Extended Data Fig. 4: Protein expression levels of developmentally important transcription factors are sensitive to Runx factor dosage in Phase 1.
From: Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms
a, Representative histograms display Runx1 (top) or Runx3 (bottom) expression levels at 2 days (left) or 4 days (right) after transducing Runx1 OE or empty-control vectors. Cells were gated on live alternative lineage- infection+ cells, then separated as cKithi CD25− (DN1) and cKithi CD25+ (DN2a) populations. Graph summarizes results from 4–8independent experiments. Mann-Whitney test. *** P = 0.0002 for Runx1 gMFI on d2pi (n = 8), *** P < 0.0006 and ** P = 0.007 for Runx1 gMFI on d4pi (n = 6). For comparing Runx3 gMFI, * P = 0.0411 for d2pi (n = 6) and * P = 0.0286 for d4pi (n = 4). b, Runx1, Runx2, and Runx3 mRNA expression levels in Runx1 OE, Runx1/Runx3 knock-out (KO), and control cells. Runx transcript levels were measured using single cell RNA-seq described in Fig. 3. Differentially expressed gene statistics were calculated using a Wilcoxon test employing a Seurat algorithm. Bounds of box extend from 25th to 75th percentiles, median is indicated using horizontal line, and whiskers show 1.5⨯ the interquartile range. Points show outliers. c, Flow cytometry analysis strategy to test Runx1 dosage effects on regulating other TFs. Runx1 expression levels in control vs. Runx1 OE cells were compared using histogram overlay, then cells expressing low or mid-levels of Runx1 (Runx1low/mid) and cells with high levels of Runx1 (Runx1hi) were determined. d, TCF1, GATA3, and PU.1 expression levels in Runx1low/mid vs. Runx1hi cells in control and Runx1 OE cells were compared. Numbers inside the histograms are gMFI values. Graphs summarize 6 experiments. Mann-Whitney test. * P = 0.04 for Runx3, ** P = 0.0079 and * P = 0.0321 for TCF1, * P = 0.04 for GATA3, ** P = 0.0079 and * P = 0.0159 for PU.1, ns = not significant.
