Figure 4
Figure 4. Hif1α contributes to β-catenin transcription directly. (A) Flow cytometric analysis of β-catenin protein levels. 7TGC-transduced mouse NOTCH1-ΔE leukemias were transduced with Hif1αTM (normoxia-stable, active Hif1α P402A/P577A/N813A triple mutant) or empty virus control, and cultured in vitro for 2 days on MS5 stromal feeders. Leukemia cells carrying the Hif1αTM or empty lentivirus were FACS-sorted by the linked hCD8 marker, then stained for intracellular β-catenin protein and analyzed by flow cytometry. Results depicted are representative of 3 different leukemias. (B) Flow cytometric analysis for Wnt reporter GFP expression. 7TGC-transduced leukemia cells were transduced with Hif1αTM/hCD8 or empty lentivirus and cultured in vitro for 2 days on MS5 stromal feeders. Cells were then harvested and assayed by flow cytometry. Gated Cherry+ hCD8+ leukemia cells are shown. Results depicted are representative of 3 different leukemias. (C) Real-time quantitative reverse transcription PCR analysis of β-catenin mRNA levels. The 144CLP mouse T-ALL cell line and 2 independent mouse leukemias were transduced with Hif1αTM or empty virus control, FACS-sorted for the linked GFP marker 2 days later, and then lysed for RNA. Mean ± SD values of triplicate assays are plotted. *P < .05; ***P < .001 (Student t test). (D) Map of the mouse Ctnnb1 locus with putative Hif1α binding sites indicated with asterisks. Primer pairs used for quantitative local ChIP assays are indicated by arrowheads with associated map coordinates. (E) Alignment of putative Hif1α binding sites within the mouse Ctnnb1 locus. Six regions containing Hif1α consensus binding sites are shown with the core RCGTGC motif indicated by underlined text in large font. Map coordinates corresponding to the positions assayed by local ChIP-PCR in (F) are indicated by underlined numbers in large font. Locus coordinates are from the GRCm38/mm10 genome assembly. (F) ChIP for Hif1α occupancy over the mouse Ctnnb1 locus. The 144CLP cell line was transduced with Myc epitope-tagged Hif1αTM or empty virus control, and FACS-sorted for the linked GFP marker 3 days later. Sheared chromatin was immunoprecipitated with anti-Myc tag (TAG) or IgG control antibody. Quantitative PCR was performed using primer pairs flanking putative Hif1α binding sites as indicated in (C). Data were normalized to their respective input DNA controls. Mean ± SD values of triplicate assays are plotted. **P < .01; ****P < .0001 (Student t test). FSC, forward light scatter.

Hif1α contributes to β-catenin transcription directly. (A) Flow cytometric analysis of β-catenin protein levels. 7TGC-transduced mouse NOTCH1-ΔE leukemias were transduced with Hif1αTM (normoxia-stable, active Hif1α P402A/P577A/N813A triple mutant) or empty virus control, and cultured in vitro for 2 days on MS5 stromal feeders. Leukemia cells carrying the Hif1αTM or empty lentivirus were FACS-sorted by the linked hCD8 marker, then stained for intracellular β-catenin protein and analyzed by flow cytometry. Results depicted are representative of 3 different leukemias. (B) Flow cytometric analysis for Wnt reporter GFP expression. 7TGC-transduced leukemia cells were transduced with Hif1αTM/hCD8 or empty lentivirus and cultured in vitro for 2 days on MS5 stromal feeders. Cells were then harvested and assayed by flow cytometry. Gated Cherry+ hCD8+ leukemia cells are shown. Results depicted are representative of 3 different leukemias. (C) Real-time quantitative reverse transcription PCR analysis of β-catenin mRNA levels. The 144CLP mouse T-ALL cell line and 2 independent mouse leukemias were transduced with Hif1αTM or empty virus control, FACS-sorted for the linked GFP marker 2 days later, and then lysed for RNA. Mean ± SD values of triplicate assays are plotted. *P < .05; ***P < .001 (Student t test). (D) Map of the mouse Ctnnb1 locus with putative Hif1α binding sites indicated with asterisks. Primer pairs used for quantitative local ChIP assays are indicated by arrowheads with associated map coordinates. (E) Alignment of putative Hif1α binding sites within the mouse Ctnnb1 locus. Six regions containing Hif1α consensus binding sites are shown with the core RCGTGC motif indicated by underlined text in large font. Map coordinates corresponding to the positions assayed by local ChIP-PCR in (F) are indicated by underlined numbers in large font. Locus coordinates are from the GRCm38/mm10 genome assembly. (F) ChIP for Hif1α occupancy over the mouse Ctnnb1 locus. The 144CLP cell line was transduced with Myc epitope-tagged Hif1αTM or empty virus control, and FACS-sorted for the linked GFP marker 3 days later. Sheared chromatin was immunoprecipitated with anti-Myc tag (TAG) or IgG control antibody. Quantitative PCR was performed using primer pairs flanking putative Hif1α binding sites as indicated in (C). Data were normalized to their respective input DNA controls. Mean ± SD values of triplicate assays are plotted. **P < .01; ****P < .0001 (Student t test). FSC, forward light scatter.

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