Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: Representative expression pattern of ARRB1 in non-neoplastic and malignant prostate cancer tissues. Non-neoplastic tissue shows weak nuclear and moderate cytoplasmic staining in luminal and basal cells. Staining is also present in stromal cells (s). Moderate to intense cytoplasmic and intense nuclear staining is noted in Gleason 4 (G4) areas of the tumour. Intense staining is noted in scattered bone metastatic prostate cancer cells. Quantification of ARRB1 staining in non-neoplastic and malignant prostate tissue shown in (A) (Porto TMA, see Supplementary information for details). NN=non-neoplastic, TT=tumour tissue. P < 0.001 for total positive ARRB1 cases in TT versus NN. Nuclear (solely nuclear + cytoplasmic and nuclear) or solely cytoplasmic ARRB1 staining in non-neoplastic and tumour tissue. P < 0.001 for positive nuclear ARRB1 in TT versus NN. Assessment of association between ARRB1 expressions (total expression versus only cytoplasmic versus nuclear) in prostate cancer samples and clinicopathological data. The comparisons were examined for statistical significance using Pearson's chi-square (χ 2 ) test, P < 0.05 being the threshold for significance. Distribution of nuclear ARRB1 in low (< 7) and high (≥ 7) grade tumours.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Expressing, Staining

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: Cytoplasmic versus nuclear levels of ARRB1 in a panel of prostate cancer cell lines. GFP control, nucARRB1 and wtARRB1 proliferation. Proliferation of ARRB1 KD C4-2 cells compared to control. Anchorage-independent growth (left) and migration/invasion (right) potential of C4-2 cells expressing GFP control or nucARRB1. Migration/invasion potential of low ARRB1 LNCaP cells expressing GFP control or nucARRB1. Data information: (B and C) N = 3, (D and E) N = 6, values are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available online for this figure.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Migration, Expressing

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: A CEAS-generated genomic distribution of ARRB1, p300, H3K4me1 and H3K4me3 in C4-2s compared to the genomic frequency of the regions considered. The pie chart shows ARRB1-binding sites distribution relative to proximal promoter regions. B Venn diagram showing the overlap (minimum 1 bp) between functional endogenous ARRB1 and p300-binding sites in C4-2 cells. C Distribution of ARRB1-binding sites, either alone or shared with p300, relative to H3K4me1 and H3K4me3 regions in C4-2s. D, E Distance distribution of ARRB1 and p300 peak centres relative to the nearest TSS (D) or RNAPolII site (E). F Venn diagram showing the overlap (minimum 1 bp) between functional endogenous ARRB1 binding sites in human prostate tissue and cell lines. G Integrated Genome Browser view of ChIP-Seq enrichment profiles of ARRB1, H3K4me3, H3K4me1 and RNAPII in parental C4-2, nucARRB1 cell lines and human prostate tissue.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Generated, Binding Assay, Functional Assay, ChIP-sequencing

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: Gene expression heatmap showing ARRB1-regulated genes in control GFP, wtARRB1 or nucARRB1 versus parental C4-2 control. Overlap between DEG in wtARRB1 and nucARRB1. GSEA-enrichment analysis for hypoxia-responsive genes between normoxic nucARRB1 DEG and DU145 prostate cancer cells incubated for 1, 2, 4, 8 and 12 h in hypoxic conditions. Ingenuity Pathway Analysis (IPA) of the 854 direct ARRB1 transcriptional targets. The small Venn diagram cartoon shows the number and overlap of genes in the different categories. IPA analyses of the p300/ARRB1- or ARRB1 alone-regulated genes subgroups are also shown.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Expressing, Incubation

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: GFP control and nucARRB1 (top) or scramble shRNA and ARRB1 shRNA (bottom) cells were incubated in hypoxia (1% O 2 ) for 0, 2, 4 or 8 h. Heatmap showing expression levels of HIF1A metabolic target genes VEGFA, LDHA, MXI1 and BNIP3L measured by qRT-PCR. Expression of HIF1A target genes in nucARRB1 cells transiently transfected with scramble (scr) or two different HIF1A siRNAs (siRNA1 and siRNA2) and grown in hypoxia (1% O 2 for 8 h) 48 h post-transfection. TF motif over-representation using DREME. The ARRB1-associated matched motifs (left) are compared to motifs in the JASPAR_CORE database (right). TF name, P -value (probability that the match occurred by random chance according to the null model), E -value (expected number of false positives in the matches) and Q -value (minimum false discovery rate required to include the match) are shown. HIF1A and ARRB1 recruitment to the chromatin was assessed by ChIP followed by qRT-PCR. NucARRB1 cells transfected with scramble or two different HIF1A siRNAs. Fourty-eight hours after transfections, the cells were incubated in normoxia or hypoxia (1% O 2 for 12 h). Left: ChIP-seq-enrichment profiles of endogenous ARRB1, p300 and H3K4me1 and H3K4me3 in C4-2s indicating the genomic location of HIF1A-binding sites (black boxes) as reported in Lofstead et al . The red boxes indicate the genomic region selected for designing the primers used to amplify the immunoprecipitated chromatin. MXI1-L and MXI1-S indicate HIF1A-binding sites regulating the two different MXI1 isoforms. Transcriptional regulation of the MXI1-L-associated isoform has been shown to be HIF1A dependent, whereas that of the MXI1-S-associated isoform is HIF1A independent. Right: ARRB1- and HIF1A-enrichment normalised to input and amount of chromatin at the VEGFA and MXI1-L sites. Data information: For all graphs, N = 3, values are mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: shRNA, Incubation, Expressing, Quantitative RT-PCR, Transfection, ChIP-sequencing, Binding Assay, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: Left: immunoblot showing increased levels of HIF1A in three nucARRB1 cell lines compared to GFP control. Right: ImageJ quantification of the bands on the left. Immunoblot of GFP control and nucARRB1 incubated under hypoxic conditions (1% O 2 ) for increasing periods of time. Left: Immunoblot of two control shRNA and three ARRB1 shRNA cell lines showing a reduction in HIF1A levels in ARRB1 KD cell lines. ARRB1 and β-actin loading control are also shown. Right: ImageJ quantification of the bands on the left. HIF1A stabilisation in control and ARRB1 knock-down cell lines incubated in hypoxia (1% O 2 ) for increasing periods of time. NucARRB1 cells were transfected with scramble or two different HIF1A siRNAs. Cell numbers were determined after 2, 4, 6 and 8 days. Schematic diagram of the regulation of HIF1A by various stimuli including oxygen tension, oncometabolites succinate and fumarate and PHD activators me2OG and R59949. ARRB1 may regulate HIF1A stability through modulation of FH and SDH expression. SDHA and FH mRNA expression levels of in nucARRB1 cells relative to control. Normoxic levels of HIF1A, FH and SDHA in GFP and nucARRB1 cell extracts. HIF1A, FH and SDHA levels in nucARRB1 cells transiently transfected with empty vector (EV), FH, SDHA or both FH and SDHA expression vectors. EV-transfected GFP cells were used a baseline control. Extracts were harvested 48 h post-transfection. NucARRB1 cell numbers described in G above were determined 24, 48 and 72 h after transfection. Relative mRNA expression levels of HIF1A metabolic targets from cells described in (G) were measured by qRT-PCR 48 h after transfection. Data information: For all graphs, N = 3, values are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available online for this figure.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Western Blot, Incubation, shRNA, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

doi: 10.15252/embj.201386874

Figure Lengend Snippet: Schematic diagram illustrating metabolites of the glycolytic pathway and the TCA cycle that are altered by ARRB1. In red are metabolites or genes showing increased concentration or expression, respectively, in nucARRB1; in blue are those with lower concentration or expression, respectively. Reductive carboxylation pathway of glutamine is indicated in green. Modulation of HIF1A signalling by ARRB1 occurs at two levels: (i) ARRB1 indirectly alters expression levels of metabolic enzymes FH and SDHA, resulting in accumulation of oncometabolites fumarate and succinate and pseudohypoxic stabilisation of HIF1A, and (ii) ARRB1 acts as a co-factor of HIF1A to enhance its transcriptional activity both in normoxic and hypoxic conditions.

Article Snippet: A humanised sequence of ARRB1 tagged with 3XNLS sequences was obtained from Sloning BioTechnology and supplied in a pPCRScript vector. nucARRB1 was generated by cloning the (XhoI-EcoRV) fragment into pAcGFP-C1 digested with XhoI and EcoRV to generate pAcGFP-ARRB1-3NLS. wtARRB1 was generated by digesting pAcGFP-ARRB1-3NLS with NheI and cloning the (NheI-NheI) fragment into pAcGFP-C1 (Clontech) digested with NheI and XbaI.

Techniques: Concentration Assay, Expressing, Activity Assay

Journal:

Article Title: Identification and Characterization of a Novel Uropathogenic Escherichia coli -Associated Fimbrial Gene Cluster

doi: 10.1128/IAI.72.7.3890-3901.2004

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Antibiotics were used at the following concentrations unless otherwise indicated: ampicillin, 50 μg/ml; chloramphenicol, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 50 μg/ml; and rifampin, 50 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 E. coli strain or plasmid Relevant characteristics Source or reference E. coli strains CFT073 Pyelonephritis isolate, fim + pap + hly + 31 CFT073 Rif Spontaneous rifampin-resistant mutant of CFT073 This study UMD100 CFT073 aufC ::pSSP104, ampicillin resistant This study UMD132 CFT073 Δ( aufABCDEFG ), nalidixic and chloramphenicol resistant This study UMD133 CFT073 Δ( aufABCDEFG ), nalidixic resistant This study ORN172 Δ( fimBEACDFGH ), kanamycin resistant 43 HB101 F − ( gpt-proA ) 62 leu supe44 ara-14 galK2 lacY1 ( mcrC-mrr ) rpsL20 xyl-5 mtl-1 recA13 37 TOP10F′ F′[ lacI q Tn 10 (Tet R )] mrcA Δ( mrr-hsdRMS-mrcrBC ) φ80 lacZ ΔM15 Δφ lacX74 recA1 araD139 Δ( ara-leu )7697 galU galk rpsL (Str r ) endA1 nupG Invitrogen BL21-AI F − ompT hsdS B (r B − m B − ) gal dcm (DE3) araB :: T7RNAP-tet A Invitrogen BL21 (DE3)pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3) pLysS (Cam r ) Invitrogen S17-λ pir pro thi recA hsdR; chromosomal RP4-2 (Tn 1 ::ISR 1 tet ::Mu Km::Tn 7 ); λpir 17 Plasmids pcosmid 12A Cosmid clone of CFT073 that contains the entire auf operon pELB1 pCRScript Cam (SK+) with the 8.6-kb BsiWI/KasI fragment containing the auf operon This study pELB2 pBlueScript-II KS(+) with the 9-kb NotI/XhoI fragment containing auf operon under T7 promoter control This study pAufA pCR T7/NT-TOPO with the aufA gene lacking the codons for the signal sequence This study pPCRScript Cam (SK+) Cloning vector, chloramphenicol resistant Stratagene pBlueScript-II KS (+) Cloning vector, ampicillin resistant Stratagene pCR T7/NT-TOPO Cloning vector, ampicillin resistant Invitrogen pGP704 Suicide vector, ampicillin resistant, R6K origin 29 pT-Aadv Cloning vector, ampicillin resistant Clonetech pSSP102 pT-Adv with the 1.2-kb aufC fragment Stratagene pSSP104 pGP704 with the 1.2-kb XabI/KpnI aufC fragment, ampicillin resistant This study PKD46 Low-copy-number plasmid encoding the phage λ Red recombinase expressing γ, β, and exo from the arabinose-inducible Para B 8 pFT-A Helper plasmid, carrying the FLP recombinase, ampicillin resistant 34 pKD3 Plasmid containing an FRT-flanked cat gene, ampicillin and chloramphenicol resistant 8 Open in a separate window Strains and plasmids used in this study Recombinant DNA methods.

Techniques: Plasmid Preparation, Mutagenesis, Sequencing, Clone Assay, Expressing