Journal: bioRxiv

Article Title: NOTUM-MEDIATED WNT SILENCING DRIVES EXTRAVILLOUS TROPHOBLAST CELL LINEAGE DEVELOPMENT

doi: 10.1101/2024.02.13.579974

Figure Lengend Snippet: Upstream regulation of NOTUM in EVT cells. ( A ) Time course of EPAS1 and NOTUM from stem state (Day 0) and days 3, 6, and 8 of EVT cell differentiation. Graphs represent mean values ± SEM, one-way ANOVA, Tukey’s post hoc test, n=4, **P<0.01, ****P<0.0001 (B) Immunofluorescence of EPAS1/HIF2A (red) and NOTUM (green) in TS cells induced to differentiate into EVT cells following transduction with lentivirus containing control shRNA or EPAS1 shRNA. DAPI positive nuclei labeling is shown in grey. Merged fluorescence images EPAS1, NOTUM and DAPI. Scale bar: 500 μm. Effects of EPAS1 shRNA knockdown on EPAS1 and NOTUM expression assessed by ( C ) RT-qPCR, graphs represent mean values ± SEM, n=3, unpaired t -test ****P<0.0001 and ( D ) western blotting.

Article Snippet: After transfer, membranes were blocked with 5% non-fat milk/BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed with primary antibodies to NOTUM (1:1500, SAB3500082, Sigma-Aldrich), HLA-G (1:1000. ab52455, Abcam), total CTNNB1 (1:1000, clone D10A8, 8480, Cell Signaling), active-CTNNB1 (1:1000, clone 8E7, 05-665, Sigma-Aldrich), phosphorylated CTNNB1 (1:1000, Ser33/37/Thr41, 9561 Cell Signaling), EPAS1 (1:300, 66731-1-Ig, Proteintech) and glyceraldehyde-3-phosphate dehydrogenase (ab9485; Abcam) overnight at 4°C.

Techniques: Cell Differentiation, Immunofluorescence, Transduction, Control, shRNA, Labeling, Fluorescence, Knockdown, Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Hypoxia-induced histone methylation and NF-κB activation in pancreas cancer fibroblasts promote EMT-supportive growth factor secretion

doi: 10.1101/2025.01.30.635486

Figure Lengend Snippet: A) 0082T cells were transfected with siRNA against HIF1A , siRNA against EPAS1 (gene encoding HIF2α), or a control siRNA and cultured in 1% O 2 for 4 hr before lysing. Western blotting was performed for the indicated proteins. Image representative of n=3. B) and C) 0082T CAFs were transfected with the siRNAs described in (A) and grown for 120 hr at 1% O 2 . Immunofluorescence for HGF or IGF-2 was performed. Data are presented as mean ± SEM. Throughout the panels, ** p < 0.01, *** p < 0.001.

Article Snippet: siRNAs targeting HIF1A (sc-35561) or EPAS1 (sc-35316) and a control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Cell Culture, Western Blot, Immunofluorescence

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: (A) Knockdown of HIF-2α downregulates B-Myb protein levels in pVHL-deficient 786-O cells. Cell lysates of control-, B-Myb-, or HIF-2α-knockdown 786-O cell lines were immunoblotted with anti-B-Myb or anti-HIF-2α antibodies to determine protein levels of B-Myb and HIF-2α, respectively. Hsp90 was used as loading control. Representative data from three independent experiments are shown. (B) Knockdown of HIF-2α does not affect B-Myb mRNA levels. Total RNA was isolated from control- or HIF-2α-knockdown 786-O cells and analyzed by quantitative RT-PCR analysis. Data represent the mean ± SD of three independent experiments. (C) B-Myb downregulation by HIF-2α knockdown in pVHL-deficient cells is proteasome-dependent. Control or HIF-2α-knockdown 786-O cells were cultured in the presence or absence of MG132 (10 μM for 6 h) and subjected to immunoblotting with anti-B-Myb or anti-HIF-2α antibodies. Hsp90 was used as loading control. Representative data from three independent experiments are shown. (D) Stabilization of HIF-2α and B-Myb by MG132 and lactacystin. 786-O cells were cultured in the presence or absence of MG132 (10 μM for 6 h) or lactacystin (5 μM for 6 h) and immunoblotted with anti-B-Myb or anti-HIF-2α antibodies. Hsp90 was used as loading control. Representative data from three independent experiments are shown. (E) Stability of B-Myb with or without HIF-2α. Control 786-O cells or HIF-2α knockdown cells were exposed to cycloheximide (CHX, 50 μg/ml) for 30 or 60 min. The lysates were subjected to western blot with antibodies against B-Myb (short and long exposure are shown), HIF-2α, or Hsp90. Hsp90 was used as loading control. Representative data from three independent experiments are shown. (F) The intensities of the B-Myb bands in (D) were normalized to those of the corresponding Hsp90 bands and plotted as ratio of the normalized value against control cells at 0 min. Data are presented as the mean ± SD of three independent experiments. (G) The intensities of B-Myb bands in (D) at 0 min were set as 1.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Knockdown, Control, Isolation, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: (A) Control- or 3×FLAG-pVHL-expressing 786-O cell lines were cultured in the presence or absence of CoCl 2 (400 μM) for 7 h, and cell lysates were immunoblotted with anti-B-Myb, anti-HIF-2α, or anti-pVHL antibodies. Forced expression of 3×FLAG-pVHL downregulates HIF-2α and B-Myb protein levels under normoxic conditions, and CoCl 2 treatment rescues HIF-2α and B-Myb protein levels. Hsp90 was used as loading control. Representative data from three independent experiments are shown. (B) Knockdown of HIF-2α suppresses the accumulation of B-Myb under hypoxic-like conditions. Control or pVHL-expressing 786-O cell lines were transfected with non-specific or HIF-2α-targeting siRNA and cultured for 2 days, and cell lysates were immunoblotted with anti-B-Myb, pVHL, or anti-HIF-2α antibodies. Cells were incubated in the presence or absence of CoCl 2 (400 μM) for 7 h before they were harvested. Hsp90 was used as a loading control. Representative data from three independent experiments are shown.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Control, Expressing, Cell Culture, Knockdown, Transfection, Incubation

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: (A) Endogenous interaction between HIF-2α and B-Myb. 786-O cell lysates were immunoprecipitated (IP) with an anti-HIF-2α antibody and immunoblotted with anti-B-Myb or anti-HIF-2α antibodies. Representative data from three independent experiments are shown. (B) Cytoplasmic and nuclear fractions were prepared from 786-O cell lysates and utilized for co-immunoprecipitation assays as in (A). Endogenous HIF-2α and B-Myb are enriched in the nucleus but also localize to the cytoplasm. An interaction between HIF-2α and B-Myb was detected in the nuclear fraction but not the cytoplasmic fraction. Hsp90 and Lamin B1 were used as cytoplasmic and nuclear protein markers, respectively. Representative data from two independent experiments are shown.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: (A) Schematic representation of the HIF-2α deletion mutants used in this study. (B) Co-immunoprecipitation between full-length B-Myb and deletion mutants of HIF-2α. Wild-type or deletion mutants of 3×FLAG-HIF-2α were coexpressed with full-length 3×HA-B-Myb in HEK293T cells, and cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody and immunoblotted with anti-FLAG or anti-HA antibodies. Deletion mutants of HIF-2α containing a central region between residues 436 to 600, which fully spans the transactivation domain and partially spans the oxygen-dependent degradation domain, interact with full length B-Myb. Representative data from three independent experiments are shown.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: (A) Schematic representation of the B-Myb deletion mutants used in this study. (B) Co-immunoprecipitation between full-length HIF-2α and deletion mutants of B-Myb. Wild-type or deletion mutants of 3×HA-B-Myb were coexpressed with full length 3×FLAG-HIF-2α in HEK293T cells, and cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody and immunoblotted with anti-FLAG or anti-HA antibodies. Deletion mutants of B-Myb containing the C-terminal region (starting at residue 468), which comprises the conserved region and the negative regulatory domain, interact with full-length HIF-2α. Representative data from three independent experiments are shown.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Residue

Journal: PLoS ONE

Article Title: Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2

doi: 10.1371/journal.pone.0175593

Figure Lengend Snippet: Relative mRNA levels of AGXT, RBPJL, CFB, FKBP1B, and SEPT6 were quantified after non-specific control knockdown or knockdown of B-Myb or HIF-2α in 786-O cells. Total RNA was purified from parental 786-O cells, non-specific control, or B-Myb- or HIF-2α-knockdown 786-O cells and analyzed by quantitative RT-PCR. Data represent the mean ± SD of four independent experiments.

Article Snippet: Antibodies against the following were used in this study: FLAG (1 μg/mL; M2, Sigma-Aldrich), HA (1 μg/mL; 12CA5, Sigma-Aldrich), B-Myb (1 μg/mL; sc-81192, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HIF-2α (1 μg/mL; sc-13596, Santa Cruz Biotechnology).

Techniques: Control, Knockdown, Purification, Quantitative RT-PCR