Structured Review

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Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either <t>HIF1α</t> or ARNT results in decreased heterodimer formation. B, This results in decreased binding
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1) Product Images from "Impaired Fetoplacental Angiogenesis in Growth-Restricted Fetuses With Abnormal Umbilical Artery Doppler Velocimetry Is Mediated by Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)"

Article Title: Impaired Fetoplacental Angiogenesis in Growth-Restricted Fetuses With Abnormal Umbilical Artery Doppler Velocimetry Is Mediated by Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)

Journal: The Journal of Clinical Endocrinology and Metabolism

doi: 10.1210/jc.2014-2385

Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding
Figure Legend Snippet: Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding

Techniques Used: Binding Assay

2) Product Images from "Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo"

Article Title: Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1471

E2F7 expression is induced in response to hypoxia by HIF1. ( A ) Westernblot analysis of E2F7, E2F8 and HIF1α expression in lysates from HeLa cells transfected with control (scrambled, scr) siRNA, or one of three different E2F7 or E2F8-specific siRNAs (numbered 1, 2 and 3), as indicated. Cells were maintained in normoxia (−) or hypoxia (+) as indicated. Non-specific and background (‘b’) bands serve as loading controls. ( B ) Graphs showing E2F7, E2F8 or VEGFA mRNA levels (depicted as fold change compared to normoxic mRNA levels) isolated from HeLa cells grown in normoxia or hypoxia, and determined by qPCR. In this and all subsequent figures black bars present normoxic (N), and white bars hypoxic (H) conditions. ( C ) ChIP assay, using normoxic or hypoxic HeLa cells, showing enrichment of HIF1α, E2F1 (positive control) or IgG (negative control) on the E2F7 promoter (element 2). ( D ) Graphs showing E2F7 or E2F8 mRNA levels as determined by qPCR. RNA was isolated from HeLa cells grown in normoxia or hypoxia, transfected with control (scr) or HIF1α siRNA as indicated. Lower panels are Western blots showing E2F7, HIF1α and HDAC1 (loading) protein levels. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.
Figure Legend Snippet: E2F7 expression is induced in response to hypoxia by HIF1. ( A ) Westernblot analysis of E2F7, E2F8 and HIF1α expression in lysates from HeLa cells transfected with control (scrambled, scr) siRNA, or one of three different E2F7 or E2F8-specific siRNAs (numbered 1, 2 and 3), as indicated. Cells were maintained in normoxia (−) or hypoxia (+) as indicated. Non-specific and background (‘b’) bands serve as loading controls. ( B ) Graphs showing E2F7, E2F8 or VEGFA mRNA levels (depicted as fold change compared to normoxic mRNA levels) isolated from HeLa cells grown in normoxia or hypoxia, and determined by qPCR. In this and all subsequent figures black bars present normoxic (N), and white bars hypoxic (H) conditions. ( C ) ChIP assay, using normoxic or hypoxic HeLa cells, showing enrichment of HIF1α, E2F1 (positive control) or IgG (negative control) on the E2F7 promoter (element 2). ( D ) Graphs showing E2F7 or E2F8 mRNA levels as determined by qPCR. RNA was isolated from HeLa cells grown in normoxia or hypoxia, transfected with control (scr) or HIF1α siRNA as indicated. Lower panels are Western blots showing E2F7, HIF1α and HDAC1 (loading) protein levels. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.

Techniques Used: Expressing, Transfection, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Negative Control, Western Blot

In vitro and in vivo validation of NRP1 regulation by HIF1α and E2F7. ( A ) ChIP-seq signal (y-axis: peak height) shown for E2F7 (N and HYP) and HIF1α (HYP) on the NRP1 promoter (indicated in grey). Input DNA was sequenced as a control. Lines underneath the graphs indicate annotated genes, boxes present exons and lines with arrows indicate introns. Arrow indicates direction of transcription. ( B ) Validation of HIF1α, E2F7 and E2F1 enrichment on the NRP1 promoter as analyzed by ChIP-qPCR in normoxic or hypoxic HeLa cells. Isotype matched IgG served as a negative control. ( C ) Positive controls for ChIP: binding of E2F7 to the E2F1 promoter, and HIF1α binding to the BNIP3L promoter. A non-specific region 700 bp upstream of the E2F binding site in the E2F1 promoter served as a negative control. ( D ) Graph shows NRP1 mRNA levels as determined by qPCR in lysates isolated from HeLa cells transfected with control (scr), E2F7 and E2F8 (7/8), E2F7 (7), HIF1α (1α) or E2F7 and HIF1α (7/1α). ( E ) Same as (D) but now for E2F1 and NIX mRNA levels, positive controls for E2F7 and HIF1α, respectively. ( F ) In situ hybridizations (ISH) for nrp1a using non-injected control (NIC), e2f7/8 MO (5 + 5 ng, n = 46), or hif1ab MO (5 ng, n = 70) injected zebrafish embryos, obtained from three independent experiments. e2f7/8 MO and NIC littermates were analyzed at 26 hpf, hif1ab MO injected and NIC littermate embryos at 28 hpf. 100% of the e2f7/8 MO injected embryos, and 69% of the hif1ab MO injected embryos showed enhanced nrp1a expression in MN. All panels show lateral views. Panels 2, 3, 7 and 8 show magnifications of the head region. Panels 4, 5, 9 and 10 show magnifications of the trunk region. Asterisks show examples of spinal motorneurons. ( G ) Graphs show fold change of epo or e2f1 mRNA expression in hif1ab MO (1α) or e2f7/8 (7/8) injected zebrafish embyros, respectively, compared to NIC. mRNA levels were determined in > 30 embryos from three independent experiments. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.
Figure Legend Snippet: In vitro and in vivo validation of NRP1 regulation by HIF1α and E2F7. ( A ) ChIP-seq signal (y-axis: peak height) shown for E2F7 (N and HYP) and HIF1α (HYP) on the NRP1 promoter (indicated in grey). Input DNA was sequenced as a control. Lines underneath the graphs indicate annotated genes, boxes present exons and lines with arrows indicate introns. Arrow indicates direction of transcription. ( B ) Validation of HIF1α, E2F7 and E2F1 enrichment on the NRP1 promoter as analyzed by ChIP-qPCR in normoxic or hypoxic HeLa cells. Isotype matched IgG served as a negative control. ( C ) Positive controls for ChIP: binding of E2F7 to the E2F1 promoter, and HIF1α binding to the BNIP3L promoter. A non-specific region 700 bp upstream of the E2F binding site in the E2F1 promoter served as a negative control. ( D ) Graph shows NRP1 mRNA levels as determined by qPCR in lysates isolated from HeLa cells transfected with control (scr), E2F7 and E2F8 (7/8), E2F7 (7), HIF1α (1α) or E2F7 and HIF1α (7/1α). ( E ) Same as (D) but now for E2F1 and NIX mRNA levels, positive controls for E2F7 and HIF1α, respectively. ( F ) In situ hybridizations (ISH) for nrp1a using non-injected control (NIC), e2f7/8 MO (5 + 5 ng, n = 46), or hif1ab MO (5 ng, n = 70) injected zebrafish embryos, obtained from three independent experiments. e2f7/8 MO and NIC littermates were analyzed at 26 hpf, hif1ab MO injected and NIC littermate embryos at 28 hpf. 100% of the e2f7/8 MO injected embryos, and 69% of the hif1ab MO injected embryos showed enhanced nrp1a expression in MN. All panels show lateral views. Panels 2, 3, 7 and 8 show magnifications of the head region. Panels 4, 5, 9 and 10 show magnifications of the trunk region. Asterisks show examples of spinal motorneurons. ( G ) Graphs show fold change of epo or e2f1 mRNA expression in hif1ab MO (1α) or e2f7/8 (7/8) injected zebrafish embyros, respectively, compared to NIC. mRNA levels were determined in > 30 embryos from three independent experiments. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.

Techniques Used: In Vitro, In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Binding Assay, Isolation, Transfection, In Situ, In Situ Hybridization, Injection, Expressing

The HIF1α-E2F7 complex regulates NRP1 through an E2F-hub. ( A ) Left panels show confocal images of the head region of Tg( nrp1a:gfp ) js12 zebrafish at 28 and 52 hpf. Fish are divided in three groups (high, medium, low) based on the level of transgene expression in the eye (showing 2 examples per group). Graphs present quantification of the three groups at the indicated times after injection of control MO, e2f7/8 MO (5 + 5 ng) or hif1α MO (5 ng) in zebrafish embryos (n ≥ 154 embryos in ≥4 independent experiments for each specific condition). ( B ) Schematic figure of the cloned 1532 bp human NRP1 promoter, and promoter regions subcloned from it, using NcoI and SacI restriction sites. ( C ) Luciferase reporter assays showing the fold induction of normalized relative luciferase units (NRLU) of different NRP1 promoter constructs by E2F1 compared to controls. ( D ) Similar as in (C) but now for the E2F1 control promoters E2F7 and E2F1 . ( E ) Reporter assay showing the dose-dependent induction of the NRP1 promoter activity by different amounts of E2F1. ( F ) Reporter assay comparing the activity of the indicated NRP1 promoter constructs in hypoxia compared normoxia. ( G ) Representation of the 246 bp NRP1 promoter and putative E2F binding sites as identified by MatInspector. HIF binding sites were not identified in the 246 bp fragment. ( H ) Reporter assay showing activity of the 1532 bp NRP1 promoter in the presence of E2F1 alone (set at 100%) or together with E2F7 or HIF1α (1α). ( I ) Control reporter assays showing regulation of the E2F7 promoter (left panel) promoter in the presence of E2F1 alone (set at 100%), or together with E2F7. Middle and right panels show positive controls to check for functional E2F7 or HIF1 activity using E2F1 or VEGFA promoter constructs respectively. ( J ) Reporter assay showing induction of the wild-type and the ΔE2F-hub NRP1 promoter by E2F1 compared to controls. ( K ) Similar as in (H) but now for the wildtype or 41 bp E2F-hub deleted (ΔE2F) 246 bp NRP1 promoter. All quantified data present the average ± S.D. (except for (A) in which the average ± S.E.M. as shown) compared to the indicated controls in at least three independent experiments.
Figure Legend Snippet: The HIF1α-E2F7 complex regulates NRP1 through an E2F-hub. ( A ) Left panels show confocal images of the head region of Tg( nrp1a:gfp ) js12 zebrafish at 28 and 52 hpf. Fish are divided in three groups (high, medium, low) based on the level of transgene expression in the eye (showing 2 examples per group). Graphs present quantification of the three groups at the indicated times after injection of control MO, e2f7/8 MO (5 + 5 ng) or hif1α MO (5 ng) in zebrafish embryos (n ≥ 154 embryos in ≥4 independent experiments for each specific condition). ( B ) Schematic figure of the cloned 1532 bp human NRP1 promoter, and promoter regions subcloned from it, using NcoI and SacI restriction sites. ( C ) Luciferase reporter assays showing the fold induction of normalized relative luciferase units (NRLU) of different NRP1 promoter constructs by E2F1 compared to controls. ( D ) Similar as in (C) but now for the E2F1 control promoters E2F7 and E2F1 . ( E ) Reporter assay showing the dose-dependent induction of the NRP1 promoter activity by different amounts of E2F1. ( F ) Reporter assay comparing the activity of the indicated NRP1 promoter constructs in hypoxia compared normoxia. ( G ) Representation of the 246 bp NRP1 promoter and putative E2F binding sites as identified by MatInspector. HIF binding sites were not identified in the 246 bp fragment. ( H ) Reporter assay showing activity of the 1532 bp NRP1 promoter in the presence of E2F1 alone (set at 100%) or together with E2F7 or HIF1α (1α). ( I ) Control reporter assays showing regulation of the E2F7 promoter (left panel) promoter in the presence of E2F1 alone (set at 100%), or together with E2F7. Middle and right panels show positive controls to check for functional E2F7 or HIF1 activity using E2F1 or VEGFA promoter constructs respectively. ( J ) Reporter assay showing induction of the wild-type and the ΔE2F-hub NRP1 promoter by E2F1 compared to controls. ( K ) Similar as in (H) but now for the wildtype or 41 bp E2F-hub deleted (ΔE2F) 246 bp NRP1 promoter. All quantified data present the average ± S.D. (except for (A) in which the average ± S.E.M. as shown) compared to the indicated controls in at least three independent experiments.

Techniques Used: Fluorescence In Situ Hybridization, Expressing, Injection, Clone Assay, Luciferase, Construct, Reporter Assay, Activity Assay, Binding Assay, Functional Assay

Binding and regulation of the common targets by the HIF1α and E2F7. ( A ) Graphs show ChIP-qPCR analysis of E2F7 (left panels), HIF1α (middle panels) and E2F1 (right panels) enrichment on the common repressed target promoters. Non-specific, isotype matched IgG serve as a negative control. ( B ) Graphs showing mRNA levels as determined by qPCR and presented as fold change comparing to scr normoxia, of the common repressed targets. Messenger RNA levels were analyzed in HeLa cells transfected with control (scr), E2F7 and E2F8 (7/8), E2F7 ( 7 ), HIF1α (1α) or E2F7 and HIF1α (7/1α) siRNAs and grown in normoxia or hypoxia, as indicated. ( C ) Similar as in (A) but now for the HIF1α-E2F7 induced targets. ( D ) Similar as in (B) but now for the HIF1α-E2F7 induced targets. ( E ). Upper graphs present E2F7 binding to the E2F1 promoter, to a control region (in the E2F1 promoter) and the E2F3 promoter. Lower graphs show E2F7, HIF1α and E2F1 enrichment to the MCM2 promoter. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.
Figure Legend Snippet: Binding and regulation of the common targets by the HIF1α and E2F7. ( A ) Graphs show ChIP-qPCR analysis of E2F7 (left panels), HIF1α (middle panels) and E2F1 (right panels) enrichment on the common repressed target promoters. Non-specific, isotype matched IgG serve as a negative control. ( B ) Graphs showing mRNA levels as determined by qPCR and presented as fold change comparing to scr normoxia, of the common repressed targets. Messenger RNA levels were analyzed in HeLa cells transfected with control (scr), E2F7 and E2F8 (7/8), E2F7 ( 7 ), HIF1α (1α) or E2F7 and HIF1α (7/1α) siRNAs and grown in normoxia or hypoxia, as indicated. ( C ) Similar as in (A) but now for the HIF1α-E2F7 induced targets. ( D ) Similar as in (B) but now for the HIF1α-E2F7 induced targets. ( E ). Upper graphs present E2F7 binding to the E2F1 promoter, to a control region (in the E2F1 promoter) and the E2F3 promoter. Lower graphs show E2F7, HIF1α and E2F1 enrichment to the MCM2 promoter. All quantified data present the average ± S.D. compared to the indicated controls in at least three independent experiments.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Transfection

Genome-wide analysis of HIF1α and E2F7 common targets by ChIP-seq and microarray analysis. ( A ) Flow chart showing the applied approach. The E2F7 ChIP-seq was performed both in normoxia (N) and hypoxia (H), all other experiments only in hypoxia. ( B ) Table that summarizes the overlap between the E2F7 and HIF1α targets identified in the microarray data (cut off: ≥ 2 fc; P
Figure Legend Snippet: Genome-wide analysis of HIF1α and E2F7 common targets by ChIP-seq and microarray analysis. ( A ) Flow chart showing the applied approach. The E2F7 ChIP-seq was performed both in normoxia (N) and hypoxia (H), all other experiments only in hypoxia. ( B ) Table that summarizes the overlap between the E2F7 and HIF1α targets identified in the microarray data (cut off: ≥ 2 fc; P

Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Microarray, Flow Cytometry

Dualistic functions of the HIF1α-E2F7 complex in gene regulation, and biological implications. Hypoxia induces E2F7 expression through transcriptional activation by HIF1. The almost complete absence of differential gene regulation of common targets by the classified transcriptional activator HIF1α and the repressor E2F7, as observed in our microarray data (Supplementary Figure S2C and D), as well as in the combined ChIP-seq and microarray data (Figure 2C and D ; Supplementary Figure S2F), unequivocally demonstrated the existence of the transcriptional network regulated by the HIF1α-E2F7 complex, in which the complex can either function as a repressor or activator. We reveal a direct role for HIF1α in transcriptional repression by acting independent of HIF-binding sites, but instead through an E2F-hub, as we show for NRP1 . We expect that the HIF1α-E2F7 complex stimulates gene expression through HIF-binding sites, as we recently showed for VEGFA ( 14 ). Although not shown, NRP1 is also repressed, and CYR61 stimulated by HIF1β/ARNT (Supplementary Figure S4B,C). The HIF1α-E2F7 complex may counterbalance the expression of common repressed targets by replacing E2F1 from these promoters in hypoxia, when expression of HIF1α and E2F7 is induced. This mechanism regulates MN axon guidance during normal development, but could also serve neuroprotective functions, as growth cone collapse may eventually result in neuronal death. Whether HIF1-E2F7 induction of VEGFA expression also serves neuroprotective functions remains to be shown, which is also true for the potential role of the HIF1α-E2F7/ NRP1 pathway in regulating (tumor) angiogenesis.
Figure Legend Snippet: Dualistic functions of the HIF1α-E2F7 complex in gene regulation, and biological implications. Hypoxia induces E2F7 expression through transcriptional activation by HIF1. The almost complete absence of differential gene regulation of common targets by the classified transcriptional activator HIF1α and the repressor E2F7, as observed in our microarray data (Supplementary Figure S2C and D), as well as in the combined ChIP-seq and microarray data (Figure 2C and D ; Supplementary Figure S2F), unequivocally demonstrated the existence of the transcriptional network regulated by the HIF1α-E2F7 complex, in which the complex can either function as a repressor or activator. We reveal a direct role for HIF1α in transcriptional repression by acting independent of HIF-binding sites, but instead through an E2F-hub, as we show for NRP1 . We expect that the HIF1α-E2F7 complex stimulates gene expression through HIF-binding sites, as we recently showed for VEGFA ( 14 ). Although not shown, NRP1 is also repressed, and CYR61 stimulated by HIF1β/ARNT (Supplementary Figure S4B,C). The HIF1α-E2F7 complex may counterbalance the expression of common repressed targets by replacing E2F1 from these promoters in hypoxia, when expression of HIF1α and E2F7 is induced. This mechanism regulates MN axon guidance during normal development, but could also serve neuroprotective functions, as growth cone collapse may eventually result in neuronal death. Whether HIF1-E2F7 induction of VEGFA expression also serves neuroprotective functions remains to be shown, which is also true for the potential role of the HIF1α-E2F7/ NRP1 pathway in regulating (tumor) angiogenesis.

Techniques Used: Expressing, Activation Assay, Microarray, Chromatin Immunoprecipitation, Binding Assay

The HIF1α-E2F7 complex regulates MN development in an NRP1-dependent manner. ( A ) Confocal images of MN in the trunk regions above the yolk sac extension of Tg( nrp1a:gfp ) js12 zebrafish at 48 hpf. Zebrafish embryos were non-injected (NIC: non-injected control), or injected with e2f7/8 (5 + 5 ng), hif1ab (5 ng), or control (10 ng) MO. Stunted MN are indicated with an asterisks, truncations resulting in the absence of the hinge are indicated with an arrow. Black bar presents 50 μM. ( B ) Quantification of MN defects in all MN analyzed, as described under (A). Left graph shows quantification of MN defects in e2f7/8 MO or hif1ab MO injected or non-injected wild-type Tg ( nrp1a:gfp ) js12 zebrafish. The two right graphs show quantification of MN defects in e2f7/8 MO or hif1ab MO injected, and non-injected wild-type (black bars) or nrp1a hu10012 mutant (white bars) Tg( nrp1a:gfp ) js12 zebrafish. The numbers in the graphs present the number of analyzed zebrafish (obtained from at least three independent experiments). Per fish, all MN above the yolk sac extension (10–11 MN) were analyzed. ( C ) Analysis of MN defects in wild-type Tg( nrp1a:gfp ) js12 zebrafish embryos, or in embryos obtained from crossing e2f7 A207/A207 ; e2f8 A196/A196 ; Tg( nrp1a:gfp ) js12 zebrafish with e2f7 A207/A207 ; e2f8 WT/A196 ; Tg( nrp1a:gfp ) js12 zebrafish. MN defects were analyzed in the trunk regions above the yolk sac extension at 48 hpf. Left panels show representative confocal images of analyzed MN for both groups. Graph presents quantification of the number of fish with MN defects (presented as%), analyzed in 137 wild-type, or 220 e2f7/8 mutant zebrafish embryos. All quantified data present the average ± S.E.M. compared to the indicated controls in at least three independent experiments. * P
Figure Legend Snippet: The HIF1α-E2F7 complex regulates MN development in an NRP1-dependent manner. ( A ) Confocal images of MN in the trunk regions above the yolk sac extension of Tg( nrp1a:gfp ) js12 zebrafish at 48 hpf. Zebrafish embryos were non-injected (NIC: non-injected control), or injected with e2f7/8 (5 + 5 ng), hif1ab (5 ng), or control (10 ng) MO. Stunted MN are indicated with an asterisks, truncations resulting in the absence of the hinge are indicated with an arrow. Black bar presents 50 μM. ( B ) Quantification of MN defects in all MN analyzed, as described under (A). Left graph shows quantification of MN defects in e2f7/8 MO or hif1ab MO injected or non-injected wild-type Tg ( nrp1a:gfp ) js12 zebrafish. The two right graphs show quantification of MN defects in e2f7/8 MO or hif1ab MO injected, and non-injected wild-type (black bars) or nrp1a hu10012 mutant (white bars) Tg( nrp1a:gfp ) js12 zebrafish. The numbers in the graphs present the number of analyzed zebrafish (obtained from at least three independent experiments). Per fish, all MN above the yolk sac extension (10–11 MN) were analyzed. ( C ) Analysis of MN defects in wild-type Tg( nrp1a:gfp ) js12 zebrafish embryos, or in embryos obtained from crossing e2f7 A207/A207 ; e2f8 A196/A196 ; Tg( nrp1a:gfp ) js12 zebrafish with e2f7 A207/A207 ; e2f8 WT/A196 ; Tg( nrp1a:gfp ) js12 zebrafish. MN defects were analyzed in the trunk regions above the yolk sac extension at 48 hpf. Left panels show representative confocal images of analyzed MN for both groups. Graph presents quantification of the number of fish with MN defects (presented as%), analyzed in 137 wild-type, or 220 e2f7/8 mutant zebrafish embryos. All quantified data present the average ± S.E.M. compared to the indicated controls in at least three independent experiments. * P

Techniques Used: Injection, Mutagenesis, Fluorescence In Situ Hybridization

3) Product Images from "Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1"

Article Title: Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1

Journal: Molecular carcinogenesis

doi: 10.1002/mc.22802

Hypoxia does not cause persistent ZEB1 upregulation (A) Western blotting analysis showing HIF1α and ZEB1 protein levels in BEAS-2B cells. Control (normoxia): cells cultured at 21% O 2 (atmospheric O 2 levels) for 2 weeks; Hypoxia: cells cultured at 3% O 2 for 2 weeks; Normoxia Revert: cells cultured at 3% O 2 for 2 weeks, followed by culturing at 21% O 2 for 2 weeks. While significant increase in HIF1α and ZEB1 protein levels occurred under hypoxia, subsequent reverting the cells to normoxia reduced their levels to that of control cells that were never exposed to hypoxia. (B) Western blotting analysis showing HIF1α and ZEB1 protein levels in BEAS-2B cells. All the cells were cultured at 21% O 2 (atmospheric O 2 levels). Untreated: untreated cells; Ni-exposed: cells exposed to Ni for 2 weeks; Ni-washed-out: cells exposed to Ni for 2 weeks and subsequently cultured in Ni-free medium for 2 more weeks. Both HIF1α and ZEB1 protein levels increased in Ni-exposed cells. While HIF1α levels decreased upon Ni-wash-out, ZEB1 levels remained persistently high.
Figure Legend Snippet: Hypoxia does not cause persistent ZEB1 upregulation (A) Western blotting analysis showing HIF1α and ZEB1 protein levels in BEAS-2B cells. Control (normoxia): cells cultured at 21% O 2 (atmospheric O 2 levels) for 2 weeks; Hypoxia: cells cultured at 3% O 2 for 2 weeks; Normoxia Revert: cells cultured at 3% O 2 for 2 weeks, followed by culturing at 21% O 2 for 2 weeks. While significant increase in HIF1α and ZEB1 protein levels occurred under hypoxia, subsequent reverting the cells to normoxia reduced their levels to that of control cells that were never exposed to hypoxia. (B) Western blotting analysis showing HIF1α and ZEB1 protein levels in BEAS-2B cells. All the cells were cultured at 21% O 2 (atmospheric O 2 levels). Untreated: untreated cells; Ni-exposed: cells exposed to Ni for 2 weeks; Ni-washed-out: cells exposed to Ni for 2 weeks and subsequently cultured in Ni-free medium for 2 more weeks. Both HIF1α and ZEB1 protein levels increased in Ni-exposed cells. While HIF1α levels decreased upon Ni-wash-out, ZEB1 levels remained persistently high.

Techniques Used: Western Blot, Cell Culture

4) Product Images from "FRAX597, a PAK1 inhibitor, synergistically reduces pancreatic cancer growth when combined with gemcitabine"

Article Title: FRAX597, a PAK1 inhibitor, synergistically reduces pancreatic cancer growth when combined with gemcitabine

Journal: BMC Cancer

doi: 10.1186/s12885-016-2057-z

PAK1 knock-down (KD) inhibits expression of AKT and HIF1α. Expression of phospho-AKT (pAKT) was significantly reduced in the PAK1 KD clones: 2.05 and 2.10 (PANC-1 ( a )); and 3.09 and 3.12 (MiaPaCa-2 ( b )), compared to the negative controls (NC), as assessed by western blot. HIF1α expression was reduced in PANC-1 ( c ) and MiaPaCa-2 ( d ) PAK1 KD clones under normoxia and hypoxia (1 % O 2 ) conditions. The data represent mean ± SEM, summarised from three independent experiments. * p
Figure Legend Snippet: PAK1 knock-down (KD) inhibits expression of AKT and HIF1α. Expression of phospho-AKT (pAKT) was significantly reduced in the PAK1 KD clones: 2.05 and 2.10 (PANC-1 ( a )); and 3.09 and 3.12 (MiaPaCa-2 ( b )), compared to the negative controls (NC), as assessed by western blot. HIF1α expression was reduced in PANC-1 ( c ) and MiaPaCa-2 ( d ) PAK1 KD clones under normoxia and hypoxia (1 % O 2 ) conditions. The data represent mean ± SEM, summarised from three independent experiments. * p

Techniques Used: Expressing, Clone Assay, Western Blot

5) Product Images from "A compact VEGF signature associated with distant metastases and poor outcomes"

Article Title: A compact VEGF signature associated with distant metastases and poor outcomes

Journal: BMC Medicine

doi: 10.1186/1741-7015-7-9

VEGF profile, glycolysis and HIF1α gene expression analyses . A) Gene expression for the VEGF profile (plus average values), for the six glycolysis genes and glycolysis centroid, HIF1α and fibroblast centroids are shown across the 146 patient UNC training data set with the tumors ordered according to their VEGF profile average values. B) Similar analysis as presented in A except the data set is the NKI patient test set.
Figure Legend Snippet: VEGF profile, glycolysis and HIF1α gene expression analyses . A) Gene expression for the VEGF profile (plus average values), for the six glycolysis genes and glycolysis centroid, HIF1α and fibroblast centroids are shown across the 146 patient UNC training data set with the tumors ordered according to their VEGF profile average values. B) Similar analysis as presented in A except the data set is the NKI patient test set.

Techniques Used: Expressing

6) Product Images from "EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate"

Article Title: EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate

Journal: Angiogenesis

doi: 10.1007/s10456-011-9217-1

Expression of HIF1α in prostate and liver. a HIF1α immunostaining of vessels in transverse sections of prostate ventral lobes from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/−
Figure Legend Snippet: Expression of HIF1α in prostate and liver. a HIF1α immunostaining of vessels in transverse sections of prostate ventral lobes from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/−

Techniques Used: Expressing, Immunostaining

7) Product Images from "A HIF1? Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas"

Article Title: A HIF1? Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0010008

HIF1α Attenuates SDHB Levels (A) HIF1α expression was induced by treatment of mouse pheochromocytoma MPC 9/3L cells with 150 μM cobalt chloride for the indicated times. SDHB expression decreased in treated cells. Glut1 indicates increased activity of HIF1α, and β-actin was used as a loading control. (B) Transient expression in HEK293 cells of a HIF1α double mutant PA (P402A/P564A) that is resistant to VHL-mediated degradation reduced expression of SDHB. (C) A2058 cell lines stably expressing HIF1α shRNA do not show change in SDHB after cobalt chloride exposure, while SDHB is downregulated in control GFP shRNA cells treated with cobalt chloride. (D) Proposed model of HIF1α and SDHB interregulation. HIF1α downregulates SDHB, which leads to complex II dysfunction. High succinate levels resulting from loss of complex II, in turn, inhibit prolyl hydroxylase (PHD) activity [ 19 ]. Non-hydroxylated HIF1α is resistant to VHL-mediated targeting for degradation and can therefore activate downstream genes, such as angiogenic factors. “E3 complex” indicates the E3 ubiquitin ligase complex for which VHL is the substrate recognition factor.
Figure Legend Snippet: HIF1α Attenuates SDHB Levels (A) HIF1α expression was induced by treatment of mouse pheochromocytoma MPC 9/3L cells with 150 μM cobalt chloride for the indicated times. SDHB expression decreased in treated cells. Glut1 indicates increased activity of HIF1α, and β-actin was used as a loading control. (B) Transient expression in HEK293 cells of a HIF1α double mutant PA (P402A/P564A) that is resistant to VHL-mediated degradation reduced expression of SDHB. (C) A2058 cell lines stably expressing HIF1α shRNA do not show change in SDHB after cobalt chloride exposure, while SDHB is downregulated in control GFP shRNA cells treated with cobalt chloride. (D) Proposed model of HIF1α and SDHB interregulation. HIF1α downregulates SDHB, which leads to complex II dysfunction. High succinate levels resulting from loss of complex II, in turn, inhibit prolyl hydroxylase (PHD) activity [ 19 ]. Non-hydroxylated HIF1α is resistant to VHL-mediated targeting for degradation and can therefore activate downstream genes, such as angiogenic factors. “E3 complex” indicates the E3 ubiquitin ligase complex for which VHL is the substrate recognition factor.

Techniques Used: Expressing, Activity Assay, Mutagenesis, Stable Transfection, shRNA

8) Product Images from "Mechanism of hypoxia-inducible factor 1?-mediated Mcl1 regulation in Helicobacter pylori-infected human gastric epithelium"

Article Title: Mechanism of hypoxia-inducible factor 1?-mediated Mcl1 regulation in Helicobacter pylori-infected human gastric epithelium

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00372.2010

HIF1α binds to hif1 α HBS. A : chromatin immunoprecipitation (ChIP) analysis of HIF1α immunocomplex for HRE-containing mcl1 promoter. Chromatins from H. pylori -infected or CoCl 2 -treated cells were subjected to ChIP assay by use of
Figure Legend Snippet: HIF1α binds to hif1 α HBS. A : chromatin immunoprecipitation (ChIP) analysis of HIF1α immunocomplex for HRE-containing mcl1 promoter. Chromatins from H. pylori -infected or CoCl 2 -treated cells were subjected to ChIP assay by use of

Techniques Used: Chromatin Immunoprecipitation, Infection

A multiprotein complex is formed at mcl1 HRE following H. pylori infection. A : EMSA shows the formation of a complex at mcl1 HRE after 3-h MOI 300 H. pylori infection (indicated by an arrow). B : a representative ( n = 3) Western blot shows that p300, HIF1α,
Figure Legend Snippet: A multiprotein complex is formed at mcl1 HRE following H. pylori infection. A : EMSA shows the formation of a complex at mcl1 HRE after 3-h MOI 300 H. pylori infection (indicated by an arrow). B : a representative ( n = 3) Western blot shows that p300, HIF1α,

Techniques Used: Infection, Western Blot

H. pylori infection upregulates APE1, HIF1α, and Mcl1 in the human gastric epithelium. A : representative immunohistochemistry showing greater expression of APE-1, Hif1α, and Mcl1 in H. pylori -infected ( n = 2) gastric epithelium and lamina
Figure Legend Snippet: H. pylori infection upregulates APE1, HIF1α, and Mcl1 in the human gastric epithelium. A : representative immunohistochemistry showing greater expression of APE-1, Hif1α, and Mcl1 in H. pylori -infected ( n = 2) gastric epithelium and lamina

Techniques Used: Infection, Immunohistochemistry, Expressing

Helicobacter pylori induce hypoxia-inducible factor (HIF)-1α and HIF1α-regulated Mcl1 expression in native and cultured gastric epithelial cells (GEC). A : Western blot analysis of whole cell lysates prepared from AGS cells infected with
Figure Legend Snippet: Helicobacter pylori induce hypoxia-inducible factor (HIF)-1α and HIF1α-regulated Mcl1 expression in native and cultured gastric epithelial cells (GEC). A : Western blot analysis of whole cell lysates prepared from AGS cells infected with

Techniques Used: Expressing, Cell Culture, Western Blot, Infection

APE1 and p300 regulate HIF1α expression and activity in GEC. A : representative Western blot ( n = 3) demonstrating the expression of HIF1α in pSIREN and short hairpin RNA (shRNA) cells infected for 3 h with different MOI of H. pylori .
Figure Legend Snippet: APE1 and p300 regulate HIF1α expression and activity in GEC. A : representative Western blot ( n = 3) demonstrating the expression of HIF1α in pSIREN and short hairpin RNA (shRNA) cells infected for 3 h with different MOI of H. pylori .

Techniques Used: Expressing, Activity Assay, Western Blot, shRNA, Infection

APE1 is required for induction of HIF1α expression and transcriptional activity by H. pylori. A : effect of wild-type (WT) APE1 and nuclear localization signal-deficient mutant of APE1 (NΔ41) expression in shRNA cells infected with MOI
Figure Legend Snippet: APE1 is required for induction of HIF1α expression and transcriptional activity by H. pylori. A : effect of wild-type (WT) APE1 and nuclear localization signal-deficient mutant of APE1 (NΔ41) expression in shRNA cells infected with MOI

Techniques Used: Expressing, Activity Assay, Mutagenesis, shRNA, Infection

9) Product Images from "Near-infrared fluorescence imaging of cancer mediated by tumor hypoxia and HIF1α/OATPs signaling axis"

Article Title: Near-infrared fluorescence imaging of cancer mediated by tumor hypoxia and HIF1α/OATPs signaling axis

Journal: Biomaterials

doi: 10.1016/j.biomaterials.2014.05.073

NIRF imaging of clinical RCC samples. (A, B) Representative NIRF images of excised kidney by complete nephrectomy (A) as well as small cuts of normal (B, top) and RCC tissues (B, bottom) from an individual patient. Scale bars represent x10 9 and x10 8 for (A) and (B), respectively, in the unit of radiant efficiency. (C) Quantitation of MHI-148 dye uptake in normal and tumor tissues from three RCC patients, presented as the ratio of dye intensity to tissue weight from five cuts of tissues of each patient (mean ± SEM). (D) H E stain and IHC analysis of HIF1α and OATP1B3 expression in normal and RCC samples. Representative images are shown. Original magnification, x200 (H E) and x400 (IHC); scale bars represent 20 µm. * p
Figure Legend Snippet: NIRF imaging of clinical RCC samples. (A, B) Representative NIRF images of excised kidney by complete nephrectomy (A) as well as small cuts of normal (B, top) and RCC tissues (B, bottom) from an individual patient. Scale bars represent x10 9 and x10 8 for (A) and (B), respectively, in the unit of radiant efficiency. (C) Quantitation of MHI-148 dye uptake in normal and tumor tissues from three RCC patients, presented as the ratio of dye intensity to tissue weight from five cuts of tissues of each patient (mean ± SEM). (D) H E stain and IHC analysis of HIF1α and OATP1B3 expression in normal and RCC samples. Representative images are shown. Original magnification, x200 (H E) and x400 (IHC); scale bars represent 20 µm. * p

Techniques Used: Imaging, Quantitation Assay, Staining, Immunohistochemistry, Expressing

MHI-148, a NIRF dye, assessed tumor hypoxia. (A) Chemical structure of NIR heptamethine carbocyanine MHI-148 dye. (B) Generation and validation of human PCa PC-3 cells that stably expressed the 5HRE-pODD-Luc construct (top). Stable cells were treated with hypoxia (1% O 2 ) for different times as indicated and then subjected to either luminescence imaging (middle) or immunoblotting of HIF1α protein expression (bottom). (C) In vivo and ex vivo images of PC-3 tumor xenografts that expressed 5HRE-pODD-Luc construct by both bioluminescence (top) and NIRF (bottom) imaging, which showed superimposed signal distribution. Scale bars represent x10 5 and x10 9 for in vivo bioluminescence and NIRF, respectively; x10 4 and x10 9 for ex vivo bioluminescence and NIRF, respectively, in the unit of radiant efficiency.
Figure Legend Snippet: MHI-148, a NIRF dye, assessed tumor hypoxia. (A) Chemical structure of NIR heptamethine carbocyanine MHI-148 dye. (B) Generation and validation of human PCa PC-3 cells that stably expressed the 5HRE-pODD-Luc construct (top). Stable cells were treated with hypoxia (1% O 2 ) for different times as indicated and then subjected to either luminescence imaging (middle) or immunoblotting of HIF1α protein expression (bottom). (C) In vivo and ex vivo images of PC-3 tumor xenografts that expressed 5HRE-pODD-Luc construct by both bioluminescence (top) and NIRF (bottom) imaging, which showed superimposed signal distribution. Scale bars represent x10 5 and x10 9 for in vivo bioluminescence and NIRF, respectively; x10 4 and x10 9 for ex vivo bioluminescence and NIRF, respectively, in the unit of radiant efficiency.

Techniques Used: Stable Transfection, Construct, Imaging, Expressing, In Vivo, Ex Vivo

Hypoxia and HIF1α mediated the uptake of MHI-148 dye by cancer cells. (A) Determination of MHI-148 dye uptake in multiple cancer cell lines under either normoxic or hypoxic (1% O 2 , 4 hr) conditions (N=3, mean ± SEM). Normal human prostatic epithelial PrEC cells were used as a negative control. Dye uptake by cells under normoxia was set as 100%. (B) Determination of MHI-148 dye uptake in select cancer cell lines under CoCI 2 (200 µM) treatment for different time periods as indicated (N=3, mean ± SEM). The CoCI 2 effect in PC-3 cells as a representative was examined by immunoblotting HIF1α protein expression. (C, D) Determination of MHI-148 dye uptake in cancer cells either overexpressing constitutively active HIF1α (C) or with stable knockdown of HIF1α (D) (N=3, mean ± SEM). The efficacy of overexpression or knockdown of HIF1α in PC-3 cells as a representative was examined by immunoblotting HIF1α protein expression. * p
Figure Legend Snippet: Hypoxia and HIF1α mediated the uptake of MHI-148 dye by cancer cells. (A) Determination of MHI-148 dye uptake in multiple cancer cell lines under either normoxic or hypoxic (1% O 2 , 4 hr) conditions (N=3, mean ± SEM). Normal human prostatic epithelial PrEC cells were used as a negative control. Dye uptake by cells under normoxia was set as 100%. (B) Determination of MHI-148 dye uptake in select cancer cell lines under CoCI 2 (200 µM) treatment for different time periods as indicated (N=3, mean ± SEM). The CoCI 2 effect in PC-3 cells as a representative was examined by immunoblotting HIF1α protein expression. (C, D) Determination of MHI-148 dye uptake in cancer cells either overexpressing constitutively active HIF1α (C) or with stable knockdown of HIF1α (D) (N=3, mean ± SEM). The efficacy of overexpression or knockdown of HIF1α in PC-3 cells as a representative was examined by immunoblotting HIF1α protein expression. * p

Techniques Used: Negative Control, Expressing, Over Expression

Hypoxia and HIF1α mediated the uptake of MHI-148 dye by tumor xenografts. (A) Representative in vivo and ex vivo NIRF images of control (left flank) vs. HIF1α-overexpressing (right flank) (left panel) and control (left flank) vs. HIFIα-knockdown (right flank) (right panel) PC-3 tumor xenografts. Scale bars represent x10 8 for both in vivo and ex vivo NIRF in the unit of radiant efficiency. (B) Quantitation of tumor uptake of MHI-148 dye (N=5, mean ± SEM) presented as the ratio of dye intensity to tumor weight. * p
Figure Legend Snippet: Hypoxia and HIF1α mediated the uptake of MHI-148 dye by tumor xenografts. (A) Representative in vivo and ex vivo NIRF images of control (left flank) vs. HIF1α-overexpressing (right flank) (left panel) and control (left flank) vs. HIFIα-knockdown (right flank) (right panel) PC-3 tumor xenografts. Scale bars represent x10 8 for both in vivo and ex vivo NIRF in the unit of radiant efficiency. (B) Quantitation of tumor uptake of MHI-148 dye (N=5, mean ± SEM) presented as the ratio of dye intensity to tumor weight. * p

Techniques Used: In Vivo, Ex Vivo, Quantitation Assay

Co-expression and regulation of HIF1α and OATPs in human PCa. (A) Heat map depicting OATPs mRNA expression profiling in constitutively active HIF1α-overexpressing PC-3 cells by qPCR. (B, C) IHC (B) and double QDL (C, left panel) staining of HIF1α and OATP1B3 expression in clinical specimens of normal prostatic epithelium, Gleason grade 3 and 5 PCa (N=15 for each). Representative images are shown. Original magnification, x400; scale bars represent 20 µm. Cell-based average intensity counts of HIF1α and OATP1B3 stain from 1,000 each of normal, grade 3 and 5 samples were quantified using in Form software (C, middle panel). Intensity of HIF1α and OATP1B3 co-expression in 100 single cells from a representative high-grade individual patient was analyzed for gene expression correlation (C, right panel). (D, E) Determination of OATP1B3 protein (D) and mRNA (E) expression in PC-3 and ARCaP E cells in response to hypoxia (1% O 2 ) for 24 hrs and 4 hrs, respectively. (F) Sequences of the canonical HRE (top sequence) and a potential HRE in OATP1B3 promoter (bottom sequence). (G) Chromatin from either vehicle- or CoCI 2 -treated (200 µM, 16 hr) PC-3 cells was immunoprecipitated using anti-HIF1α or IgG antibodies followed by qPCR using 1 primer set for the HRE in OATP1B3 promoter and 1 control primer set for 0ATP1B3 exonl. Data represent the mean ± SEM of three separate experiments. * p
Figure Legend Snippet: Co-expression and regulation of HIF1α and OATPs in human PCa. (A) Heat map depicting OATPs mRNA expression profiling in constitutively active HIF1α-overexpressing PC-3 cells by qPCR. (B, C) IHC (B) and double QDL (C, left panel) staining of HIF1α and OATP1B3 expression in clinical specimens of normal prostatic epithelium, Gleason grade 3 and 5 PCa (N=15 for each). Representative images are shown. Original magnification, x400; scale bars represent 20 µm. Cell-based average intensity counts of HIF1α and OATP1B3 stain from 1,000 each of normal, grade 3 and 5 samples were quantified using in Form software (C, middle panel). Intensity of HIF1α and OATP1B3 co-expression in 100 single cells from a representative high-grade individual patient was analyzed for gene expression correlation (C, right panel). (D, E) Determination of OATP1B3 protein (D) and mRNA (E) expression in PC-3 and ARCaP E cells in response to hypoxia (1% O 2 ) for 24 hrs and 4 hrs, respectively. (F) Sequences of the canonical HRE (top sequence) and a potential HRE in OATP1B3 promoter (bottom sequence). (G) Chromatin from either vehicle- or CoCI 2 -treated (200 µM, 16 hr) PC-3 cells was immunoprecipitated using anti-HIF1α or IgG antibodies followed by qPCR using 1 primer set for the HRE in OATP1B3 promoter and 1 control primer set for 0ATP1B3 exonl. Data represent the mean ± SEM of three separate experiments. * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Software, Sequencing, Immunoprecipitation

10) Product Images from "Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia"

Article Title: Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia

Journal: International Journal of Oncology

doi: 10.3892/ijo.2015.3010

Hypoxia influences the response of vemurafenib in 518A2 melanoma cells and effects mRNA levels and protein expression of HIF1α and CAIX. (A) Time lapse measurements of the inhibitory effect of vemurafenib in normoxic and hypoxic conditions by the x-CELLigence system. (B) Real-time PCR analysis of HIF1α and CA9 mRNA levels, normalized to the internal control ( β-actin ). Error bars represent ± SD from three different experiments. (C) Expression of HIF1α and CAIX proteins in vemurafenib-treated 518A2 cells was evaluated by immunoblot analyses.
Figure Legend Snippet: Hypoxia influences the response of vemurafenib in 518A2 melanoma cells and effects mRNA levels and protein expression of HIF1α and CAIX. (A) Time lapse measurements of the inhibitory effect of vemurafenib in normoxic and hypoxic conditions by the x-CELLigence system. (B) Real-time PCR analysis of HIF1α and CA9 mRNA levels, normalized to the internal control ( β-actin ). Error bars represent ± SD from three different experiments. (C) Expression of HIF1α and CAIX proteins in vemurafenib-treated 518A2 cells was evaluated by immunoblot analyses.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Immunohistochemistry of two representative 518A2 xenograft tumor samples stained with HIF1α and CAIX antibodies from corresponding tumor regions. Overview images of two representative melanoma tumors are shown in the top row (a-1, b-1 and c-1, d-1); staining for CAIX (a-1, a-2, c-1 and c-2) and HIF1α (b-1, b-2, d-1 and d-2) shows regions with high expression. Scale bars, 200 μm (a-1, b-1, c-1 and d-1), and 100 μm (a-2, b-2, c-2 and d-2). Arrows point to the area of magnification.
Figure Legend Snippet: Immunohistochemistry of two representative 518A2 xenograft tumor samples stained with HIF1α and CAIX antibodies from corresponding tumor regions. Overview images of two representative melanoma tumors are shown in the top row (a-1, b-1 and c-1, d-1); staining for CAIX (a-1, a-2, c-1 and c-2) and HIF1α (b-1, b-2, d-1 and d-2) shows regions with high expression. Scale bars, 200 μm (a-1, b-1, c-1 and d-1), and 100 μm (a-2, b-2, c-2 and d-2). Arrows point to the area of magnification.

Techniques Used: Immunohistochemistry, Staining, Expressing

11) Product Images from "EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate"

Article Title: EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate

Journal: Angiogenesis

doi: 10.1007/s10456-011-9217-1

Expression of HIF1α in prostate and liver. a HIF1α immunostaining of vessels in transverse sections of prostate ventral lobes from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/− mice at age 20–24 mos. b HIF1α immunostaining of vessels in transverse sections of liver from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/− mice at age 20–24 mos. VHL +/− mice displayed cytoplasmic staining of hepatocytes and sinusoidal lining cells proximal to portal areas ( black arrow ). EAF2 −/− VHL +/− mice displayed cytoplasmic staining of hepatocytes and sinusoidal lining cells ( black arrows ) in areas of increased oval cell proliferation ( dashed arrow ). c Western blot analysis of HIF1α expression in liver extracts of wild-type or EAF2 −/− mice at age 12 mos. Blots were reprobed with GAPDH antibody to confirm equal protein loading. d Modulation of HIF1α expression by EAF2 in MEF cells. EAF2 −/− MEF cells transfected with GFP-EAF2 had reduced HIF1α expression. Blots were reprobed with β-actin antibody to confirm equal protein loading. Western blot images are representative of at least 3 experiments
Figure Legend Snippet: Expression of HIF1α in prostate and liver. a HIF1α immunostaining of vessels in transverse sections of prostate ventral lobes from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/− mice at age 20–24 mos. b HIF1α immunostaining of vessels in transverse sections of liver from wild-type control (WT), EAF2 −/− , VHL +/− and EAF2 −/− VHL +/− mice at age 20–24 mos. VHL +/− mice displayed cytoplasmic staining of hepatocytes and sinusoidal lining cells proximal to portal areas ( black arrow ). EAF2 −/− VHL +/− mice displayed cytoplasmic staining of hepatocytes and sinusoidal lining cells ( black arrows ) in areas of increased oval cell proliferation ( dashed arrow ). c Western blot analysis of HIF1α expression in liver extracts of wild-type or EAF2 −/− mice at age 12 mos. Blots were reprobed with GAPDH antibody to confirm equal protein loading. d Modulation of HIF1α expression by EAF2 in MEF cells. EAF2 −/− MEF cells transfected with GFP-EAF2 had reduced HIF1α expression. Blots were reprobed with β-actin antibody to confirm equal protein loading. Western blot images are representative of at least 3 experiments

Techniques Used: Expressing, Immunostaining, Mouse Assay, Staining, Western Blot, Transfection

12) Product Images from "Mode of Cell Death Induction by Pharmacological Vacuolar H+-ATPase (V-ATPase) Inhibition *"

Article Title: Mode of Cell Death Induction by Pharmacological Vacuolar H+-ATPase (V-ATPase) Inhibition *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.412007

Archazolid induces changes in metabolism. SKBR3 cells were treated with different concentrations of archazolid for varying durations. A , expression of HIF1α in the lysates or the nucleus was analyzed via Western blotting. B , cells were treated
Figure Legend Snippet: Archazolid induces changes in metabolism. SKBR3 cells were treated with different concentrations of archazolid for varying durations. A , expression of HIF1α in the lysates or the nucleus was analyzed via Western blotting. B , cells were treated

Techniques Used: Expressing, Western Blot

Schematic overview of V-ATPase inhibition and cell death. Inhibition of the V-ATPase by archazolid leads to a decrease of the ATP level and thereby to an activation of several stress responses like autophagy, HIF1α induction, phosphorylation of
Figure Legend Snippet: Schematic overview of V-ATPase inhibition and cell death. Inhibition of the V-ATPase by archazolid leads to a decrease of the ATP level and thereby to an activation of several stress responses like autophagy, HIF1α induction, phosphorylation of

Techniques Used: Inhibition, Activation Assay

13) Product Images from "Productive Parvovirus B19 Infection of Primary Human Erythroid Progenitor Cells at Hypoxia Is Regulated by STAT5A and MEK Signaling but not HIF?"

Article Title: Productive Parvovirus B19 Infection of Primary Human Erythroid Progenitor Cells at Hypoxia Is Regulated by STAT5A and MEK Signaling but not HIF?

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002088

Regulation of B19V infection of CD36 + EPCs by HIFα under hypoxia vs. normoxia. ( A ) HIF1α levels in CD36 + EPCs cultured under normoxia (N) or hypoxia (H). Cell lysates were prepared on the indicated days and were analyzed by Western blotting with anti-HIF1α and anti-β-actin. ( B C ) Effects of the putative HIF-HBS in the P6 promoter. Day 7 hypoxia- or normoxia-cultured CD36 + EPCs were transduced with Lenti-P6-GFP or Lenti-P6(ΔHBS)-GFP (B), and then were either maintained under normoxia (→N) or hypoxia (→H). At 48 hrs post-transduction, level of GFP expression (as MFI) was measured by flow cytometry. ( D ) Effects of R59949 on B19V infection. Day 8 hypoxia-cultured CD36 + EPCs were treated with R59949 at the final concentrations shown. At 24 hrs post-treatment, cells were collected for HIF1α detection by Western blotting, or infected with B19V at an MOI of 1,000 gc/cell. At 48 and 72 hrs p.i., the levels of VP2-encoding mRNA per β-actin mRNA were quantified. ( E F ) Effects of HIF1α and PHD knockdown on B19V infection. Day 7 CD36 + EPCs cultured under each condition were transduced with the indicated lentiviruses. At 48 hrs post-transduction, cells were infected with B19V (at MOIs of 2,000 and 4,000 gc/cell for hypoxia- and normoxia-cultured cells, respectively). At 48 hrs p.i., the expression levels of NS1 (as % of positive cells) and HIF1α (as MFI) were analyzed by flow cytometry in lentivirus-transduced (GFP + ) cells. Dashed reference lines were selected arbitrarily to show the relative position of the peaks, and Bkg (background) represents the secondary antibody only control.
Figure Legend Snippet: Regulation of B19V infection of CD36 + EPCs by HIFα under hypoxia vs. normoxia. ( A ) HIF1α levels in CD36 + EPCs cultured under normoxia (N) or hypoxia (H). Cell lysates were prepared on the indicated days and were analyzed by Western blotting with anti-HIF1α and anti-β-actin. ( B C ) Effects of the putative HIF-HBS in the P6 promoter. Day 7 hypoxia- or normoxia-cultured CD36 + EPCs were transduced with Lenti-P6-GFP or Lenti-P6(ΔHBS)-GFP (B), and then were either maintained under normoxia (→N) or hypoxia (→H). At 48 hrs post-transduction, level of GFP expression (as MFI) was measured by flow cytometry. ( D ) Effects of R59949 on B19V infection. Day 8 hypoxia-cultured CD36 + EPCs were treated with R59949 at the final concentrations shown. At 24 hrs post-treatment, cells were collected for HIF1α detection by Western blotting, or infected with B19V at an MOI of 1,000 gc/cell. At 48 and 72 hrs p.i., the levels of VP2-encoding mRNA per β-actin mRNA were quantified. ( E F ) Effects of HIF1α and PHD knockdown on B19V infection. Day 7 CD36 + EPCs cultured under each condition were transduced with the indicated lentiviruses. At 48 hrs post-transduction, cells were infected with B19V (at MOIs of 2,000 and 4,000 gc/cell for hypoxia- and normoxia-cultured cells, respectively). At 48 hrs p.i., the expression levels of NS1 (as % of positive cells) and HIF1α (as MFI) were analyzed by flow cytometry in lentivirus-transduced (GFP + ) cells. Dashed reference lines were selected arbitrarily to show the relative position of the peaks, and Bkg (background) represents the secondary antibody only control.

Techniques Used: Infection, Cell Culture, Western Blot, Transduction, Expressing, Flow Cytometry, Cytometry

14) Product Images from "Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis"

Article Title: Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI70982

The HIF1α/VEGF-A/FOXO1/TWIST1 pathway is manifested in high–Gleason grade PCa.
Figure Legend Snippet: The HIF1α/VEGF-A/FOXO1/TWIST1 pathway is manifested in high–Gleason grade PCa.

Techniques Used:

MAOA regulates HIF1α stability.
Figure Legend Snippet: MAOA regulates HIF1α stability.

Techniques Used:

MAOA regulates HIF1α stability through ROS.
Figure Legend Snippet: MAOA regulates HIF1α stability through ROS.

Techniques Used:

15) Product Images from "Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer"

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041171

Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA. Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.
Figure Legend Snippet: Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA. Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.

Techniques Used: In Vivo, Mouse Assay, Western Blot

16) Product Images from "Improving the metabolic fidelity of cancer models with a physiological cell culture medium"

Article Title: Improving the metabolic fidelity of cancer models with a physiological cell culture medium

Journal: Science Advances

doi: 10.1126/sciadv.aau7314

Plasmax induces cell line–specific transcriptomic alterations and prevents the pseudohypoxic gene expression signature of cells cultured in commercial media. ( A ) PCA of gene expression obtained from RNA-seq data of BT549, CAL-120, and MDA-MB-468 cells cultured in Plasmax or DMEM-F12, in normoxia. n = 3. Each symbol represents an independent experiment. ( B ) Venn diagram showing the number of genes that were differentially regulated in these cell lines when cultured in Plasmax and DMEM-F12. ( C ) Heat map of genes that were significantly [false discovery rate (FDR) of 10%] and coherently regulated (absolute log 2 fold change ≥ 0.585) by culturing cells in Plasmax, in normoxia, in at least two of three cell lines. n = 3, log 2 (mean fold change). ( D ) Correlation analysis of genes regulated by hypoxia in Plasmax ( y axis) and genes regulated by Plasmax in normoxia ( x axis). Each dot represents the mean of three independent experiments. ( E ) Expression levels of HIF1α target genes CA9 , TXNIP , PDK1 , and BNIP3 in BT549 cells relative to control condition (normoxia, DMEM-F12). Means ± SEM; n = 3, each dot represents an independent experiment, and P values refer to a two-tailed t test for unpaired homoscedastic samples. ( F ) Western blot showing HIF1α levels in BT549 cells in Plasmax (P) or DMEM-F12 (D), in normoxia (N) and hypoxia (H). ( G ) Western blot showing HIF1α levels in BT549, CAL-120, and MDA-MB-468 cells cultured in DMEM-F12 or Plasmax in normoxia. ( H ) Western blot showing HIF1α levels in BT549 cells cultured in normoxia in DMEM-F12, Plasmax, DMEM, and RPMI 1640. ( I ) Western blot showing pyruvate-dependent HIF1α levels in BT549 cells, cultured in DMEM-F12, Plasmax, and DMEM in normoxia. (F to I) Images are representative of three independent experiments. P values refer to a two-tailed t test for unpaired homoscedastic samples.
Figure Legend Snippet: Plasmax induces cell line–specific transcriptomic alterations and prevents the pseudohypoxic gene expression signature of cells cultured in commercial media. ( A ) PCA of gene expression obtained from RNA-seq data of BT549, CAL-120, and MDA-MB-468 cells cultured in Plasmax or DMEM-F12, in normoxia. n = 3. Each symbol represents an independent experiment. ( B ) Venn diagram showing the number of genes that were differentially regulated in these cell lines when cultured in Plasmax and DMEM-F12. ( C ) Heat map of genes that were significantly [false discovery rate (FDR) of 10%] and coherently regulated (absolute log 2 fold change ≥ 0.585) by culturing cells in Plasmax, in normoxia, in at least two of three cell lines. n = 3, log 2 (mean fold change). ( D ) Correlation analysis of genes regulated by hypoxia in Plasmax ( y axis) and genes regulated by Plasmax in normoxia ( x axis). Each dot represents the mean of three independent experiments. ( E ) Expression levels of HIF1α target genes CA9 , TXNIP , PDK1 , and BNIP3 in BT549 cells relative to control condition (normoxia, DMEM-F12). Means ± SEM; n = 3, each dot represents an independent experiment, and P values refer to a two-tailed t test for unpaired homoscedastic samples. ( F ) Western blot showing HIF1α levels in BT549 cells in Plasmax (P) or DMEM-F12 (D), in normoxia (N) and hypoxia (H). ( G ) Western blot showing HIF1α levels in BT549, CAL-120, and MDA-MB-468 cells cultured in DMEM-F12 or Plasmax in normoxia. ( H ) Western blot showing HIF1α levels in BT549 cells cultured in normoxia in DMEM-F12, Plasmax, DMEM, and RPMI 1640. ( I ) Western blot showing pyruvate-dependent HIF1α levels in BT549 cells, cultured in DMEM-F12, Plasmax, and DMEM in normoxia. (F to I) Images are representative of three independent experiments. P values refer to a two-tailed t test for unpaired homoscedastic samples.

Techniques Used: Expressing, Cell Culture, RNA Sequencing Assay, Multiple Displacement Amplification, Two Tailed Test, Western Blot

17) Product Images from "Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells"

Article Title: Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells

Journal: Aging Cell

doi: 10.1111/acel.12181

Werner syndrome protein (WRN) knockdown affects the levels of hypoxia-inducible factor 1 (HIF1α). Nuclear extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or green fluorescence protein (GFP) (CTR) that were grown in the absence or presence of doxycycline (+dox) for 3 or 5 days under atmosphere of 1% (A) or 21% (B) oxygen, and analyzed by immunoblotting using antibodies against WRN, HIF1α and α-tubulin as loading control. (C) Nuclear extracts from shCTR and shWRN Hela cells treated with or without MG-132 or dimethyloxalylglycine as indicated were analyzed by immunoblotting with antibodies specific to hydroxylated HIF1 α (hydroxyl-HIF1α) or total HIF1α (D), Reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) analysis of mRNA steady-state levels for HIF1α in Hela cells grown in 1% oxygen 3 days after WRN knockdown. mRNA expression levels were normalized to tubulin mRNA and are shown as relative to shCTR cells. RNA was isolated from at least three biological samples of Hela cells with shCTR or shWRN, and RT–qPCR assays were carried out in triplicate samples. The graph and statistics were generated using Excel. (E) Extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP (CTR) that were grown in the absence or presence of doxycycline (+dox) for the indicated days, and analyzed by immunoblotting using antibodies against WRN, phosphorylated 4E binding protein 1 (4EBP1) (P-4EBP1), total 4EBP1, and α-tubulin as loading control.
Figure Legend Snippet: Werner syndrome protein (WRN) knockdown affects the levels of hypoxia-inducible factor 1 (HIF1α). Nuclear extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or green fluorescence protein (GFP) (CTR) that were grown in the absence or presence of doxycycline (+dox) for 3 or 5 days under atmosphere of 1% (A) or 21% (B) oxygen, and analyzed by immunoblotting using antibodies against WRN, HIF1α and α-tubulin as loading control. (C) Nuclear extracts from shCTR and shWRN Hela cells treated with or without MG-132 or dimethyloxalylglycine as indicated were analyzed by immunoblotting with antibodies specific to hydroxylated HIF1 α (hydroxyl-HIF1α) or total HIF1α (D), Reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) analysis of mRNA steady-state levels for HIF1α in Hela cells grown in 1% oxygen 3 days after WRN knockdown. mRNA expression levels were normalized to tubulin mRNA and are shown as relative to shCTR cells. RNA was isolated from at least three biological samples of Hela cells with shCTR or shWRN, and RT–qPCR assays were carried out in triplicate samples. The graph and statistics were generated using Excel. (E) Extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP (CTR) that were grown in the absence or presence of doxycycline (+dox) for the indicated days, and analyzed by immunoblotting using antibodies against WRN, phosphorylated 4E binding protein 1 (4EBP1) (P-4EBP1), total 4EBP1, and α-tubulin as loading control.

Techniques Used: Transduction, Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Generated, Binding Assay

18) Product Images from "The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition"

Article Title: The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition

Journal: Nature cell biology

doi: 10.1038/ncb3264

NNMT affects naïve to primed hESC transition by repressing Wnt pathway and activating HIF pathway A–B: Sequencing trace files, DNA sequences, protein sequences and 3D protein structures predicted from sequence (Pymol) of various NNMT CRISPR-Cas9 KO mutant clones (gNNMT 7.2.1, A; gNNMT 6.2.4, B). Green color represents the truncated NNMT protein in the CRISPR-Cas9 mutant. C: Schematic representation of wild type NNMT protein and proteins resulting from the CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4. D: Elf1 NNMT CRISPR-Cas9 KO cells have higher amounts of SAM than wild type Elf1 cells (n=6; s.e.m.; p=1.23E-05 for gNNMT7.2.1, p=5.47E-06 for gNNMT6.2.4; 2-tailed t-test). E: Western blot analysis reveals higher HIF1α expression and H3K27me3 marks in Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4 compared to control Elf1 (iCas9) cells. F: qPCR analysis of the naïve marker DNMT3L in wild type Elf1 cells (n=6) and Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 (n=5) and gNNMT 6.2.4 (n=3). s.e.m.; p=0.0009 for gNNMT 6.2.4 vs. Elf1, p=0.027 for gNNMT 7.2.1 vs. Elf1; 2-tailed t-test. G: log2 fold expression change of NNMT, WNT ligands and HIF target genes in Elf1 CRISPR-Cas9 KO gNNMT 7.2.1 compared to wild type Elf1 cells (RNAseq). H: PCA plot of CRISPR NNMT knockout line and different naïve and primed lines sequenced in this study. gNNMT 6.2.2 and gNNM 7.3.5 are heterozogous controls. PC1 (x-axis) explains majority of the variation in the data (61%), and the gNNMT 7.2.1 knockdown line moved along x-axis substiantially away from other naïve lines and toward the primed state. I: Hypergeometric test p-values for the overlap between genes expressed higher (lower) in gNNMT 7.2.1 compared to Elf1 and genes expressed higher (lower) in primed lines compared to naïve lines from multiple studies. Color shade is proportional to negative log10 of p-values. gNNMT 7.2.1 transcriptomic signature has significant overlap with all published primed transcriptomic datasets, supporting its transition toward a primed stage. J: Model of the intricate relationship between metabolism and epigenetic in hESCs. Unprocessed original scans of blots are shown in Supplementary Fig.9 . For raw data, see Supplementary Table 4 . n= number of biological replicates.
Figure Legend Snippet: NNMT affects naïve to primed hESC transition by repressing Wnt pathway and activating HIF pathway A–B: Sequencing trace files, DNA sequences, protein sequences and 3D protein structures predicted from sequence (Pymol) of various NNMT CRISPR-Cas9 KO mutant clones (gNNMT 7.2.1, A; gNNMT 6.2.4, B). Green color represents the truncated NNMT protein in the CRISPR-Cas9 mutant. C: Schematic representation of wild type NNMT protein and proteins resulting from the CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4. D: Elf1 NNMT CRISPR-Cas9 KO cells have higher amounts of SAM than wild type Elf1 cells (n=6; s.e.m.; p=1.23E-05 for gNNMT7.2.1, p=5.47E-06 for gNNMT6.2.4; 2-tailed t-test). E: Western blot analysis reveals higher HIF1α expression and H3K27me3 marks in Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4 compared to control Elf1 (iCas9) cells. F: qPCR analysis of the naïve marker DNMT3L in wild type Elf1 cells (n=6) and Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 (n=5) and gNNMT 6.2.4 (n=3). s.e.m.; p=0.0009 for gNNMT 6.2.4 vs. Elf1, p=0.027 for gNNMT 7.2.1 vs. Elf1; 2-tailed t-test. G: log2 fold expression change of NNMT, WNT ligands and HIF target genes in Elf1 CRISPR-Cas9 KO gNNMT 7.2.1 compared to wild type Elf1 cells (RNAseq). H: PCA plot of CRISPR NNMT knockout line and different naïve and primed lines sequenced in this study. gNNMT 6.2.2 and gNNM 7.3.5 are heterozogous controls. PC1 (x-axis) explains majority of the variation in the data (61%), and the gNNMT 7.2.1 knockdown line moved along x-axis substiantially away from other naïve lines and toward the primed state. I: Hypergeometric test p-values for the overlap between genes expressed higher (lower) in gNNMT 7.2.1 compared to Elf1 and genes expressed higher (lower) in primed lines compared to naïve lines from multiple studies. Color shade is proportional to negative log10 of p-values. gNNMT 7.2.1 transcriptomic signature has significant overlap with all published primed transcriptomic datasets, supporting its transition toward a primed stage. J: Model of the intricate relationship between metabolism and epigenetic in hESCs. Unprocessed original scans of blots are shown in Supplementary Fig.9 . For raw data, see Supplementary Table 4 . n= number of biological replicates.

Techniques Used: Sequencing, CRISPR, Mutagenesis, Clone Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Marker, Knock-Out

HIF1α is required for naïve to primed hESC transition A: screen shot of RNA expression and H3K27me3 marks of EGLN1 (PHD2) in naïve hESCs [Elf1 12 , WIRB3 naïve and BGO1 naïve 8 )], primed hESCs [WIRB3 primed 8 , H1 and H9 64 and Elf1 treated with STAT3 inhbitor (100 µM) for 6h. B: HIFα is hydroxylated on prolyl residues by EGLN1 (PHD2), leading to VHL-mediated proteolysis. C-D: Sequencing trace files, DNA sequences and protein sequences of HIF1α CRISPR-Cas9 knock-out (KO) mutant clones (gHIF1 6.2.1, C; gHIF1 6.3.1, D). E: schematic representation of wild type HIF1α protein and proteins resulting from CRISPR-Cas9 knock-out (KO) mutants gHIF1 6.2.1 and gHIF1 6.3.1. bHLH= basic helix-loop-helix domain, PAS= Per-Arnt-Sim domain, NTAD= N-terminus transcriptional activation domain, CTAD= C-terminus transcriptional activation domain. F: HIF1α is not expressed in CRISPR-Cas9 KO mutants. Western blot analysis of HIF1α expression in cells pushed toward the primed stage by culture in TeSR1 for 5 days in wild type Elf1 cells (iCas9 Elf1), and two CRISPR-Cas9 KO mutants of HIF1α (gHIF1 6.2.1 and gHIF1 6.3.1). G: qPCR analysis of hESCs transitioning to primed reveals that naïve markers (DNMT3L and NNMT) are still expressed higher in Elf1 HIF1α CRISPR-Cas9 KO cells compared to wild type Elf1, while primed marker IDO1 and HIF target genes (PDK1 and VEGFA) are downregulated (n=3; s.e.m.; p=0.024 for DNMT3L, p=0.0005 for NNMT, p=0.001 for IDO1, p=0.12 for PDK1, p=0.004 for VEGFA; 2-tailed t-test). H: KO of HIF1α prevents the metabolic switch occurring during the transition of hESCs from naïve to primed state as shown by measuring OCR after FCCP addition using SeaHorse. n=3 for gHIF1 6.3.1 2iLIF and AF and n=4 for Elf iCas9 and gHIF1 6.2.1 2iLIF and AF; s.e.m.; p=0.0117 for gHIF1 6.2.1 vs. Elf iCas9, p=0.0032 for gHIF1 6.3.1 vs. Elf iCas9; 2-tailed t-test. Unprocessed original scans of blots are shown in Supplementary Fig.9 . For raw data, see Supplementary Table 4 . n= number of biological replicates.
Figure Legend Snippet: HIF1α is required for naïve to primed hESC transition A: screen shot of RNA expression and H3K27me3 marks of EGLN1 (PHD2) in naïve hESCs [Elf1 12 , WIRB3 naïve and BGO1 naïve 8 )], primed hESCs [WIRB3 primed 8 , H1 and H9 64 and Elf1 treated with STAT3 inhbitor (100 µM) for 6h. B: HIFα is hydroxylated on prolyl residues by EGLN1 (PHD2), leading to VHL-mediated proteolysis. C-D: Sequencing trace files, DNA sequences and protein sequences of HIF1α CRISPR-Cas9 knock-out (KO) mutant clones (gHIF1 6.2.1, C; gHIF1 6.3.1, D). E: schematic representation of wild type HIF1α protein and proteins resulting from CRISPR-Cas9 knock-out (KO) mutants gHIF1 6.2.1 and gHIF1 6.3.1. bHLH= basic helix-loop-helix domain, PAS= Per-Arnt-Sim domain, NTAD= N-terminus transcriptional activation domain, CTAD= C-terminus transcriptional activation domain. F: HIF1α is not expressed in CRISPR-Cas9 KO mutants. Western blot analysis of HIF1α expression in cells pushed toward the primed stage by culture in TeSR1 for 5 days in wild type Elf1 cells (iCas9 Elf1), and two CRISPR-Cas9 KO mutants of HIF1α (gHIF1 6.2.1 and gHIF1 6.3.1). G: qPCR analysis of hESCs transitioning to primed reveals that naïve markers (DNMT3L and NNMT) are still expressed higher in Elf1 HIF1α CRISPR-Cas9 KO cells compared to wild type Elf1, while primed marker IDO1 and HIF target genes (PDK1 and VEGFA) are downregulated (n=3; s.e.m.; p=0.024 for DNMT3L, p=0.0005 for NNMT, p=0.001 for IDO1, p=0.12 for PDK1, p=0.004 for VEGFA; 2-tailed t-test). H: KO of HIF1α prevents the metabolic switch occurring during the transition of hESCs from naïve to primed state as shown by measuring OCR after FCCP addition using SeaHorse. n=3 for gHIF1 6.3.1 2iLIF and AF and n=4 for Elf iCas9 and gHIF1 6.2.1 2iLIF and AF; s.e.m.; p=0.0117 for gHIF1 6.2.1 vs. Elf iCas9, p=0.0032 for gHIF1 6.3.1 vs. Elf iCas9; 2-tailed t-test. Unprocessed original scans of blots are shown in Supplementary Fig.9 . For raw data, see Supplementary Table 4 . n= number of biological replicates.

Techniques Used: RNA Expression, Sequencing, CRISPR, Knock-Out, Mutagenesis, Clone Assay, Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Marker

19) Product Images from "DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase"

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01342-12

CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.
Figure Legend Snippet: CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, In Vitro

DCNL1 modulates HIF1α activity. (A) HEK293 cells were transfected with nontargeting scrambled siRNA (siSCR) or DCNL1-targeting siRNA (siDCNL1). An equivalent amount of protein lysate was resolved by SDS-PAGE and detected by immunoblotting using the indicated antibodies. (B) Stable cell lines in the indicated cellular backgrounds were generated through lentivirus-mediated infection of two different shRNA constructs targeting DCNL1. Cells were treated with MG132 for 4 h and lysed, and an equivalent amount of protein lysate was resolved by SDS-PAGE. The indicated proteins were detected by immunoblotting. (C) The indicated stable cell lines were transiently transfected with plasmids encoding firefly luciferase under the control of a hypoxia response element and a Renilla luciferase control. Cells were treated with MG132 for 4 h prior to a dual-luciferase assay. pGIPZ was arbitrarily set to 100 to represent normal HIF1α activity. Error bars represent standard deviations of the mean. An unpaired t test was performed to assess the statistical significance.
Figure Legend Snippet: DCNL1 modulates HIF1α activity. (A) HEK293 cells were transfected with nontargeting scrambled siRNA (siSCR) or DCNL1-targeting siRNA (siDCNL1). An equivalent amount of protein lysate was resolved by SDS-PAGE and detected by immunoblotting using the indicated antibodies. (B) Stable cell lines in the indicated cellular backgrounds were generated through lentivirus-mediated infection of two different shRNA constructs targeting DCNL1. Cells were treated with MG132 for 4 h and lysed, and an equivalent amount of protein lysate was resolved by SDS-PAGE. The indicated proteins were detected by immunoblotting. (C) The indicated stable cell lines were transiently transfected with plasmids encoding firefly luciferase under the control of a hypoxia response element and a Renilla luciferase control. Cells were treated with MG132 for 4 h prior to a dual-luciferase assay. pGIPZ was arbitrarily set to 100 to represent normal HIF1α activity. Error bars represent standard deviations of the mean. An unpaired t test was performed to assess the statistical significance.

Techniques Used: Activity Assay, Transfection, SDS Page, Stable Transfection, Generated, Infection, shRNA, Construct, Luciferase

DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.
Figure Legend Snippet: DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.

Techniques Used: Construct, In Vitro, Incubation, Purification, SDS Page, Transfection, Immunoprecipitation, Generated

20) Product Images from "Hypoxia-inducible factor (HIF1α) gene expression in human shock states"

Article Title: Hypoxia-inducible factor (HIF1α) gene expression in human shock states

Journal: Critical Care

doi: 10.1186/cc11414

Expression of HIF1α over time in survivors (white circles) and non survivors (black circles) . The horizontal bar indicates the median for each group.
Figure Legend Snippet: Expression of HIF1α over time in survivors (white circles) and non survivors (black circles) . The horizontal bar indicates the median for each group.

Techniques Used: Expressing

Expression of different HIF1α variants in shock patients (black bars) and controls (grey bars) .
Figure Legend Snippet: Expression of different HIF1α variants in shock patients (black bars) and controls (grey bars) .

Techniques Used: Expressing

21) Product Images from "The Hypoxia-inducible Factor Renders Cancer Cells More Sensitive to Vitamin C-induced Toxicity *"

Article Title: The Hypoxia-inducible Factor Renders Cancer Cells More Sensitive to Vitamin C-induced Toxicity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.538157

Activation of HIF increases the susceptibility to Vc-induced cell toxicity. A , left panel , representative Western blot analysis showing normalization of HIF1α and HIF2α protein levels after reintroduction of VHL into RCC10 cells. An empty
Figure Legend Snippet: Activation of HIF increases the susceptibility to Vc-induced cell toxicity. A , left panel , representative Western blot analysis showing normalization of HIF1α and HIF2α protein levels after reintroduction of VHL into RCC10 cells. An empty

Techniques Used: Activation Assay, Western Blot

22) Product Images from "D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2"

Article Title: D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2

Journal: Nature Communications

doi: 10.1038/ncomms8768

α-KG mediates the cellular effects of wild-type D2HGDH expression. ( a ) Methylation of H3 lysine residues was determined by western blot in HEK-293 exposed to 0.5 mM or 1 mM of octyl-α-KG (or vehicle control) for 6 h. Octyl-α-KG suppressed K4, K9, K27, K36 methylation in a dose-dependent manner. No changes were found in H3K79me2 levels confirming that this residue is not regulated by an α-KG-dependent HDM. ( b ) Exposure to octyl-α-KG (or vehicle control) in cells grown under hypoxia (1% O 2 ) led to an increase in HIF1α hydroxylation (Pro-402) and consequent decrease in total HIF1α levels. Densitometric quantification is shown at the bottom of the western blots. ( c ) Octyl-α-KG significantly increased the abundance of 5hmC marks in the DNA ( P =0.0001, two-tailed Student's t -test, left panel) and decreased that of 5mC marks (global DNA methylation) ( P =0.0001, two-sided Student's t -test, right panel), in a dose-dependent fashion. The data shown in c represent the mean and s.d. of an assay performed in triplicate. Data shown in a – c were confirmed with at least one independent biological replicate. ( d ) HEK-293 cells stably expressing WT D2HGDH were exposed to 1 mM of dimethyloxalylglycine (DMOG) for 6 h, and the methylation levels of H3 lysines verified by western blot. Exposure to DMOG restored K4me3 and K9me2 in D2HGDH-WT cells to the levels found in MSCV control. ( e ) Hypoxia (1% O 2 for 18 h) increases HIF1α hydroxylation (Pro-402) and decreases its stability and activity, defined by GLUT1 expression, in cells expressing WT D2HGDH when compared with isogenic control cells (MSCV); exposure to DMOG fully countered the effects of WT D2HGDH on HIF1α and GLUT1. ( f ) D2HGDH-WT expressing cells display significantly higher and low abundance of 5hmC (left panel) and 5mC (right panel) marks, respectively, than its isogenic controls expressing an empty MSCV vector ( P =0.0008 and P =0.0016 ANOVA). Exposure to DMOG (1 mM for 6 h) reversed the effects of WT D2HGDH back to the MSCV baseline. Experiments shown in d and e were repeated twice, the data in f represent the mean and s.d. of a representative experiment (from two biological replicates) performed in triplicate.
Figure Legend Snippet: α-KG mediates the cellular effects of wild-type D2HGDH expression. ( a ) Methylation of H3 lysine residues was determined by western blot in HEK-293 exposed to 0.5 mM or 1 mM of octyl-α-KG (or vehicle control) for 6 h. Octyl-α-KG suppressed K4, K9, K27, K36 methylation in a dose-dependent manner. No changes were found in H3K79me2 levels confirming that this residue is not regulated by an α-KG-dependent HDM. ( b ) Exposure to octyl-α-KG (or vehicle control) in cells grown under hypoxia (1% O 2 ) led to an increase in HIF1α hydroxylation (Pro-402) and consequent decrease in total HIF1α levels. Densitometric quantification is shown at the bottom of the western blots. ( c ) Octyl-α-KG significantly increased the abundance of 5hmC marks in the DNA ( P =0.0001, two-tailed Student's t -test, left panel) and decreased that of 5mC marks (global DNA methylation) ( P =0.0001, two-sided Student's t -test, right panel), in a dose-dependent fashion. The data shown in c represent the mean and s.d. of an assay performed in triplicate. Data shown in a – c were confirmed with at least one independent biological replicate. ( d ) HEK-293 cells stably expressing WT D2HGDH were exposed to 1 mM of dimethyloxalylglycine (DMOG) for 6 h, and the methylation levels of H3 lysines verified by western blot. Exposure to DMOG restored K4me3 and K9me2 in D2HGDH-WT cells to the levels found in MSCV control. ( e ) Hypoxia (1% O 2 for 18 h) increases HIF1α hydroxylation (Pro-402) and decreases its stability and activity, defined by GLUT1 expression, in cells expressing WT D2HGDH when compared with isogenic control cells (MSCV); exposure to DMOG fully countered the effects of WT D2HGDH on HIF1α and GLUT1. ( f ) D2HGDH-WT expressing cells display significantly higher and low abundance of 5hmC (left panel) and 5mC (right panel) marks, respectively, than its isogenic controls expressing an empty MSCV vector ( P =0.0008 and P =0.0016 ANOVA). Exposure to DMOG (1 mM for 6 h) reversed the effects of WT D2HGDH back to the MSCV baseline. Experiments shown in d and e were repeated twice, the data in f represent the mean and s.d. of a representative experiment (from two biological replicates) performed in triplicate.

Techniques Used: Expressing, Methylation, Western Blot, Two Tailed Test, DNA Methylation Assay, Stable Transfection, Activity Assay, Plasmid Preparation

Partial knockdown of D2HGDH in B-cell lymphoma cell lines significantly modifies histone/DNA methylation and HIF1α hydroxylation. ( a ) SiRNA-mediated partial knockdown of D2HGDH with two targeting oligonucleotides increased the methylation levels of H3K4me3 in comparison with cells transfected with a control siRNA. ( b ) Under hypoxia (1% O 2 , 16 h), B lymphoma cells with partial suppression of D2HGDH expression displayed lower HIF1α hydroxylation and consequent stabilization of total HIF1α. In a and b , the extent of D2HGDH suppression is shown by western blotting and densitometry quantifies all relevant changes. ( c ) The levels of 5hmC and 5mC (top and bottom panels) were significantly lower and higher, respectively, in cells with a D2HGDH KD when compared with their isogenic controls ( P
Figure Legend Snippet: Partial knockdown of D2HGDH in B-cell lymphoma cell lines significantly modifies histone/DNA methylation and HIF1α hydroxylation. ( a ) SiRNA-mediated partial knockdown of D2HGDH with two targeting oligonucleotides increased the methylation levels of H3K4me3 in comparison with cells transfected with a control siRNA. ( b ) Under hypoxia (1% O 2 , 16 h), B lymphoma cells with partial suppression of D2HGDH expression displayed lower HIF1α hydroxylation and consequent stabilization of total HIF1α. In a and b , the extent of D2HGDH suppression is shown by western blotting and densitometry quantifies all relevant changes. ( c ) The levels of 5hmC and 5mC (top and bottom panels) were significantly lower and higher, respectively, in cells with a D2HGDH KD when compared with their isogenic controls ( P

Techniques Used: DNA Methylation Assay, Methylation, Transfection, Expressing, Western Blot

IDH2 mediates D2HGDH effects on histone and DNA methylation and HIF1α hydroxylation. ( a ) Western blot analysis of IDH1 and IDH2 in subcellular fractions of D2HGDH models (knockdown, left panel; ectopic expression, right panel) shows modulation of IDH2 levels. Densitometric quantification is shown at the bottom. ( b ) Top—western blot analysis of IDH2 KD cells expressing an empty-MSCV vector or WT D2HGDH. Middle—Expression of WT D2HGDH decreased H3K methylation (compare lanes marked with a red star); IDH2 KD restored the H3K methylation levels in these cells. Bottom—expression of WT D2HGDH increased HIF1α hydroxylation and decreased total HIF1α levels (compare lanes marked with a red star); IDH2 KD reversed the increase in HIF1α hydroxylation (and decrease in total HIF1α) associated with expression of D2HGDH WT. ( c ) Cells expressing D2HGDH-WT (and an empty pLKO.1) displayed a significantly higher abundance of 5hmC marks (top) and a concomitant decrease in global DNA methylation (botton) than MSCV-pLKO.1 controls (marked by red star; P
Figure Legend Snippet: IDH2 mediates D2HGDH effects on histone and DNA methylation and HIF1α hydroxylation. ( a ) Western blot analysis of IDH1 and IDH2 in subcellular fractions of D2HGDH models (knockdown, left panel; ectopic expression, right panel) shows modulation of IDH2 levels. Densitometric quantification is shown at the bottom. ( b ) Top—western blot analysis of IDH2 KD cells expressing an empty-MSCV vector or WT D2HGDH. Middle—Expression of WT D2HGDH decreased H3K methylation (compare lanes marked with a red star); IDH2 KD restored the H3K methylation levels in these cells. Bottom—expression of WT D2HGDH increased HIF1α hydroxylation and decreased total HIF1α levels (compare lanes marked with a red star); IDH2 KD reversed the increase in HIF1α hydroxylation (and decrease in total HIF1α) associated with expression of D2HGDH WT. ( c ) Cells expressing D2HGDH-WT (and an empty pLKO.1) displayed a significantly higher abundance of 5hmC marks (top) and a concomitant decrease in global DNA methylation (botton) than MSCV-pLKO.1 controls (marked by red star; P

Techniques Used: DNA Methylation Assay, Western Blot, Expressing, Plasmid Preparation, Methylation

Cellular effects of D2HGDH mutations in DLBCL. ( a ) Methylation of H3 lysine residues and HIF1α hydroxylation/total levels (under hypoxic conditions) were determined by western blot in 14 DLBCL cell lines. H3K4 and K9 methylation were higher, while HIF1α hydroxylation (Hy-HIF1α) was lower (and its total levels consequently higher) in D2HGDH-mutant cell lines when compared with those expressing the WT enzyme. Densitometric quantifications are shown at the bottom of the western blots, and for H3K4me3 and H3K9me2 also in graphic display (mean and s.e.m., Mann–Whitney test). The WB at the bottom displays the expression of D2HGDH across these cell lines. ( b ) The levels of 5hmC (left) and the 5mC (right) were significantly lower and higher, respectively, in DLBCL cell lines expressing a mutant D2HGDH gene than in the WT cells ( P =0.002, two-tailed Mann–Whitney test). The data shown are mean of 3 data points for each cell line (four mutant and ten WT) derived from three independent biological replicates. ( c ) Western blot analysis of 12 primary DLBCLs (four mutant and eight D2HGDH WT) shows higher H3K4 and H3K9 methylation in mutant lymphomas. Densitometric quantification of H3K4me3 and H3K9me2 (normalized by total H3) is shown below the blots and in graphic display (mean and s.e.m., Mann–Whitney test). The WB at the bottom shows the expression of D2HGDH across these biopsies. ( d ) The levels of 5hmC (top) and 5Mc marks (bottom) were significantly lower and higher, respectively, in DLBCLs expressing a mutant D2HGDH gene than in the WT tumours ( P =0.004 or 0.008, two-tailed Mann–Whitney test). The data shown are mean of three independent measurements for each tumour.
Figure Legend Snippet: Cellular effects of D2HGDH mutations in DLBCL. ( a ) Methylation of H3 lysine residues and HIF1α hydroxylation/total levels (under hypoxic conditions) were determined by western blot in 14 DLBCL cell lines. H3K4 and K9 methylation were higher, while HIF1α hydroxylation (Hy-HIF1α) was lower (and its total levels consequently higher) in D2HGDH-mutant cell lines when compared with those expressing the WT enzyme. Densitometric quantifications are shown at the bottom of the western blots, and for H3K4me3 and H3K9me2 also in graphic display (mean and s.e.m., Mann–Whitney test). The WB at the bottom displays the expression of D2HGDH across these cell lines. ( b ) The levels of 5hmC (left) and the 5mC (right) were significantly lower and higher, respectively, in DLBCL cell lines expressing a mutant D2HGDH gene than in the WT cells ( P =0.002, two-tailed Mann–Whitney test). The data shown are mean of 3 data points for each cell line (four mutant and ten WT) derived from three independent biological replicates. ( c ) Western blot analysis of 12 primary DLBCLs (four mutant and eight D2HGDH WT) shows higher H3K4 and H3K9 methylation in mutant lymphomas. Densitometric quantification of H3K4me3 and H3K9me2 (normalized by total H3) is shown below the blots and in graphic display (mean and s.e.m., Mann–Whitney test). The WB at the bottom shows the expression of D2HGDH across these biopsies. ( d ) The levels of 5hmC (top) and 5Mc marks (bottom) were significantly lower and higher, respectively, in DLBCLs expressing a mutant D2HGDH gene than in the WT tumours ( P =0.004 or 0.008, two-tailed Mann–Whitney test). The data shown are mean of three independent measurements for each tumour.

Techniques Used: Methylation, Western Blot, Mutagenesis, Expressing, MANN-WHITNEY, Two Tailed Test, Derivative Assay

23) Product Images from "Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells"

Article Title: Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

Journal: Toxicology and applied pharmacology

doi: 10.1016/j.taap.2012.08.003

CoCl 2 increases HIF1α protein level
Figure Legend Snippet: CoCl 2 increases HIF1α protein level

Techniques Used:

CoCl 2 -mediated FGF21 down-regulation is independent of HIF1α/2 α
Figure Legend Snippet: CoCl 2 -mediated FGF21 down-regulation is independent of HIF1α/2 α

Techniques Used:

24) Product Images from "Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein"

Article Title: Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171616

HIF1α and HIF2α regulate mRNA and protein levels of Spry2. (A) Cells transfected with siRNA against HIF1α, HIF2α or both isoforms were incubated under hypoxic conditions (3% O 2 ) for 24 hours. RNA was isolated and mRNA levels of HIF1α , HIF2α (right panels) and SPRY2 (left panel) were monitored by qRT-PCR with specific primers/probe and normalized with 18S rRNA. Cells transfected with mutant siRNA were used as control. Graphs are mean + SEM of 5 independent experiments. (B) Cells were treated same as in (A) except hypoxic incubation was for 32 hours. The protein levels of HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as loading control. Graph is mean + SEM from six independent experiments. (C) HEK293T cells transfected with vector alone or HIF1β along with vector, HIF1α, HIF2α, or both HIF1α and HIF2α were incubated under normoxic conditions for 40 hours after transfection. The mRNA amounts of SPRY2 (left panel) or PGK1 (right panel) were monitored by qRT- PCR and normalized with 18S rRNA. Graphs are mean + SEM from four independent experiments. Each group was compared with cells transfected with pcDNA3-HIF1β only. Statistical significance was assessed using unpaired Student t-tests (A B) or one-way ANOVA with Dunnett’s multiple comparison test (C) **: p
Figure Legend Snippet: HIF1α and HIF2α regulate mRNA and protein levels of Spry2. (A) Cells transfected with siRNA against HIF1α, HIF2α or both isoforms were incubated under hypoxic conditions (3% O 2 ) for 24 hours. RNA was isolated and mRNA levels of HIF1α , HIF2α (right panels) and SPRY2 (left panel) were monitored by qRT-PCR with specific primers/probe and normalized with 18S rRNA. Cells transfected with mutant siRNA were used as control. Graphs are mean + SEM of 5 independent experiments. (B) Cells were treated same as in (A) except hypoxic incubation was for 32 hours. The protein levels of HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as loading control. Graph is mean + SEM from six independent experiments. (C) HEK293T cells transfected with vector alone or HIF1β along with vector, HIF1α, HIF2α, or both HIF1α and HIF2α were incubated under normoxic conditions for 40 hours after transfection. The mRNA amounts of SPRY2 (left panel) or PGK1 (right panel) were monitored by qRT- PCR and normalized with 18S rRNA. Graphs are mean + SEM from four independent experiments. Each group was compared with cells transfected with pcDNA3-HIF1β only. Statistical significance was assessed using unpaired Student t-tests (A B) or one-way ANOVA with Dunnett’s multiple comparison test (C) **: p

Techniques Used: Transfection, Incubation, Isolation, Quantitative RT-PCR, Mutagenesis, Western Blot, Plasmid Preparation

HIF1α and HIF2α do not regulate the stability of SPRY2 mRNA, but they bind to the proximal promoter and intron of SPRY2 . (A) Hep3B cells transfected with control or HIF1α/HIF2α siRNAs were incubated in hypoxia for 24 hours and then treated with actinomycin D (3 μg/mL). Total RNA was extracted at the indicated times and the mRNA levels of SPRY2 were monitored using qRT-PCR. (B) Schematic of SPRY2 from -3850 to 3395 encompassing the promoter, transcription start site (+1), exon 1 (Ex1), intron, and exon 2 (Ex2). Each grey rectangle labeled with a letter represents a putative HRE and the location of each HRE is labeled underneath. Each numbered line above shows the location of a primer pair designed to amplify a region of DNA with specific putative HREs in a ChIP. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control rabbit IgG or HIF1β antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Graphs are the mean + SEM from five independent experiments. (D) Hep3B cells transfected with control, HIF1α, HIF2α, or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. ChIP assays were performed as stated in (C) except primers were used that encompass the HREs located in the promoter of the HIF1α-responsive gene PFK-1 or the HIF2α-responsive gene EPO . Graph shows the mean + SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test (C D) *: p
Figure Legend Snippet: HIF1α and HIF2α do not regulate the stability of SPRY2 mRNA, but they bind to the proximal promoter and intron of SPRY2 . (A) Hep3B cells transfected with control or HIF1α/HIF2α siRNAs were incubated in hypoxia for 24 hours and then treated with actinomycin D (3 μg/mL). Total RNA was extracted at the indicated times and the mRNA levels of SPRY2 were monitored using qRT-PCR. (B) Schematic of SPRY2 from -3850 to 3395 encompassing the promoter, transcription start site (+1), exon 1 (Ex1), intron, and exon 2 (Ex2). Each grey rectangle labeled with a letter represents a putative HRE and the location of each HRE is labeled underneath. Each numbered line above shows the location of a primer pair designed to amplify a region of DNA with specific putative HREs in a ChIP. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control rabbit IgG or HIF1β antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Graphs are the mean + SEM from five independent experiments. (D) Hep3B cells transfected with control, HIF1α, HIF2α, or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. ChIP assays were performed as stated in (C) except primers were used that encompass the HREs located in the promoter of the HIF1α-responsive gene PFK-1 or the HIF2α-responsive gene EPO . Graph shows the mean + SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test (C D) *: p

Techniques Used: Transfection, Incubation, Quantitative RT-PCR, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation

Silencing DNMT1 attenuates the increase in SPRY2 mRNA and protein levels when HIF1α and HIF2α expression is silenced. (A) Hep3B and (B) HuH7 cells transfected with control, HIF1α and HIF2α, DNMT1, or DNMT1 and HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2 , HIF1α , HIF2α , and DNMT1 were quantified by qRT-PCR and normalized with (A) 18S rRNA or (B) 18S rRNA and RPLP0. Graphs show the mean + SEM from three independent experiments in duplicate or triplicate. (C) Hep3B or (D) HuH7 cells were treated as in (A B). The protein levels of DNMT1, HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as a loading control. Graph shows the mean + SEM from (C) three or (D) four independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparison test (A, B) or Sidak’s multiple comparison test (D) or unpaired student t-tests (B) *: p
Figure Legend Snippet: Silencing DNMT1 attenuates the increase in SPRY2 mRNA and protein levels when HIF1α and HIF2α expression is silenced. (A) Hep3B and (B) HuH7 cells transfected with control, HIF1α and HIF2α, DNMT1, or DNMT1 and HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2 , HIF1α , HIF2α , and DNMT1 were quantified by qRT-PCR and normalized with (A) 18S rRNA or (B) 18S rRNA and RPLP0. Graphs show the mean + SEM from three independent experiments in duplicate or triplicate. (C) Hep3B or (D) HuH7 cells were treated as in (A B). The protein levels of DNMT1, HIF1α, HIF2α and Spry2 were analyzed by Western blotting. Actin was used as a loading control. Graph shows the mean + SEM from (C) three or (D) four independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparison test (A, B) or Sidak’s multiple comparison test (D) or unpaired student t-tests (B) *: p

Techniques Used: Expressing, Transfection, Incubation, Isolation, Quantitative RT-PCR, Western Blot

HIF1α and HIF2α repress SPRY2 mRNA levels by enhancing the methylation of the SPRY2 promoter. (A) Upper panel : Hep3B cells treated with vehicle (V) or decitabine (DAC) were incubated in hypoxia for 24 hours. DNA was extracted, bisulfite-converted, and the methylation status was assessed with methylation specific (M) and unmethylated specific (U) PCR primers. The amount of β-actin DNA was monitored to control for DNA amount loaded into each PCR. The amounts of methylated and unmethylated SPRY2 promoter DNA were quantified by densitometry and normalized to β-actin. Graph shows the mean + SEM for three independent experiments. Lower panel : Schematic of hSPRY2 promoter and gene showing the positions of PCR primers for both methylation specific and unmethylated specific PCRs. The arrow shows the transcription start site. Grey rectangles depict putative HREs. (B) Hep3B cells were treated with vehicle or decitabine (DAC, 5 μM), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and the mRNA amounts of SPRY2 (left panel), HIF1α and HIF2α (right panels) were monitored by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments repeated in duplicate or triplicate. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. The methylation status of the SPRY2 promoter was analyzed as in (A). Graph shows the mean + SEM from five independent experiments. Statistical significance was assessed using unpaired student t-tests (A, B C) *: p
Figure Legend Snippet: HIF1α and HIF2α repress SPRY2 mRNA levels by enhancing the methylation of the SPRY2 promoter. (A) Upper panel : Hep3B cells treated with vehicle (V) or decitabine (DAC) were incubated in hypoxia for 24 hours. DNA was extracted, bisulfite-converted, and the methylation status was assessed with methylation specific (M) and unmethylated specific (U) PCR primers. The amount of β-actin DNA was monitored to control for DNA amount loaded into each PCR. The amounts of methylated and unmethylated SPRY2 promoter DNA were quantified by densitometry and normalized to β-actin. Graph shows the mean + SEM for three independent experiments. Lower panel : Schematic of hSPRY2 promoter and gene showing the positions of PCR primers for both methylation specific and unmethylated specific PCRs. The arrow shows the transcription start site. Grey rectangles depict putative HREs. (B) Hep3B cells were treated with vehicle or decitabine (DAC, 5 μM), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and the mRNA amounts of SPRY2 (left panel), HIF1α and HIF2α (right panels) were monitored by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments repeated in duplicate or triplicate. (C) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 24 hours. The methylation status of the SPRY2 promoter was analyzed as in (A). Graph shows the mean + SEM from five independent experiments. Statistical significance was assessed using unpaired student t-tests (A, B C) *: p

Techniques Used: Methylation, Incubation, Polymerase Chain Reaction, Transfection, Isolation, Quantitative RT-PCR

DNMT1 contributes toward the suppression of SPRY2 mRNA expression by HIF1α and HIF2α. (A) Hep3B cells were treated with vehicle or laccaic acid A (LCA, 50 μg/mL), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2 (left panel), HIF1α , and HIF2α (right panels) were quantified by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments in duplicate. (B) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control mouse IgG or a DNMT1 antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Location of binding of primers is indicated in Fig 2B . Graphs show the mean + SEM from three independent experiments performed in singles or duplicates. Statistical significance was assessed using unpaired student t-tests (A) or one-way ANOVA with Dunnett’s multiple comparison test (B). *: p
Figure Legend Snippet: DNMT1 contributes toward the suppression of SPRY2 mRNA expression by HIF1α and HIF2α. (A) Hep3B cells were treated with vehicle or laccaic acid A (LCA, 50 μg/mL), transfected with control or HIF1α and HIF2α siRNAs and incubated in hypoxia for 24 hours. RNA was isolated and mRNA amounts of SPRY2 (left panel), HIF1α , and HIF2α (right panels) were quantified by qRT-PCR and normalized with 18S rRNA. Graphs show the mean + SEM from three independent experiments in duplicate. (B) Hep3B cells transfected with control or HIF1α and HIF2α siRNAs were incubated in hypoxia for 32 hours. Proteins, cross-linked to DNA, were immunoprecipitated with control mouse IgG or a DNMT1 antibody. The DNA was sheared and the amounts of co-immunoprecipitated DNA were examined by qRT-PCR with the indicated primer sets. Location of binding of primers is indicated in Fig 2B . Graphs show the mean + SEM from three independent experiments performed in singles or duplicates. Statistical significance was assessed using unpaired student t-tests (A) or one-way ANOVA with Dunnett’s multiple comparison test (B). *: p

Techniques Used: Expressing, Transfection, Incubation, Isolation, Quantitative RT-PCR, Immunoprecipitation, Binding Assay

25) Product Images from "Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells"

Article Title: Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12378

Effects of combination treatment in mutant-HIF1α expressing A549 cells ( A ) Confirmation of stable clones was done by western blot analysis using HIF1α specific antibody. Clone B was selected for further experiments. qRT-PCR showed an up-regulation of of HIF1-inducible genes (Glut1 and CAIX) in A549/HIF1 α mut cells. The A549/HIF1α mut cells showed a higher rate of proliferation ( B ) and resistance to cisplatin ( C ) compared to A549 cells with wt-HIF1α. ( D ) Migration assay using Boyden chambers showed a significant inhibition of migration in A549/HIF1mut cells in response to combination treatment than that seen in monotherapy. A= empty vector control; B, C, D, E= different selected clones. C= control, L=2μg/ml cisplatin, S1= 1μg/ml scriptaid. * indicates p
Figure Legend Snippet: Effects of combination treatment in mutant-HIF1α expressing A549 cells ( A ) Confirmation of stable clones was done by western blot analysis using HIF1α specific antibody. Clone B was selected for further experiments. qRT-PCR showed an up-regulation of of HIF1-inducible genes (Glut1 and CAIX) in A549/HIF1 α mut cells. The A549/HIF1α mut cells showed a higher rate of proliferation ( B ) and resistance to cisplatin ( C ) compared to A549 cells with wt-HIF1α. ( D ) Migration assay using Boyden chambers showed a significant inhibition of migration in A549/HIF1mut cells in response to combination treatment than that seen in monotherapy. A= empty vector control; B, C, D, E= different selected clones. C= control, L=2μg/ml cisplatin, S1= 1μg/ml scriptaid. * indicates p

Techniques Used: Mutagenesis, Expressing, Clone Assay, Western Blot, Quantitative RT-PCR, Migration, Inhibition, Plasmid Preparation

Cell death and DNA damage in response to treatment in A549/HIF1α mut cells ( A ) Confocal images of A549/wt-HIF1α and A549/HIF1α mut shows an elevated level of DNA damage in combination than single agent treatment. Increased accumulation of γ-H2AX indicates DNA damage. Nucleus= Blue (DAPI), F-actin= Red (TRITC), γ-H2AX= Green (FITC). ( B ) AnnexinV-PI dual staining and flow cytometry analysis shows significantly higher apoptosis in combination treatment than low dose cisplatin and scriptaid. Q1=necrotic population, Q2=late phase apoptosis, Q3=live cells, Q4= early phase apoptosis. C= control, L=2μg/ml cisplatin, S1= 1μg/ml scriptaid. * indicates p
Figure Legend Snippet: Cell death and DNA damage in response to treatment in A549/HIF1α mut cells ( A ) Confocal images of A549/wt-HIF1α and A549/HIF1α mut shows an elevated level of DNA damage in combination than single agent treatment. Increased accumulation of γ-H2AX indicates DNA damage. Nucleus= Blue (DAPI), F-actin= Red (TRITC), γ-H2AX= Green (FITC). ( B ) AnnexinV-PI dual staining and flow cytometry analysis shows significantly higher apoptosis in combination treatment than low dose cisplatin and scriptaid. Q1=necrotic population, Q2=late phase apoptosis, Q3=live cells, Q4= early phase apoptosis. C= control, L=2μg/ml cisplatin, S1= 1μg/ml scriptaid. * indicates p

Techniques Used: Staining, Flow Cytometry, Cytometry

26) Product Images from "Improving the metabolic fidelity of cancer models with a physiological cell culture medium"

Article Title: Improving the metabolic fidelity of cancer models with a physiological cell culture medium

Journal: Science Advances

doi: 10.1126/sciadv.aau7314

Plasmax induces cell line–specific transcriptomic alterations and prevents the pseudohypoxic gene expression signature of cells cultured in commercial media. ( A ) PCA of gene expression obtained from RNA-seq data of BT549, CAL-120, and MDA-MB-468 cells cultured in Plasmax or DMEM-F12, in normoxia. n = 3. Each symbol represents an independent experiment. ( B ) Venn diagram showing the number of genes that were differentially regulated in these cell lines when cultured in Plasmax and DMEM-F12. ( C ) Heat map of genes that were significantly [false discovery rate (FDR) of 10%] and coherently regulated (absolute log 2 fold change ≥ 0.585) by culturing cells in Plasmax, in normoxia, in at least two of three cell lines. n = 3, log 2 (mean fold change). ( D ) Correlation analysis of genes regulated by hypoxia in Plasmax ( y axis) and genes regulated by Plasmax in normoxia ( x axis). Each dot represents the mean of three independent experiments. ( E ) Expression levels of HIF1α target genes CA9 , TXNIP , PDK1 , and BNIP3 in BT549 cells relative to control condition (normoxia, DMEM-F12). Means ± SEM; n = 3, each dot represents an independent experiment, and P values refer to a two-tailed t test for unpaired homoscedastic samples. ( F ) Western blot showing HIF1α levels in BT549 cells in Plasmax (P) or DMEM-F12 (D), in normoxia (N) and hypoxia (H). ( G ) Western blot showing HIF1α levels in BT549, CAL-120, and MDA-MB-468 cells cultured in DMEM-F12 or Plasmax in normoxia. ( H ) Western blot showing HIF1α levels in BT549 cells cultured in normoxia in DMEM-F12, Plasmax, DMEM, and RPMI 1640. ( I ) Western blot showing pyruvate-dependent HIF1α levels in BT549 cells, cultured in DMEM-F12, Plasmax, and DMEM in normoxia. (F to I) Images are representative of three independent experiments. P values refer to a two-tailed t test for unpaired homoscedastic samples.
Figure Legend Snippet: Plasmax induces cell line–specific transcriptomic alterations and prevents the pseudohypoxic gene expression signature of cells cultured in commercial media. ( A ) PCA of gene expression obtained from RNA-seq data of BT549, CAL-120, and MDA-MB-468 cells cultured in Plasmax or DMEM-F12, in normoxia. n = 3. Each symbol represents an independent experiment. ( B ) Venn diagram showing the number of genes that were differentially regulated in these cell lines when cultured in Plasmax and DMEM-F12. ( C ) Heat map of genes that were significantly [false discovery rate (FDR) of 10%] and coherently regulated (absolute log 2 fold change ≥ 0.585) by culturing cells in Plasmax, in normoxia, in at least two of three cell lines. n = 3, log 2 (mean fold change). ( D ) Correlation analysis of genes regulated by hypoxia in Plasmax ( y axis) and genes regulated by Plasmax in normoxia ( x axis). Each dot represents the mean of three independent experiments. ( E ) Expression levels of HIF1α target genes CA9 , TXNIP , PDK1 , and BNIP3 in BT549 cells relative to control condition (normoxia, DMEM-F12). Means ± SEM; n = 3, each dot represents an independent experiment, and P values refer to a two-tailed t test for unpaired homoscedastic samples. ( F ) Western blot showing HIF1α levels in BT549 cells in Plasmax (P) or DMEM-F12 (D), in normoxia (N) and hypoxia (H). ( G ) Western blot showing HIF1α levels in BT549, CAL-120, and MDA-MB-468 cells cultured in DMEM-F12 or Plasmax in normoxia. ( H ) Western blot showing HIF1α levels in BT549 cells cultured in normoxia in DMEM-F12, Plasmax, DMEM, and RPMI 1640. ( I ) Western blot showing pyruvate-dependent HIF1α levels in BT549 cells, cultured in DMEM-F12, Plasmax, and DMEM in normoxia. (F to I) Images are representative of three independent experiments. P values refer to a two-tailed t test for unpaired homoscedastic samples.

Techniques Used: Expressing, Cell Culture, RNA Sequencing Assay, Multiple Displacement Amplification, Two Tailed Test, Western Blot

27) Product Images from "Impaired Fetoplacental Angiogenesis in Growth-Restricted Fetuses With Abnormal Umbilical Artery Doppler Velocimetry Is Mediated by Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)"

Article Title: Impaired Fetoplacental Angiogenesis in Growth-Restricted Fetuses With Abnormal Umbilical Artery Doppler Velocimetry Is Mediated by Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)

Journal: The Journal of Clinical Endocrinology and Metabolism

doi: 10.1210/jc.2014-2385

Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding
Figure Legend Snippet: Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding

Techniques Used: Binding Assay

28) Product Images from "Hypoxia Increases the Expression of Stem-Cell Markers and Promotes Clonogenicity in Glioblastoma Neurospheres"

Article Title: Hypoxia Increases the Expression of Stem-Cell Markers and Promotes Clonogenicity in Glioblastoma Neurospheres

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.091021

A: Constitutive expression of oxygen-stable HIF1α P402A/P564A ( left panel ) increased levels of CD133 protein expression ( right panel ) in HSR-GBM1 C5 and E11 as compared with the vector control infected parent line (P). B: Flow cytometric analysis
Figure Legend Snippet: A: Constitutive expression of oxygen-stable HIF1α P402A/P564A ( left panel ) increased levels of CD133 protein expression ( right panel ) in HSR-GBM1 C5 and E11 as compared with the vector control infected parent line (P). B: Flow cytometric analysis

Techniques Used: Expressing, Plasmid Preparation, Infection, Flow Cytometry

In vitro digoxin treatments. A: MTT [3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide] analysis for HSR-GBM1 vector control and HIF1α P402A/P564A (E11) expressing cells. Growth of HSR-GBM1 was inhibited significantly in
Figure Legend Snippet: In vitro digoxin treatments. A: MTT [3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide] analysis for HSR-GBM1 vector control and HIF1α P402A/P564A (E11) expressing cells. Growth of HSR-GBM1 was inhibited significantly in

Techniques Used: In Vitro, MTT Assay, Plasmid Preparation, Expressing

In vivo digoxin treatments. A: Western blot analysis for biopsies of HSR-GBM1 flank xenografts, taken before (0 hours) and after (2 hours) intraperitoneal digoxin injection ( n = 2), show reduced protein levels for CD133 and HIF1α. B: Mice
Figure Legend Snippet: In vivo digoxin treatments. A: Western blot analysis for biopsies of HSR-GBM1 flank xenografts, taken before (0 hours) and after (2 hours) intraperitoneal digoxin injection ( n = 2), show reduced protein levels for CD133 and HIF1α. B: Mice

Techniques Used: In Vivo, Western Blot, Injection, Mouse Assay

29) Product Images from "CDH5 is specifically activated in glioblastoma stemlike cells and contributes to vasculogenic mimicry induced by hypoxia"

Article Title: CDH5 is specifically activated in glioblastoma stemlike cells and contributes to vasculogenic mimicry induced by hypoxia

Journal: Neuro-Oncology

doi: 10.1093/neuonc/not029

CDH5 expression is regulated by HIFs in GSCs. (A) CDH5 expression was reduced in GSCs, while not significantly altered in U87, after HIF1α or HIF2α knockdown. * P
Figure Legend Snippet: CDH5 expression is regulated by HIFs in GSCs. (A) CDH5 expression was reduced in GSCs, while not significantly altered in U87, after HIF1α or HIF2α knockdown. * P

Techniques Used: Expressing

30) Product Images from "Tasquinimod (ABR-215050), a quinoline-3-carboxamide anti-angiogenic agent, modulates the expression of thrombospondin-1 in human prostate tumors"

Article Title: Tasquinimod (ABR-215050), a quinoline-3-carboxamide anti-angiogenic agent, modulates the expression of thrombospondin-1 in human prostate tumors

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-107

Tasquinimod blocks the angiogenic switch in CWR-22RH tumors . Inhibition of the
Figure Legend Snippet: Tasquinimod blocks the angiogenic switch in CWR-22RH tumors . Inhibition of the "angiogenic switch" was illustrated in treated CWR-22RH tumors. (A) Tumor growth reduction of CWR-22RH human prostate tumors inoculated into nude mice after oral treatment with tasquinimod at 10 mg/kg/day, data points represent the average ± SD, (n = 5, (**) p = 0.002; Mann-Whitney U). (B) Reduced tumor levels of VEGF, a downstream HIF1α target gene. Bars represent the mean ± SD, n = 5 and (*) p

Techniques Used: Inhibition, Mouse Assay, MANN-WHITNEY

31) Product Images from "Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis"

Article Title: Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

Journal: eLife

doi: 10.7554/eLife.02242

Metformin reduces HIF-1 activation through inhibition of mitochondrial complex I. ( A and B ) H 2 O 2 levels emitted by mitochondria isolated from Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− cells respiring on 2 mM malate and 10 mM pyruvate. Mitochondria were treated with 1 mM Metformin, 500 nM rotenone, 500 nM Antimycin, or left untreated. H 2 O 2 levels were measured using Amplex Red. ( C ) Levels of HIF1α protein in Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− cells treated with 0 or 1 mM metformin for 24 hr, then placed in normoxia (21% O 2 ), hypoxia (1.5% O 2 ) or treated with Deferoxamine (DFO) for 8 hr. ( D ) Quantification of HIF1α protein levels from panel C . ( E ) Hypoxic-induced expression of HIF target genes in Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− treated with 0, 0.5 mM or 1 mM metformin for 24 hr following treatment with normoxia or hypoxia for 16 hr. Error bars are SEM (n = 3 for Amplex Red; Blot is representative of four independent blots quantified in D , n = 4 for gene expression). * indicates significance p
Figure Legend Snippet: Metformin reduces HIF-1 activation through inhibition of mitochondrial complex I. ( A and B ) H 2 O 2 levels emitted by mitochondria isolated from Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− cells respiring on 2 mM malate and 10 mM pyruvate. Mitochondria were treated with 1 mM Metformin, 500 nM rotenone, 500 nM Antimycin, or left untreated. H 2 O 2 levels were measured using Amplex Red. ( C ) Levels of HIF1α protein in Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− cells treated with 0 or 1 mM metformin for 24 hr, then placed in normoxia (21% O 2 ), hypoxia (1.5% O 2 ) or treated with Deferoxamine (DFO) for 8 hr. ( D ) Quantification of HIF1α protein levels from panel C . ( E ) Hypoxic-induced expression of HIF target genes in Control-HCT 116 p53 −/− and NDI1-HCT 116 p53 −/− treated with 0, 0.5 mM or 1 mM metformin for 24 hr following treatment with normoxia or hypoxia for 16 hr. Error bars are SEM (n = 3 for Amplex Red; Blot is representative of four independent blots quantified in D , n = 4 for gene expression). * indicates significance p

Techniques Used: Activation Assay, Inhibition, Isolation, Expressing

32) Product Images from "Anti-leukemic effects of the V-ATPase inhibitor Archazolid A"

Article Title: Anti-leukemic effects of the V-ATPase inhibitor Archazolid A

Journal: Oncotarget

doi:

Archazolid A interferes with the iron metabolism in leukemic cells A. Archazolid A increases Hif1α. Immunoblots show Hif1α levels of Jurkat cells with/without Archazolid A (Arch) treatment at indicated concentrations for 24h. Actin indicates equal loading. B. Immunostainings for Hif1α (green) and f-actin (red) after treatment with/without Archazolid A (Arch, 10 nM, 24h) is shown. Nuclei are labeled with Hoechst33342 (blue). Scale bar 7.5 μm. C. Archazolid A mediated Hif1α increase is abrogated by iron citrate. Immunoblots show Hif1α levels of Jurkat cells with/without Archazolid A (Arch) and iron citrate (FeCit) treatment at indicated concentrations for 24h. Actin indicates equal loading. n = 3. D. Inhibition of Notch by DBZ does not influence Hif1α. Immunoblots of Jurkat cells treated with DBZ and deferoxamine (DFO) at indicated concentrations for 24h for Hif1α and actin (loading control) are shown. E. Archazolid A mediated cell death is partially rescued by iron citrate. The graph shows cell death of Jurkat cells treated with/without Archazolid A (Arch) and iron citrate (FeCit) at indicated concentrations for 48 h. Mann Whitney test, ** p = 0.0022, n = 3. F. DFO induces cell death in Jurkat cells and is enhanced by Archazolid A. Nicoletti assay of cells treated with/without Archazolid A (Arch) and DFO at indicated concentrations for 48 h is shown. One-Way ANOVA, Tukey's post test, *** p ≤ 0.001, n = 3. G. Survivin is decreased by DFO which is enhanced by Archazolid A. Immunoblots for survivin and tubulin (loading control) from cells treated with/without DFO (100 μM) and Archazolid A (Arch, 10 nM) for 48h are shown; n = 3.
Figure Legend Snippet: Archazolid A interferes with the iron metabolism in leukemic cells A. Archazolid A increases Hif1α. Immunoblots show Hif1α levels of Jurkat cells with/without Archazolid A (Arch) treatment at indicated concentrations for 24h. Actin indicates equal loading. B. Immunostainings for Hif1α (green) and f-actin (red) after treatment with/without Archazolid A (Arch, 10 nM, 24h) is shown. Nuclei are labeled with Hoechst33342 (blue). Scale bar 7.5 μm. C. Archazolid A mediated Hif1α increase is abrogated by iron citrate. Immunoblots show Hif1α levels of Jurkat cells with/without Archazolid A (Arch) and iron citrate (FeCit) treatment at indicated concentrations for 24h. Actin indicates equal loading. n = 3. D. Inhibition of Notch by DBZ does not influence Hif1α. Immunoblots of Jurkat cells treated with DBZ and deferoxamine (DFO) at indicated concentrations for 24h for Hif1α and actin (loading control) are shown. E. Archazolid A mediated cell death is partially rescued by iron citrate. The graph shows cell death of Jurkat cells treated with/without Archazolid A (Arch) and iron citrate (FeCit) at indicated concentrations for 48 h. Mann Whitney test, ** p = 0.0022, n = 3. F. DFO induces cell death in Jurkat cells and is enhanced by Archazolid A. Nicoletti assay of cells treated with/without Archazolid A (Arch) and DFO at indicated concentrations for 48 h is shown. One-Way ANOVA, Tukey's post test, *** p ≤ 0.001, n = 3. G. Survivin is decreased by DFO which is enhanced by Archazolid A. Immunoblots for survivin and tubulin (loading control) from cells treated with/without DFO (100 μM) and Archazolid A (Arch, 10 nM) for 48h are shown; n = 3.

Techniques Used: Western Blot, Labeling, Inhibition, MANN-WHITNEY, Nicoletti Assay

33) Product Images from "Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme"

Article Title: Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20101470

HK2 regulates GBM growth and invasion in an orthotopic xenograft model. (A) Survival curve of mice injected intracranially with 2 × 10 5 of U87 cells expressing HK2shRNA-RFP ( n = 10), U87 control cells ( n = 10), U87 cells overexpressing HK2-GFP ( n = 10), or a mix of U87 cells expressing HK2-GFP and HK2shRNA-RFP ( n = 6). (B) Hematoxylin and eosin stain of tumors described in A. Bars, 2,000 µm. Arrow is pointing to the small tumor in HK2shRNA-RFP tumors. (C) Hematoxylin and eosin micrographs depicting invasiveness of HK2shRNA tumors. The arrow is pointing at the invading tumor cells within the cerebellum. Bars: (top) 250 µm; (bottom) 100 µm. Bottom graph depicts the extent of invasiveness of tumors depicted in panel B, which was determined by measuring the mean distance (micrometers) of invading tumor clusters away from the main tumor mass. (D) Top immunofluorescence of GFP and RFP (blue, DAPI) in U87HK2-GFP, U87 mixed, and U87HK2shRNA-RFP tumors. Bar, 28 µm. Bottom immunofluorescence of staining of the vasculature (vWF) of tumors. Bar, 39 µm. (E) Histopathology of U87HK2-GFP, U87 control, and U87HK2shRNA intracranial tumors with antibodies specific to vWF (blood vessels labeled with arrow; bar, 100 µm), VEGF (bar, 100 µm), HIF1α (positive cells labeled with arrow; bar, 50 µm), MIB1 (bar, 100 µm), cleaved caspase 3 (bar, 100 µm). The percentage of proliferating cells (MIB1) and apoptotic cells (cleaved caspase 3) are indicated above the photomicrographs.
Figure Legend Snippet: HK2 regulates GBM growth and invasion in an orthotopic xenograft model. (A) Survival curve of mice injected intracranially with 2 × 10 5 of U87 cells expressing HK2shRNA-RFP ( n = 10), U87 control cells ( n = 10), U87 cells overexpressing HK2-GFP ( n = 10), or a mix of U87 cells expressing HK2-GFP and HK2shRNA-RFP ( n = 6). (B) Hematoxylin and eosin stain of tumors described in A. Bars, 2,000 µm. Arrow is pointing to the small tumor in HK2shRNA-RFP tumors. (C) Hematoxylin and eosin micrographs depicting invasiveness of HK2shRNA tumors. The arrow is pointing at the invading tumor cells within the cerebellum. Bars: (top) 250 µm; (bottom) 100 µm. Bottom graph depicts the extent of invasiveness of tumors depicted in panel B, which was determined by measuring the mean distance (micrometers) of invading tumor clusters away from the main tumor mass. (D) Top immunofluorescence of GFP and RFP (blue, DAPI) in U87HK2-GFP, U87 mixed, and U87HK2shRNA-RFP tumors. Bar, 28 µm. Bottom immunofluorescence of staining of the vasculature (vWF) of tumors. Bar, 39 µm. (E) Histopathology of U87HK2-GFP, U87 control, and U87HK2shRNA intracranial tumors with antibodies specific to vWF (blood vessels labeled with arrow; bar, 100 µm), VEGF (bar, 100 µm), HIF1α (positive cells labeled with arrow; bar, 50 µm), MIB1 (bar, 100 µm), cleaved caspase 3 (bar, 100 µm). The percentage of proliferating cells (MIB1) and apoptotic cells (cleaved caspase 3) are indicated above the photomicrographs.

Techniques Used: Mouse Assay, Injection, Expressing, H&E Stain, Immunofluorescence, Staining, Histopathology, Labeling

34) Product Images from "Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability"

Article Title: Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability

Journal: Oncotarget

doi: 10.18632/oncotarget.21145

Chronic hypoxia induces oxidative damage and promotes tumor cell death (a) Human colonospheres were cultured in hypoxia (0.1%) and normoxia (21%) for 72 hours. Cells were lysed and analyzed by Western blotting for PCNA (proliferation) and cleaved caspase-3 (apoptosis) (cropped gels). (b) Experiment performed as in (a) but human colonospheres overexpressing GPx2 were included. FACS analysis of PI-stained cells was then used to assess cell death. The bar graph shows the percentage of cells with sub-G1 DNA content. (c) The culture conditions were similar to a and b. Cells were pulse labeled with BrdU just prior to FACS analysis to assess the percentage of proliferating cells. (d) FACS analysis of γH2AX levels in control and GPx2-overexpressing colonospheres. Cells were exposed to hypoxia for 24 hours. (e) Colonospheres were cultured in normoxia or hypoxia for 24 hours and were subsequently embedded in agar and fixed in formalin for IHC analysis of HIF1α, γH2AX and 4-HNE levels. (f) Colonospheres were cultured in normoxia or hypoxia for 24 hours. After single cell making living cells were FACS sorted and processed for immunofluorescence analysis of 8-oxo-dG (left panel) and γH2AX (right panel). DAPI was used to visualize cell nuclei.
Figure Legend Snippet: Chronic hypoxia induces oxidative damage and promotes tumor cell death (a) Human colonospheres were cultured in hypoxia (0.1%) and normoxia (21%) for 72 hours. Cells were lysed and analyzed by Western blotting for PCNA (proliferation) and cleaved caspase-3 (apoptosis) (cropped gels). (b) Experiment performed as in (a) but human colonospheres overexpressing GPx2 were included. FACS analysis of PI-stained cells was then used to assess cell death. The bar graph shows the percentage of cells with sub-G1 DNA content. (c) The culture conditions were similar to a and b. Cells were pulse labeled with BrdU just prior to FACS analysis to assess the percentage of proliferating cells. (d) FACS analysis of γH2AX levels in control and GPx2-overexpressing colonospheres. Cells were exposed to hypoxia for 24 hours. (e) Colonospheres were cultured in normoxia or hypoxia for 24 hours and were subsequently embedded in agar and fixed in formalin for IHC analysis of HIF1α, γH2AX and 4-HNE levels. (f) Colonospheres were cultured in normoxia or hypoxia for 24 hours. After single cell making living cells were FACS sorted and processed for immunofluorescence analysis of 8-oxo-dG (left panel) and γH2AX (right panel). DAPI was used to visualize cell nuclei.

Techniques Used: Cell Culture, Western Blot, FACS, Staining, Labeling, Immunohistochemistry, Immunofluorescence

Hypoxic areas in primary tumors and liver metastases are characterized by DNA damage and low expression of the DNA repair proteins RAD51 and RIF1 (a) Immunohistochemistry (IHC) for CAIX, HIF1α and γH2AX in human primary colorectal tumours. Robust expression of all three markers was observed surrounding necrotic lesions. (b) Quantification of CAIX, HIF1α and γH2AX expression in peri-necrotic areas and reference zones in primary human colon tumors by IHC (n=20). (c) Analysis of CAIX and γH2AX expression in human liver metastases by IHC. A 20x magnification of the area depicted in the square in lower panel. (d) Quantification of CAIX and γH2AX staining (IHC) in peri-necrotic and reference tissue in liver metastases (n=30). (e) Scatterplot showing the correlation between the expression of (randomly chosen microscopic field areas of) CAIX and γH2AX, assessed with the Spearman test (rho) (n=60). (f) Patient-derived colonospheres were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, the tumor-bearing liver lobes were subjected to a vascular clamping or sham protocol [ 24 ] to induce hypoxia. After 24 hours, the livers were excised and expression of CAIX and γH2AX was examined by IHC. (g) IHC on peri-necrotic areas of clamped livers (experiment as in (f)) for CAIX, γH2AX, RIF1, RAD51, and KU70. * p
Figure Legend Snippet: Hypoxic areas in primary tumors and liver metastases are characterized by DNA damage and low expression of the DNA repair proteins RAD51 and RIF1 (a) Immunohistochemistry (IHC) for CAIX, HIF1α and γH2AX in human primary colorectal tumours. Robust expression of all three markers was observed surrounding necrotic lesions. (b) Quantification of CAIX, HIF1α and γH2AX expression in peri-necrotic areas and reference zones in primary human colon tumors by IHC (n=20). (c) Analysis of CAIX and γH2AX expression in human liver metastases by IHC. A 20x magnification of the area depicted in the square in lower panel. (d) Quantification of CAIX and γH2AX staining (IHC) in peri-necrotic and reference tissue in liver metastases (n=30). (e) Scatterplot showing the correlation between the expression of (randomly chosen microscopic field areas of) CAIX and γH2AX, assessed with the Spearman test (rho) (n=60). (f) Patient-derived colonospheres were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, the tumor-bearing liver lobes were subjected to a vascular clamping or sham protocol [ 24 ] to induce hypoxia. After 24 hours, the livers were excised and expression of CAIX and γH2AX was examined by IHC. (g) IHC on peri-necrotic areas of clamped livers (experiment as in (f)) for CAIX, γH2AX, RIF1, RAD51, and KU70. * p

Techniques Used: Expressing, Immunohistochemistry, Staining, Derivative Assay, Injection, Mouse Assay

35) Product Images from "E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1"

Article Title: E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.231

The N-terminal 80 amino acids of HIF1α are required for E2F7/8 binding. ( A ) Lysates of U2OS cells transfected with flag-tagged HIF1α or E2F8 in combination with an empty vector (−) or myc-tagged E2F7 were maintained in hypoxic
Figure Legend Snippet: The N-terminal 80 amino acids of HIF1α are required for E2F7/8 binding. ( A ) Lysates of U2OS cells transfected with flag-tagged HIF1α or E2F8 in combination with an empty vector (−) or myc-tagged E2F7 were maintained in hypoxic

Techniques Used: Binding Assay, Transfection, Plasmid Preparation

E2F7/8 stimulation of hVEGFA promoter region −1312/−140 requires direct promoter binding and the presence of HIF1. ( A ) U2OS cells were transfected with a scr, E2F7 or HIF1α siRNA in combination with a reporter plasmid containing
Figure Legend Snippet: E2F7/8 stimulation of hVEGFA promoter region −1312/−140 requires direct promoter binding and the presence of HIF1. ( A ) U2OS cells were transfected with a scr, E2F7 or HIF1α siRNA in combination with a reporter plasmid containing

Techniques Used: Binding Assay, Transfection, Plasmid Preparation

36) Product Images from "The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels"

Article Title: The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels

Journal: eLife

doi: 10.7554/eLife.22693

Disrupting V-ATPase activity decreases intracellular iron levels. ( A, B ) V-ATPase inhibition leads to intracellular iron depletion. ( A ) HeLa cells were treated with BafA (10 nM or 100 nM) or 100 µM DFO for 24 hr. HIF1α, IRP2, NCOA4 and ferritin (ferritin heavy chain 1, FTH1) levels were measured by immunoblot. ( B ) HIF1α-GFP ODD reporter cells transduced with Cas9 and sgRNA targeting V-ATPase components (TMEM199, CCDC115, ATP6V0D1 and ATP6V1A1) were sorted into GFP LOW (Lo) and GFPHIGH (Hi) populations as described. The lysates were immunoblotted for HIF1α, IRP2, or NCOA4. β-actin served as a loading control. ( C ) Iron chelation prevents HIF1α hydroxylation. In vitro prolyl hydroxylation of the HIF1α ODD protein following incubation with lysates from WT or DFO treated lysates (100 µM for 24 hr) as previously described. DMOG served as a control for PHD inhibition. ( D–F ) In vitro hydroxylation of PHD activity in DFO or BafA treated lysates supplemented with ferrous iron. Lysates from control, DFO ( D ) or BafA ( E ) treated HeLa cells were extracted as previously described, incubated with the HIF1α ODD protein, and supplemented with increasing concentrations of iron chloride (FeCl 2 , Fe(II)). Prolyl-hydroxylated HIF1α ODD levels were visualised by immunoblot and quantified by densitometry for the BafA treated lysate ( F ) (n = 3). Values are mean±SEM. *p
Figure Legend Snippet: Disrupting V-ATPase activity decreases intracellular iron levels. ( A, B ) V-ATPase inhibition leads to intracellular iron depletion. ( A ) HeLa cells were treated with BafA (10 nM or 100 nM) or 100 µM DFO for 24 hr. HIF1α, IRP2, NCOA4 and ferritin (ferritin heavy chain 1, FTH1) levels were measured by immunoblot. ( B ) HIF1α-GFP ODD reporter cells transduced with Cas9 and sgRNA targeting V-ATPase components (TMEM199, CCDC115, ATP6V0D1 and ATP6V1A1) were sorted into GFP LOW (Lo) and GFPHIGH (Hi) populations as described. The lysates were immunoblotted for HIF1α, IRP2, or NCOA4. β-actin served as a loading control. ( C ) Iron chelation prevents HIF1α hydroxylation. In vitro prolyl hydroxylation of the HIF1α ODD protein following incubation with lysates from WT or DFO treated lysates (100 µM for 24 hr) as previously described. DMOG served as a control for PHD inhibition. ( D–F ) In vitro hydroxylation of PHD activity in DFO or BafA treated lysates supplemented with ferrous iron. Lysates from control, DFO ( D ) or BafA ( E ) treated HeLa cells were extracted as previously described, incubated with the HIF1α ODD protein, and supplemented with increasing concentrations of iron chloride (FeCl 2 , Fe(II)). Prolyl-hydroxylated HIF1α ODD levels were visualised by immunoblot and quantified by densitometry for the BafA treated lysate ( F ) (n = 3). Values are mean±SEM. *p

Techniques Used: Activity Assay, Inhibition, Transduction, In Vitro, Incubation

Iron supplementation restores HIF1 activity to basal levels following V-ATPase inhibition in cell lines and primary cells. ( A–D ) Iron reconstitution in BafA or DFO treated HeLa cells. ( A, C ) HIF1α-GFP ODD reporter cells were treated with BafA (10 nM or 100 nM), or 100 µM DFO for 24 hr with 50 µM iron citrate (Fe(III)) (red), 200 µM Fe(III) (blue) or no iron (green), and GFP levels analysed by flow cytometry. ( B, D ) Wildtype HeLa cells were treated with or without BafA (10 nM or 100 nM) or DFO (100 µM) and Fe(III) (50–200 µM) as described, and endogenous HIF1α levels were measured by immunoblot. ( E–G ) HEK293T cells ( E ), human dermal fibroblasts ( F ) and RCC10 VHL null and VHL reconstituted cells ( G ) were treated with BafA (10 nM) with or without the addition of 50 µM iron citrate (Fe(III)). HIF1α levels were visualised by immunoblot. β-actin served as a loading control. ( H ) HeLa cells were treated with 20 µM MG132 for 2 hr or 10 nM BafA for 24 hr with or without the addition of 50 µM iron citrate. ( I ) EGFR degradation assay for BafA treated cells following iron treatment. HeLa cells were cultured with 10 nM BafA for 24 hr, with or without 50 µM iron citrate (Fe(III)), and stimulated with EGF as previously described. EGFR, NCOA4, and ferritin (FTH1) levels were visualised by immunoblot. β-actin was used as a loading control. ( J ) RT-qPCR analysis of HIF1α and its target genes in response to BafA and iron citrate treatment (n ≥ 2). ( K–M ) Populations of mixed CRISPR KO cells for ATP6V1A1 ( K ), TMEM199 ( L ) and CCDC115 ( M ) were treated with 50 µM iron citrate for 24 hr and HIF1α levels measured by immunoblot. Values are mean±SEM. *p
Figure Legend Snippet: Iron supplementation restores HIF1 activity to basal levels following V-ATPase inhibition in cell lines and primary cells. ( A–D ) Iron reconstitution in BafA or DFO treated HeLa cells. ( A, C ) HIF1α-GFP ODD reporter cells were treated with BafA (10 nM or 100 nM), or 100 µM DFO for 24 hr with 50 µM iron citrate (Fe(III)) (red), 200 µM Fe(III) (blue) or no iron (green), and GFP levels analysed by flow cytometry. ( B, D ) Wildtype HeLa cells were treated with or without BafA (10 nM or 100 nM) or DFO (100 µM) and Fe(III) (50–200 µM) as described, and endogenous HIF1α levels were measured by immunoblot. ( E–G ) HEK293T cells ( E ), human dermal fibroblasts ( F ) and RCC10 VHL null and VHL reconstituted cells ( G ) were treated with BafA (10 nM) with or without the addition of 50 µM iron citrate (Fe(III)). HIF1α levels were visualised by immunoblot. β-actin served as a loading control. ( H ) HeLa cells were treated with 20 µM MG132 for 2 hr or 10 nM BafA for 24 hr with or without the addition of 50 µM iron citrate. ( I ) EGFR degradation assay for BafA treated cells following iron treatment. HeLa cells were cultured with 10 nM BafA for 24 hr, with or without 50 µM iron citrate (Fe(III)), and stimulated with EGF as previously described. EGFR, NCOA4, and ferritin (FTH1) levels were visualised by immunoblot. β-actin was used as a loading control. ( J ) RT-qPCR analysis of HIF1α and its target genes in response to BafA and iron citrate treatment (n ≥ 2). ( K–M ) Populations of mixed CRISPR KO cells for ATP6V1A1 ( K ), TMEM199 ( L ) and CCDC115 ( M ) were treated with 50 µM iron citrate for 24 hr and HIF1α levels measured by immunoblot. Values are mean±SEM. *p

Techniques Used: Activity Assay, Inhibition, Flow Cytometry, Cytometry, Degradation Assay, Cell Culture, Quantitative RT-PCR, CRISPR

TMEM199 and CCDC115 are the human orthologues of the yeast Vma12p-Vma22p V-ATPase assembly complex. ( A ) Enriched gene trap insertion sites in TMEM199 identified in the forward genetic screen. (Red = sense insertions, Blue = antisense insertions). ( B ) Schematic for TMEM199 (left) and Vma12p (right) membrane topology. TMEM199 and Vma12p demonstrate 23.89% sequence identity (Clustal Omega tool (EMBL-EBI)). ( C, D ) HIF1α-GFP ODD reporter cells transduced with Cas9/TMEM199 sgRNA were sorted into GFP LOW (Lo) and GFP HIGH (Hi) populations by FACS ( C ), lysed, and immunoblotted for endogenous HIF1α and TMEM199 ( D ). PHD2 and β2m were used as positive and negative controls respectively, and β-actin served as a loading control. ( E, F ) TMEM199 reconstitution decreases HIF1α levels in TMEM199 deficient cells. TMEM199 KO clones were isolated following lentiviral transduction with sgRNA to TMEM199/Cas9 and serial dilution. Null clones were identified by immunoblot. A CRISPR resistant TMEM199 was overexpressed by lentiviral transduction in mixed populations of TMEM199 deficient cells ( E ) or clonal cells ( F ). HIF1α and TMEM199 levels were measured by immunoblot, and short and long exposures of TMEM199 levels are shown ( E ). ( G ) Co-immunoprecipitation coupled mass spectrometry. Wildtype HeLa cells and TMEM199 null cells were lysed in 1% NP-40 and immunoprecipitated for TMEM199 for 3 hr. Samples were validated by immunoblotting and submitted for mass spectrometry analysis. Proteins immunoprecipitated in wildtype HeLa compared to TMEM199 KO cells with a unique peptide count > 2 are shown. ( H ) PyMOL structural alignment of CCDC115 (pink) and Vma22p (green) based on Phyre 2 predictions. ( I ) Immunoprecipitation of FLAG-CCDC115 with endogenous TMEM199 in wildtype (+) or TMEM199 deficient (-) HeLa cells. An unrelated FLAG tagged protein (FLAG-Ct) was used as a control. The lysate inputs and immunoprecipitated samples are shown. *non-specific band. ( J, K ) HIF1α-GFP ODD reporter cells were depleted of CCDC115 by transduction with Cas9 and sgRNA. After 12 days, cells were sorted into GFP LOW (Lo, grey box, left) and GFP HIGH (Hi, grey box, right) populations by FACS ( J ), and immunoblotted for endogenous HIF1α ( K ). β-actin served as a loading control. DOI: http://dx.doi.org/10.7554/eLife.22693.004
Figure Legend Snippet: TMEM199 and CCDC115 are the human orthologues of the yeast Vma12p-Vma22p V-ATPase assembly complex. ( A ) Enriched gene trap insertion sites in TMEM199 identified in the forward genetic screen. (Red = sense insertions, Blue = antisense insertions). ( B ) Schematic for TMEM199 (left) and Vma12p (right) membrane topology. TMEM199 and Vma12p demonstrate 23.89% sequence identity (Clustal Omega tool (EMBL-EBI)). ( C, D ) HIF1α-GFP ODD reporter cells transduced with Cas9/TMEM199 sgRNA were sorted into GFP LOW (Lo) and GFP HIGH (Hi) populations by FACS ( C ), lysed, and immunoblotted for endogenous HIF1α and TMEM199 ( D ). PHD2 and β2m were used as positive and negative controls respectively, and β-actin served as a loading control. ( E, F ) TMEM199 reconstitution decreases HIF1α levels in TMEM199 deficient cells. TMEM199 KO clones were isolated following lentiviral transduction with sgRNA to TMEM199/Cas9 and serial dilution. Null clones were identified by immunoblot. A CRISPR resistant TMEM199 was overexpressed by lentiviral transduction in mixed populations of TMEM199 deficient cells ( E ) or clonal cells ( F ). HIF1α and TMEM199 levels were measured by immunoblot, and short and long exposures of TMEM199 levels are shown ( E ). ( G ) Co-immunoprecipitation coupled mass spectrometry. Wildtype HeLa cells and TMEM199 null cells were lysed in 1% NP-40 and immunoprecipitated for TMEM199 for 3 hr. Samples were validated by immunoblotting and submitted for mass spectrometry analysis. Proteins immunoprecipitated in wildtype HeLa compared to TMEM199 KO cells with a unique peptide count > 2 are shown. ( H ) PyMOL structural alignment of CCDC115 (pink) and Vma22p (green) based on Phyre 2 predictions. ( I ) Immunoprecipitation of FLAG-CCDC115 with endogenous TMEM199 in wildtype (+) or TMEM199 deficient (-) HeLa cells. An unrelated FLAG tagged protein (FLAG-Ct) was used as a control. The lysate inputs and immunoprecipitated samples are shown. *non-specific band. ( J, K ) HIF1α-GFP ODD reporter cells were depleted of CCDC115 by transduction with Cas9 and sgRNA. After 12 days, cells were sorted into GFP LOW (Lo, grey box, left) and GFP HIGH (Hi, grey box, right) populations by FACS ( J ), and immunoblotted for endogenous HIF1α ( K ). β-actin served as a loading control. DOI: http://dx.doi.org/10.7554/eLife.22693.004

Techniques Used: Sequencing, Transduction, FACS, Clone Assay, Isolation, Serial Dilution, CRISPR, Immunoprecipitation, Mass Spectrometry

V-ATPase depletion or inhibition stabilises HIF1α in a non-prolyl hydroxylated form. ( A ) HIF1α stabilisation in ATG16 null HeLa cells. HeLa cells and ATG16 null cells were treated with increasing concentrations of BafA (10 nM and 100 nM) before immunoblotting for HIF1α. ( B ) HIF1α levels following depletion of HSC70 and LAMP2A in aerobic conditions. HSC70 and LAMP2A depleted cells were generated using CRISPR-Cas9 gene editing with three individual sgRNAs (g1, g2, g3). HIF1α, LAMP2A and HSC70 levels were visualised by immunoblot. Untreated (Ct) and BafA treated HeLa cells were used as controls. ( C ) HIF1α levels following siRNA-mediated depletion of HSC70. HeLa cells were transfected with siRNA to HSC70 or an siRNA control (Ct), and HIF1α or HSC70 levels measured by immunoblot after 96 hr. Cells were treated with or without 10 nM BafA for 24 hr prior to lysis. ( D ) LAMP2A deficient HeLa cells were treated with or without 10 nM BafA for 24 hr. Three different sgRNAs were used (g1, g2, g3). ( E, F ) Immunoblot of total HIF1α and the prolyl hydroxylated form in response to MG132, DMOG, BafA and Chloroquine ( E ). Quantification of immunoblots represented using ImageJ analysis ( F ) (n = 3). ( G, H ) In vitro prolyl hydroxylation of the HIF1α ODD protein following incubation with lysates from WT, BafA and DMOG treated HeLa cells. The levels of hydroxylated HIF1α were measured using a prolyl hydroxy-HIF1α specific antibody ( G ). Quantification of the in vitro hydroxylation assay using ImageJ analysis ( H ) (n = 3). Values are mean±SEM. *p
Figure Legend Snippet: V-ATPase depletion or inhibition stabilises HIF1α in a non-prolyl hydroxylated form. ( A ) HIF1α stabilisation in ATG16 null HeLa cells. HeLa cells and ATG16 null cells were treated with increasing concentrations of BafA (10 nM and 100 nM) before immunoblotting for HIF1α. ( B ) HIF1α levels following depletion of HSC70 and LAMP2A in aerobic conditions. HSC70 and LAMP2A depleted cells were generated using CRISPR-Cas9 gene editing with three individual sgRNAs (g1, g2, g3). HIF1α, LAMP2A and HSC70 levels were visualised by immunoblot. Untreated (Ct) and BafA treated HeLa cells were used as controls. ( C ) HIF1α levels following siRNA-mediated depletion of HSC70. HeLa cells were transfected with siRNA to HSC70 or an siRNA control (Ct), and HIF1α or HSC70 levels measured by immunoblot after 96 hr. Cells were treated with or without 10 nM BafA for 24 hr prior to lysis. ( D ) LAMP2A deficient HeLa cells were treated with or without 10 nM BafA for 24 hr. Three different sgRNAs were used (g1, g2, g3). ( E, F ) Immunoblot of total HIF1α and the prolyl hydroxylated form in response to MG132, DMOG, BafA and Chloroquine ( E ). Quantification of immunoblots represented using ImageJ analysis ( F ) (n = 3). ( G, H ) In vitro prolyl hydroxylation of the HIF1α ODD protein following incubation with lysates from WT, BafA and DMOG treated HeLa cells. The levels of hydroxylated HIF1α were measured using a prolyl hydroxy-HIF1α specific antibody ( G ). Quantification of the in vitro hydroxylation assay using ImageJ analysis ( H ) (n = 3). Values are mean±SEM. *p

Techniques Used: Inhibition, Generated, CRISPR, Transfection, Lysis, Western Blot, In Vitro, Incubation

TMEM199 and CCDC115 are required for acidification of endosomal compartments. ( A ) pH clamping of HeLa cells expressing Tfnr-phl. HeLa cells were transfected with Tfnr-phl. After 48 hr the cells were clamped at the indicated pH and fluorescence measured by live cell confocal microscopy. Differential interference contrast (DIC) microscopy confirmed the presence of intact cells at pH 5 and 6. ( B ) Tfnr-phl localisation in fixed cells. HIF1α-mCherry ODD reporter cells were transfected with Tfnr-phl as described. After 24 hr the cells were plated on cover slips and treated with or without 10 nM BafA for a further 24 hr. Cells were fixed (4% paraformaldehyde (PFA)) prior to confocal microscopy. ( C ) Representative wide field images of Tfnr-phl fluorescence in HIF1α-mCherry ODD reporter cells depleted for TMEM199, CCDC115 or core V-ATPase subunits. Scale bars represent 20 µm ( A ), 5 µm ( B ), and 10 µm ( C ). DOI: http://dx.doi.org/10.7554/eLife.22693.008
Figure Legend Snippet: TMEM199 and CCDC115 are required for acidification of endosomal compartments. ( A ) pH clamping of HeLa cells expressing Tfnr-phl. HeLa cells were transfected with Tfnr-phl. After 48 hr the cells were clamped at the indicated pH and fluorescence measured by live cell confocal microscopy. Differential interference contrast (DIC) microscopy confirmed the presence of intact cells at pH 5 and 6. ( B ) Tfnr-phl localisation in fixed cells. HIF1α-mCherry ODD reporter cells were transfected with Tfnr-phl as described. After 24 hr the cells were plated on cover slips and treated with or without 10 nM BafA for a further 24 hr. Cells were fixed (4% paraformaldehyde (PFA)) prior to confocal microscopy. ( C ) Representative wide field images of Tfnr-phl fluorescence in HIF1α-mCherry ODD reporter cells depleted for TMEM199, CCDC115 or core V-ATPase subunits. Scale bars represent 20 µm ( A ), 5 µm ( B ), and 10 µm ( C ). DOI: http://dx.doi.org/10.7554/eLife.22693.008

Techniques Used: Expressing, Transfection, Fluorescence, Confocal Microscopy, Microscopy

TMEM199 and CCDC115 and are required for lysosomal degradation of EGFR and MHC Class I. ( A ) EGFR degradation assay for wildtype and BafA treated cells. HeLa cells were cultured in the presence or absence of 10 nM BafA for 24 hr. Cells were stimulated with EGF and lysed at the indicated times. Lysates were subjected to SDS-PAGE and immunoblotted for EGFR. β-actin was used as a loading control. ( B, C ) EGFR degradation assay for TMEM119 and CCDC115 deficient cells. HIF1α-GFP ODD cells were transduced with Cas9 and sgRNA to TMEM199 ( B ) or CCDC115 ( C ). After 14 days, cells were sorted into TMEM199 or CCDC115 sufficient (+/+, GFP LOW ), and TMEM199 or CCDC115 null (−/−, GFP HIGH ) populations as described. Cells were then cultured for 24 hr before stimulation with EGF (100 ng/ml), harvested at indicated times and immunoblotted for EGFR. ( D ) MHC Class I degradation in HeLa cells expressing K3. HeLa-K3 cells were transduced with Cas9 and sgRNA to TMEM199, CCDC115, ATP6V1A1 or ATP6V0D1. After 14 days, cell surface MHC Class I levels were measured by flow cytometry (mAb W6/32). Wildtype HeLa cells were used as a control for total MHC Class I. Percentages of cells with MHC Class I at the cell surface are shown. DOI: http://dx.doi.org/10.7554/eLife.22693.006
Figure Legend Snippet: TMEM199 and CCDC115 and are required for lysosomal degradation of EGFR and MHC Class I. ( A ) EGFR degradation assay for wildtype and BafA treated cells. HeLa cells were cultured in the presence or absence of 10 nM BafA for 24 hr. Cells were stimulated with EGF and lysed at the indicated times. Lysates were subjected to SDS-PAGE and immunoblotted for EGFR. β-actin was used as a loading control. ( B, C ) EGFR degradation assay for TMEM119 and CCDC115 deficient cells. HIF1α-GFP ODD cells were transduced with Cas9 and sgRNA to TMEM199 ( B ) or CCDC115 ( C ). After 14 days, cells were sorted into TMEM199 or CCDC115 sufficient (+/+, GFP LOW ), and TMEM199 or CCDC115 null (−/−, GFP HIGH ) populations as described. Cells were then cultured for 24 hr before stimulation with EGF (100 ng/ml), harvested at indicated times and immunoblotted for EGFR. ( D ) MHC Class I degradation in HeLa cells expressing K3. HeLa-K3 cells were transduced with Cas9 and sgRNA to TMEM199, CCDC115, ATP6V1A1 or ATP6V0D1. After 14 days, cell surface MHC Class I levels were measured by flow cytometry (mAb W6/32). Wildtype HeLa cells were used as a control for total MHC Class I. Percentages of cells with MHC Class I at the cell surface are shown. DOI: http://dx.doi.org/10.7554/eLife.22693.006

Techniques Used: Degradation Assay, Cell Culture, SDS Page, Transduction, Expressing, Flow Cytometry, Cytometry

Depletion or inhibition of the V-ATPase stabilises HIF1α in aerobic conditions. ( A ) Bubble plot depicting genes enriched in the forward genetic screen. Bubbles represent the genes enriched in the GFP HIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFP ODD reporter. Proteins involved in V-ATPase assembly and function (green), canonical HIF1α regulation (purple), and the oxoglutarate dehydrogenase complex (blue) are highlighted, with the number of independent gene trap insertions indicated (brackets). ( B ) Pathway analysis of enriched genes in the KBM7 forward genetic screen. The top 114 genes enriched for multiple independent gene-trapping integrations in the GFP HIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFP ODD reporter were analysed by gene ontology clustering for pathways significantly targeted in the screen. An individual gene enrichment p value
Figure Legend Snippet: Depletion or inhibition of the V-ATPase stabilises HIF1α in aerobic conditions. ( A ) Bubble plot depicting genes enriched in the forward genetic screen. Bubbles represent the genes enriched in the GFP HIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFP ODD reporter. Proteins involved in V-ATPase assembly and function (green), canonical HIF1α regulation (purple), and the oxoglutarate dehydrogenase complex (blue) are highlighted, with the number of independent gene trap insertions indicated (brackets). ( B ) Pathway analysis of enriched genes in the KBM7 forward genetic screen. The top 114 genes enriched for multiple independent gene-trapping integrations in the GFP HIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFP ODD reporter were analysed by gene ontology clustering for pathways significantly targeted in the screen. An individual gene enrichment p value

Techniques Used: Inhibition, Expressing

Disrupting transferrin uptake leads to iron-dependent HIF1 activation. ( A ) Flow cytometry of mixed populations of NCOA4 sgRNA-targeted HIF1α-GFP ODD reporter HeLa cells 8 days to 12 days post transduction. Three different sgRNA were used. ( B ) HIF1α-GFP ODD reporter and transferrin receptor levels using sgRNA2 to the transferrin receptor. ( C ) HIF1α-GFP ODD reporter and transferrin receptor levels using two different sgRNA to IRP2. ( D ) HIF1α levels following depletion of IRP2 at different time points post transduction. Mixed populations of IRP2 sgRNA-targeted HIF1α-GFP ODD reporter HeLa cells 10 or 12 days post transduction using three different sgRNA. ( E ) HIF1α-GFP ODD reporter levels in mixed populations of transferrin deficient cells (using sgRNA2 to the transferrin receptor) with or without iron citrate treatment. TFR=transferrin receptor. DOI: http://dx.doi.org/10.7554/eLife.22693.014
Figure Legend Snippet: Disrupting transferrin uptake leads to iron-dependent HIF1 activation. ( A ) Flow cytometry of mixed populations of NCOA4 sgRNA-targeted HIF1α-GFP ODD reporter HeLa cells 8 days to 12 days post transduction. Three different sgRNA were used. ( B ) HIF1α-GFP ODD reporter and transferrin receptor levels using sgRNA2 to the transferrin receptor. ( C ) HIF1α-GFP ODD reporter and transferrin receptor levels using two different sgRNA to IRP2. ( D ) HIF1α levels following depletion of IRP2 at different time points post transduction. Mixed populations of IRP2 sgRNA-targeted HIF1α-GFP ODD reporter HeLa cells 10 or 12 days post transduction using three different sgRNA. ( E ) HIF1α-GFP ODD reporter levels in mixed populations of transferrin deficient cells (using sgRNA2 to the transferrin receptor) with or without iron citrate treatment. TFR=transferrin receptor. DOI: http://dx.doi.org/10.7554/eLife.22693.014

Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Transduction

Disrupting the V-ATPase activates HIF1 and HIF2. ( A ) Immunoblot of HIF1α levels in response to the proteasome inhibitor MG132, the V-ATPase inhibitor BafA, the lysosomotropic agent Chloroquine, and the oxidative metabolism inhibitor NH 4 Cl. ( B ) Immunoblot of HIF1α levels in HeLa cells in response to BafA treatment at 0.5, 1, 2, 4, 8 and 24 hr. ( C ) Confocal immunofluorescence microscopy of WT (top) and BafA (bottom) treated HeLa cells stained for endogenous HIF1α. Cells were treated in the presence or absence of 10 nM BafA for 24 hr before immunofluorescence staining with HIF1α (white). Cells were mounted using DAPI (blue) and visualised by confocal microscopy. Scale bar represents 10 μm. ( D ) Immunocytochemical staining to examine HIF1α stabilisation in TMEM199 depleted HIF1α-GFP ODD reporter cells. HIF1α-GFP ODD reporter cells were depleted of TMEM199 using CRISPR-Cas9 genetics and stained for HIF1α (white), TMEM199 (red) and DAPI (blue). Scale bar represents 20 μm. ( E–G ) Levels of HIF1α or HIF2α and their target genes in cells depleted of V-ATPase subunits. HIF1α-GFP ODD reporter cells were transduced with sgRNA to the indicated V-ATPase subunits as described. After 14 days, cell surface CA9 was measured by flow cytometry ( E ). Levels of HIF1α, HIF2α and their targets CA9 and HO-1 were measured by immunoblot ( F, G ). DOI: http://dx.doi.org/10.7554/eLife.22693.009
Figure Legend Snippet: Disrupting the V-ATPase activates HIF1 and HIF2. ( A ) Immunoblot of HIF1α levels in response to the proteasome inhibitor MG132, the V-ATPase inhibitor BafA, the lysosomotropic agent Chloroquine, and the oxidative metabolism inhibitor NH 4 Cl. ( B ) Immunoblot of HIF1α levels in HeLa cells in response to BafA treatment at 0.5, 1, 2, 4, 8 and 24 hr. ( C ) Confocal immunofluorescence microscopy of WT (top) and BafA (bottom) treated HeLa cells stained for endogenous HIF1α. Cells were treated in the presence or absence of 10 nM BafA for 24 hr before immunofluorescence staining with HIF1α (white). Cells were mounted using DAPI (blue) and visualised by confocal microscopy. Scale bar represents 10 μm. ( D ) Immunocytochemical staining to examine HIF1α stabilisation in TMEM199 depleted HIF1α-GFP ODD reporter cells. HIF1α-GFP ODD reporter cells were depleted of TMEM199 using CRISPR-Cas9 genetics and stained for HIF1α (white), TMEM199 (red) and DAPI (blue). Scale bar represents 20 μm. ( E–G ) Levels of HIF1α or HIF2α and their target genes in cells depleted of V-ATPase subunits. HIF1α-GFP ODD reporter cells were transduced with sgRNA to the indicated V-ATPase subunits as described. After 14 days, cell surface CA9 was measured by flow cytometry ( E ). Levels of HIF1α, HIF2α and their targets CA9 and HO-1 were measured by immunoblot ( F, G ). DOI: http://dx.doi.org/10.7554/eLife.22693.009

Techniques Used: Immunofluorescence, Microscopy, Staining, Confocal Microscopy, CRISPR, Transduction, Flow Cytometry, Cytometry

37) Product Images from "Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability"

Article Title: Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability

Journal: Oncotarget

doi: 10.18632/oncotarget.21145

Chronic hypoxia induces oxidative damage and promotes tumor cell death (a) Human colonospheres were cultured in hypoxia (0.1%) and normoxia (21%) for 72 hours. Cells were lysed and analyzed by Western blotting for PCNA (proliferation) and cleaved caspase-3 (apoptosis) (cropped gels). (b) Experiment performed as in (a) but human colonospheres overexpressing GPx2 were included. FACS analysis of PI-stained cells was then used to assess cell death. The bar graph shows the percentage of cells with sub-G1 DNA content. (c) The culture conditions were similar to a and b. Cells were pulse labeled with BrdU just prior to FACS analysis to assess the percentage of proliferating cells. (d) FACS analysis of γH2AX levels in control and GPx2-overexpressing colonospheres. Cells were exposed to hypoxia for 24 hours. (e) Colonospheres were cultured in normoxia or hypoxia for 24 hours and were subsequently embedded in agar and fixed in formalin for IHC analysis of HIF1α, γH2AX and 4-HNE levels. (f) Colonospheres were cultured in normoxia or hypoxia for 24 hours. After single cell making living cells were FACS sorted and processed for immunofluorescence analysis of 8-oxo-dG (left panel) and γH2AX (right panel). DAPI was used to visualize cell nuclei.
Figure Legend Snippet: Chronic hypoxia induces oxidative damage and promotes tumor cell death (a) Human colonospheres were cultured in hypoxia (0.1%) and normoxia (21%) for 72 hours. Cells were lysed and analyzed by Western blotting for PCNA (proliferation) and cleaved caspase-3 (apoptosis) (cropped gels). (b) Experiment performed as in (a) but human colonospheres overexpressing GPx2 were included. FACS analysis of PI-stained cells was then used to assess cell death. The bar graph shows the percentage of cells with sub-G1 DNA content. (c) The culture conditions were similar to a and b. Cells were pulse labeled with BrdU just prior to FACS analysis to assess the percentage of proliferating cells. (d) FACS analysis of γH2AX levels in control and GPx2-overexpressing colonospheres. Cells were exposed to hypoxia for 24 hours. (e) Colonospheres were cultured in normoxia or hypoxia for 24 hours and were subsequently embedded in agar and fixed in formalin for IHC analysis of HIF1α, γH2AX and 4-HNE levels. (f) Colonospheres were cultured in normoxia or hypoxia for 24 hours. After single cell making living cells were FACS sorted and processed for immunofluorescence analysis of 8-oxo-dG (left panel) and γH2AX (right panel). DAPI was used to visualize cell nuclei.

Techniques Used: Cell Culture, Western Blot, FACS, Staining, Labeling, Immunohistochemistry, Immunofluorescence

Hypoxic areas in primary tumors and liver metastases are characterized by DNA damage and low expression of the DNA repair proteins RAD51 and RIF1 (a) Immunohistochemistry (IHC) for CAIX, HIF1α and γH2AX in human primary colorectal tumours. Robust expression of all three markers was observed surrounding necrotic lesions. (b) Quantification of CAIX, HIF1α and γH2AX expression in peri-necrotic areas and reference zones in primary human colon tumors by IHC (n=20). (c) Analysis of CAIX and γH2AX expression in human liver metastases by IHC. A 20x magnification of the area depicted in the square in lower panel. (d) Quantification of CAIX and γH2AX staining (IHC) in peri-necrotic and reference tissue in liver metastases (n=30). (e) Scatterplot showing the correlation between the expression of (randomly chosen microscopic field areas of) CAIX and γH2AX, assessed with the Spearman test (rho) (n=60). (f) Patient-derived colonospheres were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, the tumor-bearing liver lobes were subjected to a vascular clamping or sham protocol [ 24 ] to induce hypoxia. After 24 hours, the livers were excised and expression of CAIX and γH2AX was examined by IHC. (g) IHC on peri-necrotic areas of clamped livers (experiment as in (f)) for CAIX, γH2AX, RIF1, RAD51, and KU70. * p
Figure Legend Snippet: Hypoxic areas in primary tumors and liver metastases are characterized by DNA damage and low expression of the DNA repair proteins RAD51 and RIF1 (a) Immunohistochemistry (IHC) for CAIX, HIF1α and γH2AX in human primary colorectal tumours. Robust expression of all three markers was observed surrounding necrotic lesions. (b) Quantification of CAIX, HIF1α and γH2AX expression in peri-necrotic areas and reference zones in primary human colon tumors by IHC (n=20). (c) Analysis of CAIX and γH2AX expression in human liver metastases by IHC. A 20x magnification of the area depicted in the square in lower panel. (d) Quantification of CAIX and γH2AX staining (IHC) in peri-necrotic and reference tissue in liver metastases (n=30). (e) Scatterplot showing the correlation between the expression of (randomly chosen microscopic field areas of) CAIX and γH2AX, assessed with the Spearman test (rho) (n=60). (f) Patient-derived colonospheres were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, the tumor-bearing liver lobes were subjected to a vascular clamping or sham protocol [ 24 ] to induce hypoxia. After 24 hours, the livers were excised and expression of CAIX and γH2AX was examined by IHC. (g) IHC on peri-necrotic areas of clamped livers (experiment as in (f)) for CAIX, γH2AX, RIF1, RAD51, and KU70. * p

Techniques Used: Expressing, Immunohistochemistry, Staining, Derivative Assay, Injection, Mouse Assay

38) Product Images from "Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53"

Article Title: Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.240812

Microarray analysis revealed dose- and time-dependent repression of metabolic genes upon RITA treatment. The microarray data are presented as heat maps made using dChip (DNA chip analyzer). The rows were standardized by subtracting -fold change of control and dividing by the S.D. value. Vertical columns indicate separate arrays, and horizontal rows indicate genes. A , heat map depicting the relative mRNA levels of genes involved in regulation of metabolism in MCF7 cells treated with two concentrations of RITA over the indicated periods of time. B , comparison of changes of metabolic gene expression in MCF7 cells treated with RITA or nutlin3a over the indicated periods of time. C , schematic representation of the ATP-generating pathways, indicating the set of metabolic genes altered upon RITA treatment and their regulators. GLUT , glucose transporter; HK , hexokinase; PFKFB , phosphofructokinase fructose biphosphate; PFKM , phosphofructokinase muscles; PFKP , phosphofructokinase platelets; PGM , phosphoglycerate mutase, TIGAR , TP53-induced glycolysis and apoptosis regulator; LDH , lactate dehydrogenase; PDK , pyruvate dehydrogenase kinase; PDH , pyruvate dehydrogenase; MCT , monocarboxylate transporter; G6P ). Orange , glucose; dark orange , main metabolites produced during glycolysis and oxidative phosphorylation; dark green , targets of Myc, p53, and HIF1α; purple , p53 targets; green , Myc and HIF1α targets; blue , p53 and HIF1α targets.
Figure Legend Snippet: Microarray analysis revealed dose- and time-dependent repression of metabolic genes upon RITA treatment. The microarray data are presented as heat maps made using dChip (DNA chip analyzer). The rows were standardized by subtracting -fold change of control and dividing by the S.D. value. Vertical columns indicate separate arrays, and horizontal rows indicate genes. A , heat map depicting the relative mRNA levels of genes involved in regulation of metabolism in MCF7 cells treated with two concentrations of RITA over the indicated periods of time. B , comparison of changes of metabolic gene expression in MCF7 cells treated with RITA or nutlin3a over the indicated periods of time. C , schematic representation of the ATP-generating pathways, indicating the set of metabolic genes altered upon RITA treatment and their regulators. GLUT , glucose transporter; HK , hexokinase; PFKFB , phosphofructokinase fructose biphosphate; PFKM , phosphofructokinase muscles; PFKP , phosphofructokinase platelets; PGM , phosphoglycerate mutase, TIGAR , TP53-induced glycolysis and apoptosis regulator; LDH , lactate dehydrogenase; PDK , pyruvate dehydrogenase kinase; PDH , pyruvate dehydrogenase; MCT , monocarboxylate transporter; G6P ). Orange , glucose; dark orange , main metabolites produced during glycolysis and oxidative phosphorylation; dark green , targets of Myc, p53, and HIF1α; purple , p53 targets; green , Myc and HIF1α targets; blue , p53 and HIF1α targets.

Techniques Used: Microarray, Chromatin Immunoprecipitation, Expressing, Produced

39) Product Images from "Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia"

Article Title: Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia

Journal: The Journal of endocrinology

doi: 10.1530/JOE-17-0112

CD31-positive microvessel density in Ell2 -cko mice at age 17–20 mos. A. Quantification of CD31-positive intraductal microvessels in Ell2 -cko mice vs wild-type (WT) controls. B. Quantification of total CD31-positive microvessels Ell2 -cko mice vs wild-type (WT) controls. C. Immunostaining analysis of EAF2 and CD31-positive microvessels in prostate tissues. Original magnification 20X. D. Expression of HIF1α mRNA in Ell2 -cko and WT mice relative to GAPDH using the comparative C T method. E. Expression of HIF1α protein in C4-2 cells treated with siELL2 or siSCR. GAPDH served as loading control, and results are representative of 3 separate experiments. (*p
Figure Legend Snippet: CD31-positive microvessel density in Ell2 -cko mice at age 17–20 mos. A. Quantification of CD31-positive intraductal microvessels in Ell2 -cko mice vs wild-type (WT) controls. B. Quantification of total CD31-positive microvessels Ell2 -cko mice vs wild-type (WT) controls. C. Immunostaining analysis of EAF2 and CD31-positive microvessels in prostate tissues. Original magnification 20X. D. Expression of HIF1α mRNA in Ell2 -cko and WT mice relative to GAPDH using the comparative C T method. E. Expression of HIF1α protein in C4-2 cells treated with siELL2 or siSCR. GAPDH served as loading control, and results are representative of 3 separate experiments. (*p

Techniques Used: Mouse Assay, Immunostaining, Expressing

40) Product Images from "CDK-dependent phosphorylation of PHD1 on serine 130 alters its substrate preference in cells"

Article Title: CDK-dependent phosphorylation of PHD1 on serine 130 alters its substrate preference in cells

Journal: Journal of Cell Science

doi: 10.1242/jcs.179911

PHD1 phosphorylation at S130 modulates HIF mediated responses to hypoxia. (A) Recombinant purified PHD1, PHD1-S130A and PHD1-S130D enzymes were used in an in vitro hydroxylation assay using a peptide derived from the HIF1α ODD region (containing proline 564). Reactions were stopped with the addition of DFX, and samples were analysed by mass spectrometry. Electrospray-MS spectrum of the product of in vitro hydroxylation of the HIF1α peptide LDLEMLAPYIPMDDD showing an m/z increment of 7.99 Th (mass increment of 15.9944 Da) corresponding to proline hydroxylation of the doubly charged ion at m/z 875.8993 Th and the formation of the ion 883.8965 Th (the mass of the hydroxylated peptide) corresponding to the hydroxylation of proline 564 of HIF1α. The ion normalized level (NL) for the hydroxylated peptide is 2.89×10 7 for wild-type PHD1, 3.92×10 7 for PHD1-S130A and 4.06×10 7 for PHD1-S130D. (B) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP were transfected with PHD1 siRNA targeting the 3′-UTR (untranslated region) of endogenous PHD1 mRNA for 48 h prior to treatment with MG132 for 3 h. Whole-cell lysates were analysed by western blotting for the levels of the indicated proteins. Graph depicts western blot quantification showing mean±s.d. of a minimum of three independent experiments. (C) U2OS PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were exposed to 1% O 2 for the indicated periods of time prior to lysis. Whole-cell lysates were analysed by western blotting using the indicated antibodies. (D) Cell extracts obtained in C were analysed for the levels of the indicated HIF-dependent targets by western blotting. The graph depicts the quantification of western blots for HK2 and BNIP3, and illustrates the mean±s.d. for a minimum of three independent experiments. (E) U2OS PHD1–GFP cells were subject to a double-thymidine block release protocol prior to lysis or fixation on the indicated periods of time. For the last 3 h of each time point, 200 µM DFX was added to the cells. The left panel depicts western blot analysis for the levels of phosphorylated PHD1 at S130 and appropriate controls. The right panel represents the cell cycle profile of matching samples analysed by flow cytometry. AS, asynchronous. The graph depicts mean±s.d. of a minimum of three independent experiments. * P
Figure Legend Snippet: PHD1 phosphorylation at S130 modulates HIF mediated responses to hypoxia. (A) Recombinant purified PHD1, PHD1-S130A and PHD1-S130D enzymes were used in an in vitro hydroxylation assay using a peptide derived from the HIF1α ODD region (containing proline 564). Reactions were stopped with the addition of DFX, and samples were analysed by mass spectrometry. Electrospray-MS spectrum of the product of in vitro hydroxylation of the HIF1α peptide LDLEMLAPYIPMDDD showing an m/z increment of 7.99 Th (mass increment of 15.9944 Da) corresponding to proline hydroxylation of the doubly charged ion at m/z 875.8993 Th and the formation of the ion 883.8965 Th (the mass of the hydroxylated peptide) corresponding to the hydroxylation of proline 564 of HIF1α. The ion normalized level (NL) for the hydroxylated peptide is 2.89×10 7 for wild-type PHD1, 3.92×10 7 for PHD1-S130A and 4.06×10 7 for PHD1-S130D. (B) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP were transfected with PHD1 siRNA targeting the 3′-UTR (untranslated region) of endogenous PHD1 mRNA for 48 h prior to treatment with MG132 for 3 h. Whole-cell lysates were analysed by western blotting for the levels of the indicated proteins. Graph depicts western blot quantification showing mean±s.d. of a minimum of three independent experiments. (C) U2OS PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were exposed to 1% O 2 for the indicated periods of time prior to lysis. Whole-cell lysates were analysed by western blotting using the indicated antibodies. (D) Cell extracts obtained in C were analysed for the levels of the indicated HIF-dependent targets by western blotting. The graph depicts the quantification of western blots for HK2 and BNIP3, and illustrates the mean±s.d. for a minimum of three independent experiments. (E) U2OS PHD1–GFP cells were subject to a double-thymidine block release protocol prior to lysis or fixation on the indicated periods of time. For the last 3 h of each time point, 200 µM DFX was added to the cells. The left panel depicts western blot analysis for the levels of phosphorylated PHD1 at S130 and appropriate controls. The right panel represents the cell cycle profile of matching samples analysed by flow cytometry. AS, asynchronous. The graph depicts mean±s.d. of a minimum of three independent experiments. * P

Techniques Used: Recombinant, Purification, In Vitro, Derivative Assay, Mass Spectrometry, Transfection, Western Blot, Lysis, Blocking Assay, Flow Cytometry, Cytometry

S130 of PHD1 is important for PHD1-mediated control of cell proliferation. (A) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were transfected with siRNA oligonucleotides targeting the 3′UTR of endogenous PHD1 prior to proliferation being assessed. Total cell numbers were counted, and the graph depicts mean±s.d. of a minimum of three independent experiments. Data were normalised to proliferation in GFP cells and expressed as a percentage. (B) U2OS GFP, PHD1GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were transfected with siRNA oligonucleotides targeting the 3′UTR of endogenous PHD1 prior to fixation and immunostaining for Cep192 and pericentrin. Scale bars: 2 µm. Graph depicts box-and-whisker plots for Cep192 and Pericentrin intensity. Box-and-whisker plot, middle line shows the median value; the bottom and top of the box show the lower and upper quartiles (25-75%); whiskers extend to 10th and 90th percentiles, and all outliers are shown. n =22–38 cells per condition. (C) U2OS GFP, PHD1GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were treated with MG132 for 3 h prior to lysis. 300 µg of cell extracts were used to immunoprecipitate (IP) Cep192, with normal mouse IgG used as a control. Precipitated material was analysed by western blotting for the indicated proteins. Western blots were quantified and the graph depicts mean±s.d. of a minimum of three independent experiments. (D) Cell extracts from Fig. 3 A were analysed by western blotting for the levels of Cep192 and HIF1α. G1, S, and G2/M are the phases of the cell cycle that correspond to the indicated time points. (E) U2OS were transfected with the indicated siRNAs prior to treatment with 1% O 2 for 24 h. Whole-cell lysates were analysed by western blotting using the depicted antibodies. (F) Schematic diagram for the proposed model for PHD1 regulation by CDKs. * P
Figure Legend Snippet: S130 of PHD1 is important for PHD1-mediated control of cell proliferation. (A) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were transfected with siRNA oligonucleotides targeting the 3′UTR of endogenous PHD1 prior to proliferation being assessed. Total cell numbers were counted, and the graph depicts mean±s.d. of a minimum of three independent experiments. Data were normalised to proliferation in GFP cells and expressed as a percentage. (B) U2OS GFP, PHD1GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were transfected with siRNA oligonucleotides targeting the 3′UTR of endogenous PHD1 prior to fixation and immunostaining for Cep192 and pericentrin. Scale bars: 2 µm. Graph depicts box-and-whisker plots for Cep192 and Pericentrin intensity. Box-and-whisker plot, middle line shows the median value; the bottom and top of the box show the lower and upper quartiles (25-75%); whiskers extend to 10th and 90th percentiles, and all outliers are shown. n =22–38 cells per condition. (C) U2OS GFP, PHD1GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were treated with MG132 for 3 h prior to lysis. 300 µg of cell extracts were used to immunoprecipitate (IP) Cep192, with normal mouse IgG used as a control. Precipitated material was analysed by western blotting for the indicated proteins. Western blots were quantified and the graph depicts mean±s.d. of a minimum of three independent experiments. (D) Cell extracts from Fig. 3 A were analysed by western blotting for the levels of Cep192 and HIF1α. G1, S, and G2/M are the phases of the cell cycle that correspond to the indicated time points. (E) U2OS were transfected with the indicated siRNAs prior to treatment with 1% O 2 for 24 h. Whole-cell lysates were analysed by western blotting using the depicted antibodies. (F) Schematic diagram for the proposed model for PHD1 regulation by CDKs. * P

Techniques Used: Transfection, Immunostaining, Whisker Assay, Lysis, Western Blot

PHD1 phosphorylation at Serine 130 alters the ability of PHD1 to target HIF1α. (A) U2OS PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were exposed to 1% O 2 for 4 h prior to treatment with cycloheximide for the indicated periods of time. Whole-cell lysates were analysed by western blotting for the levels of HIF1α and appropriate controls. Western blots were quantified and the graph depicts mean±s.d. of a minimum of three independent experiments. (B) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were treated with MG132 for 3 h prior to lysis. 300 µg of cell extracts were used to immunoprecipitate (IP) HIF1α, with normal mouse IgG used as a control. Precipitated material was analysed by western blotting for the indicated proteins. Western blots were quantified, and the graph depicts the mean±s.d. of a minimum of three independent experiments. (C) Schematic diagram of the PHD1 expression constructs used in this study. Highlighted are the nuclear localization signal (NLS), S130 and the hydroxylase domain (HD). (D) HEK293 cells were transfected with 1 µg of the indicated expression constructs for 48 h prior to treatment with MG132 and processed as in B. *, non specific band. (E) HEK293 were transfected with 1 µg of the indicated expression constructs for 48 h prior to treatment with MG132 and processed as in B. Western blots were quantified, and the graph depicts mean±s.d. of a minimum of three independent experiments. * P
Figure Legend Snippet: PHD1 phosphorylation at Serine 130 alters the ability of PHD1 to target HIF1α. (A) U2OS PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were exposed to 1% O 2 for 4 h prior to treatment with cycloheximide for the indicated periods of time. Whole-cell lysates were analysed by western blotting for the levels of HIF1α and appropriate controls. Western blots were quantified and the graph depicts mean±s.d. of a minimum of three independent experiments. (B) U2OS GFP, PHD1–GFP, PHD1-S130A–GFP and PHD1-S130D–GFP cells were treated with MG132 for 3 h prior to lysis. 300 µg of cell extracts were used to immunoprecipitate (IP) HIF1α, with normal mouse IgG used as a control. Precipitated material was analysed by western blotting for the indicated proteins. Western blots were quantified, and the graph depicts the mean±s.d. of a minimum of three independent experiments. (C) Schematic diagram of the PHD1 expression constructs used in this study. Highlighted are the nuclear localization signal (NLS), S130 and the hydroxylase domain (HD). (D) HEK293 cells were transfected with 1 µg of the indicated expression constructs for 48 h prior to treatment with MG132 and processed as in B. *, non specific band. (E) HEK293 were transfected with 1 µg of the indicated expression constructs for 48 h prior to treatment with MG132 and processed as in B. Western blots were quantified, and the graph depicts mean±s.d. of a minimum of three independent experiments. * P

Techniques Used: Western Blot, Lysis, Expressing, Construct, Transfection

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Staining:

Article Title: A compact VEGF signature associated with distant metastases and poor outcomes
Article Snippet: .. Immunohistochemistry (IHC) was performed for HIF1α using Mouse Anti-Human HIF1α (BD Biosciences #610958) according to the protocol from Vleugel et al [ ]; the tumors were scored for perinecrotic and diffuse staining as described in Vleugel et al. .. Expression patterns associated with metastases To identify gene expression patterns associated with breast cancer metastases, we performed 195 microarrays representing 134 primary tumors, nine regional metastases and 18 distant metastasis specimens (146 different patients and 10 normal breast tissues).

Immunohistochemistry:

Article Title: A compact VEGF signature associated with distant metastases and poor outcomes
Article Snippet: .. Immunohistochemistry (IHC) was performed for HIF1α using Mouse Anti-Human HIF1α (BD Biosciences #610958) according to the protocol from Vleugel et al [ ]; the tumors were scored for perinecrotic and diffuse staining as described in Vleugel et al. .. Expression patterns associated with metastases To identify gene expression patterns associated with breast cancer metastases, we performed 195 microarrays representing 134 primary tumors, nine regional metastases and 18 distant metastasis specimens (146 different patients and 10 normal breast tissues).

Activity Assay:

Article Title: A HIF1? Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas
Article Snippet: .. Membranes were probed with the following antibodies: SDHB, as described above, HIF1α (BD Biosciences, San Jose, California, United States), Glut1, used as a surrogate for HIF1α activity (Alpha Diagnostic, San Antonio, Texas, United States), and FLAG (Sigma). β-actin was used as a loading control, as above. .. The lentiviral shRNA expression vector FSIPPW was used, as previously described [ ].

SDS Page:

Article Title: Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1
Article Snippet: .. The proteins separated on SDS-PAGE gels were transferred to nitrocellulose membranes (Bio-Rad) and probed with antibodies against ZEB1 (3396, Cell Signaling), SNAIL (3879, Cell Signaling), TWIST1 (ab50887, Abcam), CDH1 (NBP2-19051, Novus Biologicals) CLDN1 (Cell Signaling, 13255), FN1 (LF-MA0151, Thermo Scientific) and HIF1α (610959, BD Biosciences). .. Loading control: β actin (ab3280, Abcam).

Western Blot:

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    Becton Dickinson anti hif1α antibody
    CD86+ macrophages from human mucosa express <t>HIF1α</t> and Jag1. Quantitative analysis (static cytometry) was performed in macrophages isolated from the mucosa and the graph shows the percentages of CD86+ and CD206+ cells that expressed Jag1 (a), HIF-1α
    Anti Hif1α Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson hif1α
    Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA. Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and <t>HIF1α</t> down-regulation.
    Hif1α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson primary antibody against hif1α
    <t>HIF1α</t> could regulate ATG5 by direct binding to the promoter of ATG5. A, The schematic view of ATG5 genomic structure. The location of HIF1α responsive element (HRE) was shown. B, The transcriptional activity of reporter contain ATG5’s promoter were analyzed in PC-3 cells transfected with nonsense control or si-HIF1αexposed to normoxia or 1% O 2 for 24 h. C. The transcriptional activity of reporter contain wild type or HRE deletion ATG5’s promoter were analyzed in PC-3 cells exposed to normoxia or 1% O 2 for 24 h. D–E, ChIP analysis of ATG5 promoter was performed by using anti-HIF1α antibody in PC-3 cells exposed to 1% O 2 for 24 h. RT-PCR (D) and PCR (E) were performed with primers specific to the functional HRE in ATG5 promoter. Means ± s.e.m are shown.* P
    Primary Antibody Against Hif1α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CD86+ macrophages from human mucosa express HIF1α and Jag1. Quantitative analysis (static cytometry) was performed in macrophages isolated from the mucosa and the graph shows the percentages of CD86+ and CD206+ cells that expressed Jag1 (a), HIF-1α

    Journal: Journal of Crohn's & Colitis

    Article Title: M1 Macrophages Activate Notch Signalling in Epithelial Cells: Relevance in Crohn’s Disease

    doi: 10.1093/ecco-jcc/jjw009

    Figure Lengend Snippet: CD86+ macrophages from human mucosa express HIF1α and Jag1. Quantitative analysis (static cytometry) was performed in macrophages isolated from the mucosa and the graph shows the percentages of CD86+ and CD206+ cells that expressed Jag1 (a), HIF-1α

    Article Snippet: Immunoprecipitation was performed with anti-HIF1α antibody (BD, Madrid, Spain) or control IgG antibody.

    Techniques: Cytometry, Isolation

    Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA. Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.

    Journal: PLoS ONE

    Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

    doi: 10.1371/journal.pone.0041171

    Figure Lengend Snippet: Molecular effects of in vivo targeting of eEF-2K by liposomal siRNA. Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.

    Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: In Vivo, Mouse Assay, Western Blot

    Expression of HIF1α over time in survivors (white circles) and non survivors (black circles) . The horizontal bar indicates the median for each group.

    Journal: Critical Care

    Article Title: Hypoxia-inducible factor (HIF1α) gene expression in human shock states

    doi: 10.1186/cc11414

    Figure Lengend Snippet: Expression of HIF1α over time in survivors (white circles) and non survivors (black circles) . The horizontal bar indicates the median for each group.

    Article Snippet: Blood samples for HIF1α (italic refers to mRNA throughout the manuscript) measurements were collected on PaxgeneTM tubes (BD, Franklin Lakes, NJ, USA) and stored at -80°C until RNA extraction.

    Techniques: Expressing

    Expression of different HIF1α variants in shock patients (black bars) and controls (grey bars) .

    Journal: Critical Care

    Article Title: Hypoxia-inducible factor (HIF1α) gene expression in human shock states

    doi: 10.1186/cc11414

    Figure Lengend Snippet: Expression of different HIF1α variants in shock patients (black bars) and controls (grey bars) .

    Article Snippet: Blood samples for HIF1α (italic refers to mRNA throughout the manuscript) measurements were collected on PaxgeneTM tubes (BD, Franklin Lakes, NJ, USA) and stored at -80°C until RNA extraction.

    Techniques: Expressing

    HIF1α could regulate ATG5 by direct binding to the promoter of ATG5. A, The schematic view of ATG5 genomic structure. The location of HIF1α responsive element (HRE) was shown. B, The transcriptional activity of reporter contain ATG5’s promoter were analyzed in PC-3 cells transfected with nonsense control or si-HIF1αexposed to normoxia or 1% O 2 for 24 h. C. The transcriptional activity of reporter contain wild type or HRE deletion ATG5’s promoter were analyzed in PC-3 cells exposed to normoxia or 1% O 2 for 24 h. D–E, ChIP analysis of ATG5 promoter was performed by using anti-HIF1α antibody in PC-3 cells exposed to 1% O 2 for 24 h. RT-PCR (D) and PCR (E) were performed with primers specific to the functional HRE in ATG5 promoter. Means ± s.e.m are shown.* P

    Journal: Animal Cells and Systems

    Article Title: HIF1α promotes prostate cancer progression by increasing ATG5 expression

    doi: 10.1080/19768354.2019.1658637

    Figure Lengend Snippet: HIF1α could regulate ATG5 by direct binding to the promoter of ATG5. A, The schematic view of ATG5 genomic structure. The location of HIF1α responsive element (HRE) was shown. B, The transcriptional activity of reporter contain ATG5’s promoter were analyzed in PC-3 cells transfected with nonsense control or si-HIF1αexposed to normoxia or 1% O 2 for 24 h. C. The transcriptional activity of reporter contain wild type or HRE deletion ATG5’s promoter were analyzed in PC-3 cells exposed to normoxia or 1% O 2 for 24 h. D–E, ChIP analysis of ATG5 promoter was performed by using anti-HIF1α antibody in PC-3 cells exposed to 1% O 2 for 24 h. RT-PCR (D) and PCR (E) were performed with primers specific to the functional HRE in ATG5 promoter. Means ± s.e.m are shown.* P

    Article Snippet: Primary antibody against HIF1α (BD), ATG5 (CST), LC3 (CST), P62 (BD) and Tubulin (Sigma Aldrich) were used.

    Techniques: Binding Assay, Activity Assay, Transfection, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Functional Assay

    ATG5 is a hypoxia responsive gene. A–B, Western blotting(A) and densitometry(B) analysis of the protein levels of HIF1α, ATG5 and LC3 in PC-3 cells exposed to normoxia or 1% O 2 for 24 h. Tubulin was used as a loading control. C–D, Western blotting (C) and densitometry (D) analysis of the protein levels of ATG5, p62 and LC3 in PC-3 cells transfected the HIF1α mutant plasmids (HIF1α-M) or vector for 48 h. Tubulin was used as a loading control. E–F, Western blotting (E) and densitometry (F) analysis of the protein levels of HIF1α, ATG5 and LC3 in PC-3 cells transfected with HIF1α siRNA (si-HIF1α) or nonsense control for 48 h. Tubulin was used as a loading control. G, Relative mRNA level of ATG5 in PC-3 cells exposed to normaxia or hypoxia for 24 h. H, Relative mRNA level of ATG5 in PC-3 cells transfected with HIF1α siRNA or nonsense control for 48 h. Means ± s.e.m are shown.* P

    Journal: Animal Cells and Systems

    Article Title: HIF1α promotes prostate cancer progression by increasing ATG5 expression

    doi: 10.1080/19768354.2019.1658637

    Figure Lengend Snippet: ATG5 is a hypoxia responsive gene. A–B, Western blotting(A) and densitometry(B) analysis of the protein levels of HIF1α, ATG5 and LC3 in PC-3 cells exposed to normoxia or 1% O 2 for 24 h. Tubulin was used as a loading control. C–D, Western blotting (C) and densitometry (D) analysis of the protein levels of ATG5, p62 and LC3 in PC-3 cells transfected the HIF1α mutant plasmids (HIF1α-M) or vector for 48 h. Tubulin was used as a loading control. E–F, Western blotting (E) and densitometry (F) analysis of the protein levels of HIF1α, ATG5 and LC3 in PC-3 cells transfected with HIF1α siRNA (si-HIF1α) or nonsense control for 48 h. Tubulin was used as a loading control. G, Relative mRNA level of ATG5 in PC-3 cells exposed to normaxia or hypoxia for 24 h. H, Relative mRNA level of ATG5 in PC-3 cells transfected with HIF1α siRNA or nonsense control for 48 h. Means ± s.e.m are shown.* P

    Article Snippet: Primary antibody against HIF1α (BD), ATG5 (CST), LC3 (CST), P62 (BD) and Tubulin (Sigma Aldrich) were used.

    Techniques: Western Blot, Transfection, Mutagenesis, Plasmid Preparation

    HIF1α promotes metastasis of PC-3 cells by promoting ATG5 expression and autophagy levels. A, Relative mRNA expression of ATG5, N-cadherin and Vimentin in PC-3 cells transfected with vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. B–C, Western blotting (B) and densitometry (C) analysis of the protein levels of HIF1α, ATG5, N-cadherin and Vimentin in PC-3 cells transfected with vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. Tubulin was used as a loading control. D–E, Representative photographs (D) and number of migration cells (E) of transwell migration assay of PC-3 cells transfected with Vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. Scale bar, 200 uM.HIF1αHIF1αHIF1αHIF1αF-G, Representative photographs (F) and quantification of scratch size (G) of wound-healing assay of PC-3 cells transfected with Vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide.HIF1αHIF1α. H-I, Representative photographs (H) and number of migration cells (I) of transwell migration assay of PC-3 cells treated with Vector, HIF1α-M or both HIF1α-M and 3MA. Scale bar, 200 uM. J-K, Representative photographs (J) and number of migration cells (K) of transwell migration assay of PC-3 cells transfected with Vector, si-HIF1α or both si-HIF1α-M and ATG5 exposed to hypoxia. Scale bar, 200 uM. Means ± s.e.m are shown.* P

    Journal: Animal Cells and Systems

    Article Title: HIF1α promotes prostate cancer progression by increasing ATG5 expression

    doi: 10.1080/19768354.2019.1658637

    Figure Lengend Snippet: HIF1α promotes metastasis of PC-3 cells by promoting ATG5 expression and autophagy levels. A, Relative mRNA expression of ATG5, N-cadherin and Vimentin in PC-3 cells transfected with vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. B–C, Western blotting (B) and densitometry (C) analysis of the protein levels of HIF1α, ATG5, N-cadherin and Vimentin in PC-3 cells transfected with vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. Tubulin was used as a loading control. D–E, Representative photographs (D) and number of migration cells (E) of transwell migration assay of PC-3 cells transfected with Vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide. Scale bar, 200 uM.HIF1αHIF1αHIF1αHIF1αF-G, Representative photographs (F) and quantification of scratch size (G) of wound-healing assay of PC-3 cells transfected with Vector, HIF1α-M or both HIF1α-M and si-ATG5 oligonucleotide.HIF1αHIF1α. H-I, Representative photographs (H) and number of migration cells (I) of transwell migration assay of PC-3 cells treated with Vector, HIF1α-M or both HIF1α-M and 3MA. Scale bar, 200 uM. J-K, Representative photographs (J) and number of migration cells (K) of transwell migration assay of PC-3 cells transfected with Vector, si-HIF1α or both si-HIF1α-M and ATG5 exposed to hypoxia. Scale bar, 200 uM. Means ± s.e.m are shown.* P

    Article Snippet: Primary antibody against HIF1α (BD), ATG5 (CST), LC3 (CST), P62 (BD) and Tubulin (Sigma Aldrich) were used.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Migration, Transwell Migration Assay, Wound Healing Assay

    HIF1α promotes tumor cell proliferation in vivo by promoting ATG5 expression and autophagy levels. A–B, Representative images (A) and tumor weight (B) of tumor xenograft samples in nude mouses inoculated with wild type or HIF1α-overexpressd PC cells. C, Relative mRNA expression of HIF1α and ATG5 in harvested tumor xenograft samples of control or HIF1α-overexpressd group. D–E Western blotting (D) and densitometry (E) analysis of the protein levels of HIF1α, ATG5 and LC3 in harvested tumor xenograft samples of control or HIF1α-overexpressed group. Tubulin was used as a loading control. F–G, Representative images (F) and tumor weight (G) of tumor xenograft samples in nude mouses inoculated with wild type, HIF1α-overexpressd or both HIF1α-overexpressd and ATG5 konckdown PC cells Means ± s.e.m are shown. * P

    Journal: Animal Cells and Systems

    Article Title: HIF1α promotes prostate cancer progression by increasing ATG5 expression

    doi: 10.1080/19768354.2019.1658637

    Figure Lengend Snippet: HIF1α promotes tumor cell proliferation in vivo by promoting ATG5 expression and autophagy levels. A–B, Representative images (A) and tumor weight (B) of tumor xenograft samples in nude mouses inoculated with wild type or HIF1α-overexpressd PC cells. C, Relative mRNA expression of HIF1α and ATG5 in harvested tumor xenograft samples of control or HIF1α-overexpressd group. D–E Western blotting (D) and densitometry (E) analysis of the protein levels of HIF1α, ATG5 and LC3 in harvested tumor xenograft samples of control or HIF1α-overexpressed group. Tubulin was used as a loading control. F–G, Representative images (F) and tumor weight (G) of tumor xenograft samples in nude mouses inoculated with wild type, HIF1α-overexpressd or both HIF1α-overexpressd and ATG5 konckdown PC cells Means ± s.e.m are shown. * P

    Article Snippet: Primary antibody against HIF1α (BD), ATG5 (CST), LC3 (CST), P62 (BD) and Tubulin (Sigma Aldrich) were used.

    Techniques: In Vivo, Expressing, Western Blot