Page 116 - 2021_02-Haematologica-web

M. Ichii et al.
AB
C
DEF
Figure 5. STAP-2-dependent inflammatory signals at the pre-B stage regulate B lymphopoiesis during hema- tologic stress. (A-D) RNA-Seq experiments with pre-B cells derived from wild-type (WT) and STAP-2 transgenic (Tg) bone marrow (BM) under steady-state as well as WT and STAP-2 knockout (KO) BM one month after trans- plantation (Tx) was conducted, and the genes were grouped according to a hierarchical clustering technique with Ingenuity Pathway Analysis based on patterns of expression. (A) Number of genes extracted. (B) A color- coded heatmap allows for the visualization of the differential gene expression data categorized by their func- tions in the Ingenuity Knowledge Base. Color bar indicates the z-score for each category: the strongest pre- dicted increases (orange square) correspond to z-score > 2, the strongest predicted decreases (blue square) correspond to z-score < -2. Gray and white colors indicate categories with a -2 < z-score < 2 and without z- score, respectively. Size reflects the associated log of the calculated P-value with s significance threshold of P=0.05 (Fisher's exact test). Larger squares indicate more significant overlap among the affected genes con- tained in the dataset. (C and D) Datasets were compared, and a bioinformative approach identified shared pathways regulated under hematologic stress and by STAP-2 expression. (E) The proportion of IL7 receptor α+ in the B-lineage progenitor subsets in WT, KO, and Tg BM (pro-B; B220+ CD43+ CD19+ IgM–, pre-B; B220+ CD43– CD19+ IgM) was analyzed with flow cytometry (n=6 in each). (F) Indicated transcript levels in pro-B cells (top panel) and pre-B cells (bottom panel) were evaluated with real-time polymerase chain reaction and nor- malized to the median of WT samples (WT, n=9; KO, n=4; Tg, n=8).
430
haematologica | 2021; 106(2)