Figure 2.

NOTCH and hypoxia pathways cooperate in human HSPCs

(A) Heatmap of leading edge subset (left) and enrichment plot (right) showing relative expression of genes associated with the NOTCH pathway in CD34⁺ cells cultured in normoxia (21% O₂) or hypoxia (1%–2% O₂) with optimized densities of DXI. NES, normalized enrichment score; FDR, false discovery rate.

(B) Top: western blot analysis confirming exogenous HIF-1α expression in human CD34⁺ cells cultured under normoxic conditions with DXI after transduction with a mutant HIF-1α-expressing lentiviral vector. A control group transduced with a GFP-expressing lentiviral vector is shown for comparison. β-Tubulin was used as loading control. Bottom: HES-1 gene expression measured by qRT-PCR (n = 9 technical replicates, from three donors). Results indicate the fold increase in HES-1 levels normalized to a GFP-transduced control group.

(C) Representative ImageStream images of human CD34⁺ cells cultured under normoxic or hypoxic conditions in the absence or with optimized concentrations of DXI. BF, bright-field; NICD, NOTCH1 intracellular domain (ICD); DAPI, 4′,6-diamidino-2-phenylindole.

(D) Quantification of the mean fluorescence intensity (MFI) of NOTCH1 ICD in 10,000 cells/condition.

(E) Representative high-resolution images of individual cells assessed by confocal microscopy. The bottom panel displays a colocalization channel generated with Imaris imaging software.

(F) Quantification of NOTCH1 ICD and HIF-1α signal colocalization in individual cells for each condition by the Pearson's correlation coefficient (n = 20 cells/condition).

In (B), (D), and (F), data are displayed as mean ± SEM. Two-sided (B and F) and one-sided (D) unpaired t test were used. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗∗p ≤ 0.0001. See also Figures S2 and S3; Table S2.