|
Figure 4
(A) qRT-PCR results confirming expression changes for RNA-Seq analysis of the KD of HIKER/LINC02228 versus controls. Top 5 upregulated genes are shown. qPCR was performed on iPSC-derived CD34+ cells. n = 4 subjects per group. *P < 0.05; ***P < 0.001. t tests were performed to compare expression levels of CMS (WT) with CMS (KD of HIKER) for each gene. (B) qRT-PCR results confirming the expression changes for RNA-Seq analysis of the KD of HIKER/LINC02228 versus controls. Top 5 downregulated genes are shown. qPCR was performed on iPSC-derived CD34+ cells. n = 4 subjects per group. *P < 0.05; ***P < 0.001. t tests were performed to compare the expression levels of CMS (WT) with CMS (KD of LINC02228/HIKER) for each gene. (C) Western blot confirmation of the top 5 downregulated candidates, CSNK2B, DXO, ZNRD1, PP1R11, and TAP2. Week 1 EBs (iPSC derived) were used in this assay as described in Methods. Left: representative image for each protein candidate. Right: summary of densitometric analysis of each protein with n = 3 for each group. *P < 0.05; ***P < 0.001; t test was performed to compare the protein levels with CMS (WT) and CMS (KD of LINC02228/HIKER). (D) Functional analysis of HIKER/LINC02228 as well as CSNK2B-OE-LINC02228-KD in iPSC-derived CD34+ cells using methylcellulose colony assay. With the OE of CSNK2B gene in the background of HIKER/LINC02228 KD, mean number of BFU-E colonies/CD34+ is increased, suggesting a critical function of this gene in the mechanism of action of HIKER/LINC02228. n = 4 subjects per group. ***P < 0.001. One-way ANOVA was performed in multiple comparisons followed by Tukey’s tests.