Fig. 6

QKI5 binds to DNA and regulates target gene transcription. a Schematic diagram of RNase-ChIP assay that was applied to THP-1 cells. b ChIP-qPCR validation of QKI5 enrichment conducted on selected target genes with/without RNase treatment. Shown is the mean DNA enrichment relative to input; error bars indicate standard deviations around the means of three biological replicates. Asterisks indicate significant difference between indicated samples (***P value < 0.001, t test). c Nearby TF-binding motif prediction by SpaMo, which was used to identify putative hematopoiesis-related interaction partner TFs for QKI5. Top 5 of the identified TFs determined by e-value are shown in the figure. The number under axis represents best gap between QKI5 motif and indicated TF motifs. d Upper panel: Schematic diagram of QKI5’s interaction partner screening. The interacting proteins were identified by QKI5 co-immunoprecipitation combined with mass spectrometry analysis. Lower panel: GO functional enrichment analysis of identified QKI5 interacting proteins. e In vitro association of QKI5 with the CXCL2 promoter sequence identified by the DNA EMSA assay in which a 5′-biotin-labeled wild-type and mutant CXCL2 promoter probes were used. The corresponding unlabeled (“cold”) probes were used in the competitive assay. f Nuclear run-on assay in QKI5-knockdown THP-1 cells. Upper panel: Diagram of nuclear run-on (NRO) assay concept. Lower panel: NRO-qPCR validation of expression of selected target genes in shCtrl- or shQKI5-treated THP-1 cells. ACTB is a non-QKI5 target negative control. Error bars indicate standard deviations around the means of three biological replicates. Asterisks indicate a significant difference between the specified samples (*P value < 0.05, ***P value < 0.001, ns represents non-significant, t test). g RT-qPCR validation of the expression of selected target genes using exon-specific (left panel) and intron-specific (right panel) primers. qPCR was performed in Ctrl or QKI5/QKI5 M1-overexpressing (upper panel) and shCtrl- or shQKI5-treated (lower panel) THP-1 cells following by PMA induction for 48 h. Error bars indicate standard deviations around the means of three biological replicates. Asterisks indicate significant differences between the specified samples (*P value < 0.05, **P value < 0.01, ***P value < 0.001, n.a. represents not available, t test)