odc1 pgl3  (Addgene inc)

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma

doi: 10.1172/JCI70712

Figure Lengend Snippet: (A) Predicted transcription factors binding AZIN1 and ODC1 promoters. (B) Extracts from WI-38 cells expressing empty vector control (Ctrl) or CA-AHR probed by immunoblotting for AZIN1 and ODC1. (C) RNA from cells as in B probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (D) Schematic of conserved (black circles) or partially conserved (shaded circle) AHR binding sites in the indicated promoters. Hs, Homo sapiens; Mm, Mus musculus. (E) WI-38 DNA was immunoprecipitated with control (IgG) or AHR-specific antibodies and probed in qRT-PCR with primers for the CYP1a1 promoter (positive control), regions in AZIN1 and ODC1 promoters described in D, or GMPR (negative control). Luciferase activity for the AZIN1 and ODC1 promoter regions described in D with increasing amounts of CA-AHR (F) or BaP (G). The XRE-luc plasmid was used as a control. Data represent the average ± SEM of 2 independent experiments performed in duplicate. (H) Cell extracts of WI-38 cells expressing control shRNA (Ctrl-sh) or 2 independent shRNAs against AHR (sh1 and sh2) probed by immunoblotting with the indicated antibodies. (I) RNA from cells as in H probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 4 independent experiments performed in triplicate. (J) Polyamine content in cells as in H. Data represent the average ± SEM of 4 independent experiments. (K) Extracts of WI-38 cells treated for 2 hours with DMSO or 20 μM CH223191 probed by immunoblotting for AZIN1 and ODC1. (L) RNA from cells as in K probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (M) Polyamine content in WI-38 cells treated with CH223191 for 48 hours. Data represent the average ± SEM of 3 independent experiments. *P < 0.05 and **P < 0.001, by 2-tailed Student’s t test. AZI, AZIN1 ; CYP, CYP1a1; ODC, ODC1; TiP, TiPARP; Spd, spermidine; Put, putrescine; Spm, spermine.

Article Snippet: AZIN1 and ODC1 promoter regions containing putative AHR binding sites, as well as the ODC1 promoter including the MYC binding sites (either WT or mutated) were synthesized by Genscript and subcloned into the pGL3 promoter ( AZI1 no. 1 and (AZI1 no. 2) or pGL3 basic (ODC1) plasmids (Promega).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Positive Control, Negative Control, Luciferase, Activity Assay, shRNA

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma

doi: 10.1172/JCI70712

Figure Lengend Snippet: (A) Extracts of WI38 cells treated for 2 hours with increasing concentrations of CLF, Har, or FF probed in the immunoblot for AZIN1 and ODC1. (B) Cytoplasmic and nuclear fractions of WI38 cells treated for 2 hours with DMSO, 4 μM CLF, or 20 μM CH223191 resolved in the immunoblot and probed for AHR. Tubulin and TBP were used as positive controls. Quantification of band intensity was performed with ImageJ (NIH). (C) WI38 cells treated as in B and immunostained for AHR (red), DNA (Hoechst-33342, blue), and actin (phalloidin, green). Images are representative of 2 independent experiments. Scale bars: 20 μm. (D) Luciferase activity assay of HEK293FT cells transduced with XRE-luc, with increasing concentrations of BaP and DMSO, 4 μM CLF, or 20 μM CH223191. Data represent the average ± SEM of 2 independent experiments performed in duplicate. (E) Cytosolic fractions of Hepa 1c1c7 cells preincubated with vehicle or 16 nM TCDD and increasing concentrations of CLF or CH223191, and then incubated with XRE-containing biotinylated oligonucleotides and resolved on native gels. Protein-DNA complexes were visualized with a chemiluminescence system. (F) RNA from WI-38 cells treated for 2 hours with DMSO or 4 μM CLF probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (G) Polyamine content in WI38 cells treated with 4 μM CLF for 48 hours. Data represent the average ± SEM of 5 independent experiments. *P < 0.05 and **P < 0.001, by 2-tailed Student’s t test. (H) Proliferation of WI38 cells expressing empty vector (Ctrl) or CA-AHR, with increasing concentrations of CLF over time. Control cells were supplemented with 10 μM spermidine and 1 mM aminoguanidine. IC50 values were determined using GraphPad Prism. Data represent the average of 2 experiments performed in quadruplicate. **P < 0.001, by extra sum-of-squares F test using GraphPad Prism.

Article Snippet: AZIN1 and ODC1 promoter regions containing putative AHR binding sites, as well as the ODC1 promoter including the MYC binding sites (either WT or mutated) were synthesized by Genscript and subcloned into the pGL3 promoter ( AZI1 no. 1 and (AZI1 no. 2) or pGL3 basic (ODC1) plasmids (Promega).

Techniques: Western Blot, Luciferase, Activity Assay, Transduction, Incubation, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma

doi: 10.1172/JCI70712

Figure Lengend Snippet: (A) MM.1S cells were inoculated s.c. into both flanks of 4- to 6-week-old female SCID mice. The animals were randomized into 2 groups (6 animals/group) and treated with daily i.p. injections of vehicle in PBS or CLF (10 mg/kg). Tumor volumes were recorded every 5 days. *P < 0.05, by 2-tailed Student’s t test. (B) Protein extracts from 6 tumors/group from A were resolved by immunoblotting and probed for CYP1a1, AZIN1, or ODC1. Growth of MM.1S (C) or RPMI-8226 (D) cell xenografts was monitored as in A. Animals were randomized into 3 treatment groups: PBS, CLF, or BTZ (1 mg/kg, biweekly i.p. injections). Tumor measurement distributions were tested for normality by Shapiro-Wilk test, and the significance of individual comparisons (treated/untreated) were determined by Student’s t test with Bonferroni’s correction.

Article Snippet: AZIN1 and ODC1 promoter regions containing putative AHR binding sites, as well as the ODC1 promoter including the MYC binding sites (either WT or mutated) were synthesized by Genscript and subcloned into the pGL3 promoter ( AZI1 no. 1 and (AZI1 no. 2) or pGL3 basic (ODC1) plasmids (Promega).

Techniques: Western Blot

Journal:

Article Title: Associations of a polymorphism in the ornithine decarboxylase gene with colorectal cancer survival

doi: 10.1158/1078-0432.CCR-09-0592

Figure Lengend Snippet: Table 1

Article Snippet: For MYC experiments, ODC1 pGL3-plasmids were co-transfected with either pcDNA 3.0 or CMV- MYC expression vector (OriGene, Rockville, MD).

Techniques: