Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a , b ) CAIX expression is enhanced in glioma progression, whereas PHD3 expression is attenuated. qPCR analysis of CAIX ( a ) and PHD3 ( b ) gene expression in normal adult brain (NB), diffuse astrocytomas (WHO grade II), anaplastic astrocytomas (WHO grade III), (s)econdary and (p)rimary glioblastomas (WHO grade IV; n =76). ( c ) PHD3 promoter hypermethylation is an early event in glioma progression. Frequency of PHD3 promoter hypermethylation in NB and gliomas of different grades as determined by methylation-specific PCR. ( d , e ) PHD3 promoter hypermethylation decreases PHD3 expression. Comparison of PHD3 mRNA levels in gliomas with or without PHD3 promoter methylation as determined in b , c , respectively, within our glioma cohort ( d ). Comparison of PHD3 mRNA expression in glioblastomas with or without PHD3 promoter methylation from TCGA cohort ( n =251, tumours for which methylation data are available) ( e ). ( f ) qPCR analysis of PHD3 expression in G55, U118 and HGBM tumour cells with hypermethylated and nonmethylated PHD3 promoter, respectively, ±treatment with the demethylating agent 5-azacytidine (20 μM) for 72 h and Trichostatin A (1 μM) for the last 24 h ( n =3). All values are means+s.e.m., * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Expressing, Gene Expression, Methylation, Comparison

Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a ) Immunoblot of G55 glioblastoma cells stably transduced with control (co), PHD2 or PHD3 shRNA following exposure to 21% ( N ) or 1% O 2 ( H ) for 24 h. ( b , c ) PHD3, but not PHD2, loss promotes intracranial glioma growth. Tumour xenografts of polyclonal G55 pools expressing control, PHD2, PHD3 or PHD2/PHD3 shRNA were stained with haematoxylin and eosin (HE; n =8). ( d ) Kaplan–Meier survival curves of nude mice intracranially injected with polyclonal G55 pools expressing control or PHD3 shRNA. ( e ) Immunoblot of wild-type (WT) and PHD3 −/− astrocytoma cells following exposure to 21% ( N ) or 1% O 2 ( H ) for 24 h. ( f , g ) Genetic inactivation of PHD3 increases mouse astrocytoma growth ( n =9–10). ( h , i ) PHD3 silencing reduces tumour cell apoptosis in xenografts of polyclonal G55 pools ( n =7–8) assessed by quantifying the number of TUNEL-positive cells per tumour area. ( j , k ) Silencing of PHD3 increases cell proliferation in xenografts of polyclonal G55 pools ( n =8) quantified as the number of phospho-histone 3-positive cells per tumour area. Western blots images ( a , e ) have been cropped for presentation. Full size images are presented in . All values are means+s.e.m., * P <0.05; ** P <0.01; *** P <0.001. Scale bars, 1 mm ( b , f ), 50 μm ( h , j ).

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Western Blot, Stable Transfection, Transduction, Control, shRNA, Expressing, Staining, Injection, TUNEL Assay

Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a , b ) PHD3 silencing increases clonal cell growth in a 3D spheroid culture system. G55 cells expressing control or PHD3 shRNA were cultured as spheroids in B27-supplemented serum-free medium±EGF/FGF and the number of spheroids was quantified after 3 days ( n =6). ( c , d ) PHD3 loss protects against cell death induction following growth factor withdrawal. Apoptosis was assessed by quantifying the number of TUNEL-positive cells after 48 h of incubation in B27-supplemented serum-free medium±EGF/FGF ( n =15). ( e , f ) PHD3 loss increases cell proliferation. Cell proliferation was assessed by quantifying the number of 5-bromodeoxyuridine-positive cells after 48 h of incubation in B27-supplemented serum-free medium±EGF/FGF ( n =15). ( g ) PHD3 disruption by genetic inactivation confers a growth advantage in additional glioma cell systems. Clonal cell growth was quantified as in b in the presence or absence of EGF/FGF in PHD3 −/− astrocytoma cells ( n =6). All values are means+s.e.m., ** P <0.01; *** P <0.001. Scale bars, 50 μm.

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Expressing, Control, shRNA, Cell Culture, TUNEL Assay, Incubation, Disruption

Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a – c ) Growth inhibitory signals induce PHD3 expression. Immunoblot of G141 and HGBM tumour cells following exposure to 21% (N) or 1% O 2 (H) for 24 h, in combination with ±hypoglycaemia (treatment with 6 mM 2-deoxyglucose) ( a ), incubation with TNFα (10 ng ml −1 ) ( b ) or EGF (20 ng ml −1 ) ( c ). ( d ) PHD3 loss protects against hypoxic growth inhibition. Clonal spheroid growth ( n =6) of G141 tumour cells expressing control or PHD3 shRNA was quantified following exposure to 21% (N) or 1% O 2 (H) for 3 days. ( e , f ) PHD3 inhibits intracranial glioma growth. Tumour xenografts of polyclonal G55 pools expressing PHD3 or GFP control were stained with HE ( e ) and the tumour volume was quantified ( f ) ( n =9–10). ( g ) PHD3 decreases clonal cell growth in a 3D spheroid culture system. G55 cells expressing PHD3 or GFP control were cultured as spheroids in B27-supplemented serum-free medium±EGF/FGF and the number of spheroids was quantified after 3 days ( n =6). ( h , i ) PHD3 expression enhances apoptosis. Cell apoptosis was assessed in G55 pools expressing PHD3 or GFP control by quantifying the number of TUNEL-positive cells after 48 h of incubation in B27-supplemented serum-free medium+EGF/FGF ( n =18). ( j , k ) PHD3 inhibits proliferation. Cell proliferation was assessed in G55 pools expressing PHD3 or GFP control by quantifying the number of BrdU-positive cells after 48 h of incubation in B27-supplemented serum-free medium+EGF/FGF ( n =15). Western blot images ( a – c ) have been cropped for presentation. Full size images are presented in . All values are means+s.e.m., * P <0.05; ** P <0.01; *** P <0.001. Scale bars, 1 mm ( e ), 50 μm ( h , j ).

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Expressing, Western Blot, Incubation, Inhibition, Control, shRNA, Staining, Cell Culture, TUNEL Assay

Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a , b ) The growth-promoting effect of PHD3 loss is HIF-1/2α-independent. G55 cells expressing control or PHD3 shRNA in combination with control, HIF-1α or HIF-2α ( a ) or double HIF-1α/2α ( b ) shRNA were cultured as spheroids in B27-supplemented serum-free medium±EGF/FGF and the number of spheroids was quantified after 3–5 days ( n =6). ( c , d ) NF-κB signalling is not altered following PHD3 loss. G55 cells expressing control or PHD3 shRNA were cultured as spheroids±treatment with TNF-α (10 ng ml −1 , 30 min). Immunofluorescence with anti-p65 antibodies shows that basal and TNF-α-stimulated translocation of p65 to the nucleus is not affected by PHD3 loss ( c ). PHD3 loss does not activate NF-κB signalling. G55 cells expressing control, PHD3 or PHD2 (positive control) shRNA were transfected with an NF-κB luciferase reporter construct±treatment with TNF-α (10 ng ml −1 , 6 h) and EGF (20 ng ml −1 , 6 h; n =3) ( d ). ( e ) The hydroxylase function of PHD3 is not required for the regulation of tumour cell growth. G55 cells expressing PHD3 or control shRNA were transfected with either wild-type PHD3, the hydroxylase mutant PHD3-H196A or empty vector control, cultured as spheroids in B27-supplemented serum-free medium±EGF/FGF and the number of spheroids was quantified after 3 days ( n =6). ( f ) PHD3 wild-type and hydroxylase mutant decrease the PHD3-loss-mediated increase in intracranial tumour growth ( n =9–10). All values are means+s.e.m., * P <0.05; ** P <0.01; *** P <0.001. Scale bars, 10 μm.

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Expressing, Control, shRNA, Cell Culture, Immunofluorescence, Translocation Assay, Positive Control, Transfection, Luciferase, Construct, Mutagenesis, Plasmid Preparation

Journal: Nature Communications

Article Title: Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

doi: 10.1038/ncomms6582

Figure Lengend Snippet: ( a ) EGFR phosphorylation is enhanced following PHD3 loss. Immunoblot with two different phospho-specific antibodies against EGFR of G55 cells expressing PHD3 or scrambled control nonstimulated or stimulated with EGF (20 ng ml −1 ) for the indicated times. ( b , c ) EGFR activity is required for the growth-promoting effect of PHD3 loss. G55 cells expressing control or PHD3 shRNA were cultured as spheroids in B27-supplemented serum-free medium±EGF (20 ng ml −1 ). Where indicated, cells were pretreated with the EGFR inhibitor erlotinib (10, 20 μM) for 2 h before stimulation. The number of spheroids was quantified after 3 days ( n =6; b ). Nude mice transplanted with G55 cells expressing control or PHD3 shRNA were treated with vehicle or erlotinib and tumour growth was quantified ( n =13–18; c ). ( d , e ) PHD3 loss through deletion or promoter methylation occurs preferentially in glioblastomas without EGFR amplification. Analysis of CNA for EGFR and PHD3, as well as PHD3 promoter methylation (Meth) in glioblastomas from the TCGA cohort ( n =501, tumours for which CNA data are available). High-level EGFR amplification is shown in red , PHD3 deletion in green and PHD3 promoter methylation in blue ( d ). PHD3 deletion or promoter methylation are significantly more common in tumours without EGFR amplification ( e ). Western blot images ( a ) have been cropped for presentation. Full size images are presented in . All values are means+s.e.m., * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Activity Assay, shRNA, Cell Culture, Methylation, Amplification

Journal: American Journal of Cancer Research

Article Title: Stat5b inhibition blocks proliferation and tumorigenicity of glioblastoma stem cells derived from a de novo murine brain cancer model

doi:

Figure Lengend Snippet: Lgr5 and Stat5b expression are induced by hypoxia in GSCs. A. Representative images of immunohistochemical staining for Hif2α, Lgr5, and Stat5b in human GBM tissue. B. Stat5b mRNA levels were analyzed by qRT-PCR in GSCs cultured in hypoxic conditions for 2 days (0.5% O2; n=3; ***P<0.001). C. Western blot analysis of phospho-Stat5 and Stat5b in GSCs cultured in hypoxic conditions (0.5% O2 for 4 days, 5% O2 for 3 days). β-tubulin is shown as a loading control. D. Western blot analysis of Hif2α and Stat5b in GSCs after treatment with control-shRNA (Control) or Hif2α-shRNA (Hif2α-KD) for 4 days. GAPDH is shown as a loading control. E. Lgr5 mRNA expression levels were analyzed by qRT-PCR in GSCs after 2 days in hypoxic conditions (0.5% O2; n=3; **P<0.01). F. Lgr5 expression levels were analyzed by a flow cytometry in the GSCs after 2 days in hypoxic conditions (0.5% O2).

Article Snippet: The following antibodies were used: Stat5b (1:1000; ab178941, Abcam), Lgr5 (1:500; bs-1117R; Bioss, Woburn, MA, USA), Hif2α (1:500; NB 100-122; Novus Biologicals, Centennial, CO, USA), β-tubulin (1:200; T4026, Sigma-Aldrich), GAPDH (1:1000; 016-25523, Wako), Oct4 (1:1000; ab19875, Abcam), Survivin (1:1000; ab182132, Abcam), Cyclin E2 (1:500; ab32103, Abcam), Phospho-Rb (1:1000; #8516, Cell Signaling Technology [CST], Danvers, MA, USA), Phospho-Stat5 (1:1000; ab32364, Abcam), Caspase3 (1:1000; #9665, CST), PARP (1:1000; #9542, CST), β-Catenin (1:1000; #9587, CST).

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Cell Culture, Western Blot, shRNA, Flow Cytometry

Journal: PloS one

Article Title: ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: Figure 1. HIF integrates ECM cues and Oxygen Levels to Direct TSC Fate. (A–D) Immunofluorescence microscopy of undifferentiated control TSCs cultured on CELLstartTM with anti-CDX2 and EOMES antibodies (blue = DAPI, red = CDX2, green = Eomes). (E, G) Phase contrast microscopy of control TSCs maintained on CELLstartTM following differentiation for 7 days under normoxic (21% O2) or hypoxic (2% O2) conditions. (F, H) Immunofluoresce microscopy of control TSCs maintained on CELLstartTM following differentiation for 7 days under normoxic (21% O2) or hypoxic (2% O2) conditions with an anti-HOPX1 (red) antibody (blue = DAPI). (I) Quantitative RT-PCR analysis of Pl. I, Pl.II, Ctsq, Plf, Tfeb, SynA and SynB gene expression in wild-type (+/+) TSCs differentiated for 7 days following culture on CELLstartTM or on TC plastic in Fib-CM, compared with Arnt2/2 (2/2) TSCs differentiated following culture on TC plastic in Fib-CM. p values ,0.05 versus wild-type Fib-CM indicated by an asterisk. (J) Immunoblot of HIF- 1a and -2a protein levels in whole cell lysates of wild-type TSCs differentiated for 7 days following culture on CELLstart at 21%O2 or 2% O2. (K) Quantitative RT-PCR analysis of Pl-1, Pl-2, Plf, Tfeb, SynA and SynB expression in Vhlh+/+ and Vhlh2/2 TSCs differentiated following culture on CELLstartTM. p values ,0.05 versus wild-type indicated by an asterisk. doi:10.1371/journal.pone.0056949.g001

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF1a (R&D Systems, Minneapolis, MN), HIF-1a c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2a NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), a-Tubulin (NeoMarkers, Fremont, CA), Ac- a-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), b-catenin (Cell Signaling), cMYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Immunofluorescence, Microscopy, Control, Cell Culture, Quantitative RT-PCR, Gene Expression, Western Blot, Expressing

Journal: PloS one

Article Title: ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: Figure 3. ECM- or oxygen-dependent HIF-a subunit stabilization and TGC formation are dependent on MAP2K1/2 activity. (A) Immunoblot of whole cell lysates obtained from TSCs differentiated in 2% or 21% O2, with and without U0126, following culture on CELLstartTM, for HIF-1a, -2a or a-Tubulin. (B) Immunoblot of whole cell lysates obtained from differentiated wild-type TSCs following culture on TC plastic in Fib-CM with and without U0126 with a HIF-1a antibody. (C) (D) Immunofluorescence microscopy of TSCs maintained on CELLstartTM following differentiation for 7 days under hypoxic conditions without and with U0126 (10 uM) using anti b-Catenin antibodies (green) (blue = Dapi). (E) Quantitaive RT-PCR analysis of Plf, Pl-I, Ctsq, 4311, Mash2, Tfeb and SynA expression following differentiation of wild-type TSCs cultured on CELLstartTM under hypoxic conditions without and with U0126. p values ,0.05 versus drug free control indicated by an asterisk. (F) Immunofluorescence microscopy using anti HA (red) and b-Catenin (green) antibodies of control TSCs differentiated following culture on CELLstartTM under 21% O2 following transient tranfection with constitutively active HA:MAP2K1 or under (G) 2% O2 following transient transfection with dominant negative HA:MAP2K1. (H, I) Immunofluorescence microscopy of wild-type TSCs differentiated following culture on TC plastic in Fib-CM in 21% O2 with and without U0126 with antibodies for HDAC2 (red) and E-Cadherin (green). (J) Northern blot analysis of lineage specific marker gene expression in wild-type TSCs maintained on TC plastic in Fib-CM and differentiated with and without U0126, compared with differentiated Arnt2/2 TSCs. doi:10.1371/journal.pone.0056949.g003

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF1a (R&D Systems, Minneapolis, MN), HIF-1a c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2a NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), a-Tubulin (NeoMarkers, Fremont, CA), Ac- a-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), b-catenin (Cell Signaling), cMYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Activity Assay, Western Blot, Immunofluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Control, Transfection, Dominant Negative Mutation, Northern Blot, Marker, Gene Expression

Journal: PloS one

Article Title: ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: Figure 6. Canonical vs non-canonical HIF target gene-expression in TS cells. (A) Electrophoretic mobility shift assay (EMSA) of differentiated control TGC nuclear extracts with and without 2 different anti-HIF-1a antibodies (H1) or a HIF-2a antibody (H2) (‘‘supershift’’ SS, NS, non-specific complexes.) (B) Schematic representation of full-length HIF-1a and HIF-2a, as well as versions lacking their DNA binding basic (b) domains (HLH, Helix- loop-helix, PAS, Per-Arnt-Sim, ODDD, oxygen-dependent degradation domain). (C) Immunoblot detection of stable HA-epitope tagged HIF-1a, HIF- 1aDb, HIF-2a and HIF-2aDb protein, as well as respective target gene protein products in Hif-1/2a2/2 TSCs. (D) Integrated densitometric quantification of HIF target gene protein products relative to Actin expression in each respective cell line. doi:10.1371/journal.pone.0056949.g006

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF1a (R&D Systems, Minneapolis, MN), HIF-1a c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2a NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), a-Tubulin (NeoMarkers, Fremont, CA), Ac- a-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), b-catenin (Cell Signaling), cMYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Targeted Gene Expression, Electrophoretic Mobility Shift Assay, Control, Binding Assay, Western Blot, Expressing

Journal: PloS one

Article Title: ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

doi: 10.1371/journal.pone.0056949

Figure Lengend Snippet: Figure 7. Canonical target gene-independent HIF-2 activity drives LIMK1 expression in TS cells via c-MYC interaction. (A) Immunoblot analysis of LIMK1 protein levels in Hif-1/2a2/2 TSCs stably reconstituted with full length HIF-1a or -2a, as well as versions lacking their basic domains. (B) Immunoblot analysis of LIMK1 and LIMK2 expression in control (+), Hif-1/2a2/2 (Hif2/2), and HIF-2a and HIF-2aDb reconstituted Hif-1/2a2/2 TSCs. Integrated densitometric analysis confirmed that both HIF-2a, as well as HIF-2aDb, restored LIMK1 expression to control levels in Hif-1/2a 2/2 TSCs. (C) Immunoprecipitation with an anti-HA antibody of HA-tagged HIF-2aDb followed by immunoblot with anti-HA, c-MYC, b- Catenin, a-Tubulin and GFP antibodies. (D) Schematic representation of E-box element identified within the Limk1 promoter. Chromatin immunoprecipitation (ChIP) analysis indicated specific binding of c-MYC and HA-tagged HIF-2aDb to this element. (E) Immunoblot analysis of LIMK1 protein levels in HIF-2aDb expressing Hif-1/2a2/2 TSCs without (-) or with (+) c-MYC inhibitor. Integrated densitometric analysis confirmed reduced expression of LIMK1 relative to a-Tubulin in drug treated cells. (F) Schematic representation of HIF-2a interacting with MYC:MAX heterodimers at the Limk1 promoter. doi:10.1371/journal.pone.0056949.g007

Article Snippet: The following antibodies were used for immunoblotting, EMSA, immunoprecipitaion and immunofluoresence staining: CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF1a (R&D Systems, Minneapolis, MN), HIF-1a c-terminal (Cayman Chemical, Ann Arbor, MI), HIF-2a NB 100–122 (Novus Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1 (pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epitomics, Burlingame, CA), a-Tubulin (NeoMarkers, Fremont, CA), Ac- a-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2 (Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal), PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz Biotechnology, Santa Cruz, CA), b-catenin (Cell Signaling), cMYC (SCBT), HA (Zymed, South San Francisco, CA), GFP (Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2 (Zymed).

Techniques: Activity Assay, Expressing, Western Blot, Stable Transfection, Control, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay

Journal: Cancer research

Article Title: Stromal cell-derived factor-1alpha and CXCR4 expression in hemangioblastoma and clear cell-renal cell carcinoma: von Hippel-Lindau loss-of-function induces expression of a ligand and its receptor.

doi: 10.1158/0008-5472.CAN-04-4406

Figure Lengend Snippet: Figure 5. Analysis of RCC lines. A, immunoblot assays of HIF-1a, HIF-2a, and HIF-1h expression. VHL-null CC-RCC lines (786-0 and RCC4) and subclones transfected with a VHL expression vector (786-0+VHL and RCC4T-3-13) were exposed to 20% or 1% O2 for 24 hours. B, analysis of VEGF, SDF-1, and CXCR4 mRNA expression by quantitative real-time reverse transcription-PCR (RT-PCR). For each pair of subclones, expression of each mRNA was determined relative to that observed in VHL-expressing cells exposed to 20% O2. Note that the range of values on the Y axis differs between graphs. Columns, mean; bars, SD. *, P < 0.05, compared with VHL-expressing subclone exposed to 1% O2; **, P < 0.0001. C, expression of SDF-1a protein in cells exposed to 20% or 1% O2 for 24 hours. Supernatants were analyzed by ELISA. Data are presented as pg SDF-1a protein/100 Ag total protein. Columns, mean; bars, SD. *, P < 0.05. D, immunoblot assay of CXCR4 expression in cells exposed to 20% or 1% O2 for 48 hours.

Article Snippet: Immunoblot assays were done using mouse mAb H1a67 against HIF-1a (8) or rabbit polyclonal antibodies against HIF-2a (NB-100-122, Novus Biologicals) at 1:500 dilution for 2 hours at room temperature.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay