DU145 cells seeded in 96-well plates were transfected with either CYP1B1 minor or major allele constructs, and secreted Gauccia and ALP luciferase activities were measured. CYP1B1 promoter activities were calculated as the ratio of Gauccia to ALP. Levels were significantly higher for the rs10175368 allele A compared to major type G. Data expressed as mean±SEM for triplicates. ***P < .001, determined by Dunnett t (for rs2551188 and rs2567206) and two-tailed Student t (for rs10175368) tests. C, Binding of nuclear factor to rs10175368 motif. Left: Nuclear extract prepared from DU145 cells was incubated with biotinylated double-strand oligonucleotide probe for rs10175368 (-5346 to -5317 bp) with or without unlabelled DNA competitor, and complex separated by gel electrophoresis. Arrow denotes protein-bound probe. Right: Stronger binding of protein factor was observed for the minor allele A compared to major allele G. Data expressed as mean±SEM for triplicates. *P = .045, two-tailed Student t test
Fig 5: Comparison of male and female hippocampal CYP1B1 mRNA expression in rats subjected to either sham-surgery or pTBI. Animals in Sham received a craniotomy, while animals in pTBI received a craniotomy with a subsequent pTBI. Graphs show mRNA expression 1 dpi in males and females subjected to sham-surgery (a), mRNA expression 1 dpi in males and females subjected to pTBI (b), mRNA expression 3 dpi in males and females subjected to sham-surgery (c), mRNA expression 3 dpi in males and females subjected to pTBI (d), mRNA expression 5 dpi in males and females subjected to sham-surgery (e), mRNA expression 5 dpi in males and females subjected to pTBI (f), mRNA expression 7 dpi in males and females subjected to sham-surgery (g), mRNA expression 7 dpi in males and females subjected to pTBI (h). CYP1B1 mRNA was detected by in situ hybridization. The mean gray value is presented in the Y-axis, showing the average mean gray area calculated from each measured cell for each subregion. To calculate mean gray value, images were made binary and in-situ probes made black and separated from the white background. The gray value of the color black was set to 255 and the value for white to 0. The gray value of each cell is therefore a measurement of the area of cell marked by the in-situ probe. The gray values from each cell of each subregion were then used to calculate the mean gray value of each subregion and animal. The X-axis shows the ipsilateral subregions analyzed. Females subjected to pTBI and a 3 day survival time exhibited higher CYP1B1 mRNA expression in every CA-subregion compared to their male counterparts (d). The values are presented as mean + SD for each group, including the mean for each animal. Abbreviations: CA: Cornu Ammonis, CYP1B1: Cytochrome P450 1B1, DG: Dentate gyrus, dpi: days post-injury, pTBI: penetrating traumatic brain injury.
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