nb 100 122  (Novus Biologicals)

Journal: Science Advances

Article Title: Functional EPAS1 / HIF2A missense variant is associated with hematocrit in Andean highlanders

doi: 10.1126/sciadv.adj5661

Figure Lengend Snippet: ( A ) Conservation of rs570553380 (A>G) in a subset of 100 Species Vertebrate Multiz Alignment & Conservation (fig. S6). Conservation scores (phyloP) of nucleotides across 100 vertebrates showed significant conservation at rs570553380 [−log( P ) = 6.02; positive scores indicate slower evolutionary change while negative scores indicate accelerated evolution, greater than expected due to neutral drift ]. ( B ) Location of rs570553380 within the ribbon diagram and crystal structure of HIF-2α (white) as bound to aryl hydrocarbon receptor nuclear translocator (ARNT) (teal) and the hypoxia response element (HRE, orange) (Protein Data Bank ID: 4ZPK). His 194 (purple), shown in sphere representation, extends into one of the protein-protein interfaces between HIF-2α and ARNT. ( C ) Relative expression of HIF-2α target genes Adrenomedullin ( ADM ), Basic Helix-loop-helix Family Member e40 ( BHLE40 ), Solute Carrier Family 2 Member 1 (GLUT1 ), Hypoxia Inducible Lipid Droplet Associated ( HILPDA ), Inhibitor of Growth Family Member 4 ( ING4 ), and Vascular Endothelial Growth Factor A ( VEGF ) (table S5) in HEK293T cells nontransfected (WTNT, n = 3) and transfected wild-type (WTT, n = 3), heterozygous (HET, n = 3), and homozygous (HOM, n = 1) for rs570553380 (A>G) exposed to hypoxia (1% O2) relative to normoxia (21% O2) for 12 and 24 hours. Gene expression was determined by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Two-way analysis of variance (ANOVA) shows a significant effect of rs570553380 on gene expression of ADM , BHLE40 , HILPDA , ING4 , and VEGF after 24-hour exposure to hypoxia, as well as a transfection effect on BHLE40 gene expression. The post hoc generalized linear model shows that increased expression of ADM , BHLE40 , HILPDA , ING4 , and VEGF from 12- to 24-hour exposure to hypoxia is significantly lower in HET compared to WTT. * P < 0.05 and ** P < 0.01.

Article Snippet: Membranes were blocked for 1 hour with 5% bovine serum albumin (no. A-7030, Sigma-Aldrich, USA), diluted in PBS with 0.1% Tween 20 at room temperature, and then incubated with primary antibodies against HIF-2α (Novus, no. NB-100-122, 1/250) and β-actin (Cell Signaling Technology, no. 4970S, 1/1000) overnight at 4°C, washed, and incubated with fluorescent secondary antibodies for imaging detection (Odyssey Infrared Imager, LiCOR).

Techniques: Expressing, Transfection, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

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: 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: 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