Figure 2 | Scientific Reports

Figure 2

From: Development of Small Molecule MEIS Inhibitors that modulate HSC activity

Figure 2

(A) MEIS luciferase reporter assay. Schematic showing luciferase reporter with MEIS binding motif TGACAG in the regulatory regions that is used to test activity of Meis1 protein. (B) MEIS-Luc-Reporter 1. Two small molecules named MEISi-1 and MEISi-2 demonstrated inhibition of MEIS-p21-luciferase reporter (luciferase reporter with well characterized MEIS1 binding motif from p21 regulatory region) up to 90% at 0.1 µM concentration. (C) MEIS-Luc-Reporter 2. MEISi-1 and MEISi-2 demonstrated inhibition of MEIS-HIF-luciferase reporter (luciferase reporter with well characterized MEIS1 binding motif from Hif-1α enhancer region). (D) Effect of MEISi treatments on the Lin- cells ex vivo. Lin- cells were isolated and treated with corresponding MEIS inhibitors and doses. Post 7 days of treatment, cells were stained with Hoechst 33342 and counted using automated cell imaging platform. Hematopoietic stem and progenitor cell expansion post MEISi treatments. Lin- cells were isolated and treated with corresponding MEIS inhibitors and doses. Post 7 days of treatment, HSCs were stained with corresponding surface antigens (E) c-Kit+, (F) Sca1+, (G) LSK and (H) LSKCD34low and determined HSC content by flow cytometry. (I) Colony forming assay. Lin- cells were treated with MEIS inhibitors for seven days. Then, methocult based CFU assays were performed. Types of colonies formed post 12 days were quantified and illustrated as CFU-GEMM, CFU-G/M/GM and BFU-E colonies. (J) Expression of MEIS target genes, HIFs and CDKIs post MEISi treatments in Lin- Cells. Lin- cells were treated in vitro with MEIS inhibitors and collected RNA post 3 days of treatment for analysis of gene expression. Note that MEIS1 is known to transcriptionally regulate expression of Hif-1α, Hif-2α, p15, p19ARF and p21. n = 3, *p < 0.05.

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