Fig. 4

LINC00173 is a novel regulator of granulocytic development. a–d RNAi (shRNA)-mediated knockdown of LINC00173 in CD34⁺ HSPCs in vitro. a Granulocytic in vitro differentiation (day 14). Upper panel: May-Grünwald Giemsa (MGG) staining; scale bars 20 µm. Middle panel: neutrophil peroxidase (POX) staining; scale bar 20 µm. Lower panel: flow cytometric analysis of CD66b and CD13 surface marker expression. The bar graphs (right) show the mean ± s.d. of three independent experiments. b Percentage of bead-positive cells in a phagocytosis assay. The histogram depicts the fluorescence intensity. c Number of BFU-E and CFU-G/M (CFU-my) in methylcellulose-based CFU-assays normalized to the non-silencing shRNA control (ctrl). d Number of shRNA-transduced cells during granulocytic in vitro differentiation normalized to day 0. e Ratio of RFP657⁺ sgRNA-transduced vs. untransduced cells relative to the non-targeting control (sgRNAs against luciferase), using monoclonal NB4 cell lines stably expressing dCas-KRAB (n = 6). f Cytoplasmic to nuclear ratio of LINC00173 determined by qRT-PCR on fractionated RNA from THP-1 cells. g RNA FISH with tiled biotinylated probes in THP-1 cells; scale bars 10 µm. h GSEA results for 52 hematopoiesis-associated gene sets upon LINC00173 knockdown in CD34⁺ HSPCs. The plot shows normalized enrichment scores (NES) against nominal P-values of the normalized enrichment score¹⁸, dotted line: P = 0.05. i Heatmap showing expression of leading-edge genes from the “Fischer_DOWN IN SEVERE APLASTIC ANEMIA” gene set across the human DMAP data set¹². j RIP in NB4 cells using two different antibodies, followed by qRT-PCR to detect binding of EZH2 to LINC00173. Data are presented as percent of input in comparison to B2M. k ChIP-seq density heatmaps for H3K27me3 in promoter regions of leading-edge genes from the indicated gene sets upon LINC00173 knockdown. shRNA-transduced CD34⁺ HSPCs (sh-L173 and sh-CTRL) are compared to untransduced and uncultured CD34⁺ HSPCs. Clusters of promoters with differential H3K27me3 marks are highlighted. Representative stem cell-specific genes are shown (right). Data are presented as mean ± s.d. a–f, j, or s.e.m. c, e. *P < 0.05; **P < 0.01; ns not significant; P-values were calculated using one-way ANOVA with Dunnett’s post hoc test