Journal: bioRxiv

Article Title: Regulation of BCL11A DNA binding and expression in human erythrocyte precursor HUDEP-2 cells

doi: 10.64898/2026.02.06.704516

Figure Lengend Snippet: Inhibition of epigenetic silencing marks induces fetal globin expression in HUDEP-2 cells. ( A ) Schematic illustration of the HUDEP-2 cell culture differentiation workflow, showing compound treatment at day 0 (stage 0), day 3 (stage 1), and day 7 (stage 2). Cells at day 3 (stage 1), day 7 (stage 2), or day 9 (stage 3) were harvested for analysis. ( B-D ) HUDEP-2 cells were harvested at stage 1 after treatment with increasing concentrations of ( B ) GSK3484862 (GSK), ( C ) EML741 (EML), or ( D ) FTX6058 (FTX) for 3 days. Lower panels show the percentage of live cells quantified using a Bio-Rad cell counter and normalized to the DMSO control. Data represent mean ± SD from N=4 biological replicates. ( E-H ) HUDEP-2 cells were treated with each inhibitor (2 μM EML, 1 μM GSK, and 0.1 μM FTX). After 9 days of treatment, cells at stage 3 were harvested for western blot analysis. ( I ) Partial β-globin locus in chromosome 11 is shown above the relative mRNA expression levels (qPCR) in stage 2 HUDEP-2 cells. IP08 (protein transporter Importin 8) is used as a reference housekeeping gene. Data represent mean ± SD from N=3 independent experiments. (J) Relative mRNA expression levels of BCL11A. ( K-L ) Western blots and corresponding protein gel showing changes of H3K27me3 and BCl11A of HUDEP-2 cells at stages 1, 2 and 3.

Article Snippet: Chemical compounds GSK3484862 (HY-135146), FTX6058 (HY-139400), pomalidomide (HY-10984) and lenalidomide (HY-A0003) were purchased from MedChemExpress.

Techniques: Inhibition, Expressing, Cell Culture, Control, Western Blot

Journal: bioRxiv

Article Title: Regulation of BCL11A DNA binding and expression in human erythrocyte precursor HUDEP-2 cells

doi: 10.64898/2026.02.06.704516

Figure Lengend Snippet: (A) Relative mRNA expression levels of IGF2BP1 , HIC2 , and LIN28B after compound treatment. ( B-C ) ChIP-qPCR analysis of H3K27me3 (panel B) and H3K4me3 (panel C) at gene loci (Supplementary Figure S3) after 7-day FTX treatment (stage 2 cells) normalized to H3. Error bars represent SEM from three different experiments compared to DMSO-treated. (D) Genome-wide DNA demethylation is restricted to GSK3484862 treatment . Violin plots showing the distribution of DNA methylation levels in HUDEP-2 cells that are not differentiated (stage 1; NonDiff) and differentiated cells at stage 3 treated with DMSO, GSK, and FTX. Boxplots within the violin plots indicate the first quartile, median, and third quartile methylation values. GSK treatment results in a pronounced global reduction in DNA methylation. ( E ) Genome browser view of the ∼50-kb region on chromosome 11 covering the β-globin locus showing DNA methylation levels (reflected by line heights) at individual CpG sites. ( F ) Number of individual CpG sites that are hypo- or hypermethylated in each condition relative to DMSO. Differentially methylated CpG sites were defined by an absolute methylation difference >25% and a q-value < 0.01. Although far less extensive than with GSK treatment, FTX treatment also induces detectable changes in DNA methylation. ( G ) Genome browser views of the HBG1 (right) and HBG2 (left) loci, showing DNA methylation levels (reflected by line heights) at individual CpG sites upstream of, within, and downstream of each gene.

Article Snippet: Chemical compounds GSK3484862 (HY-135146), FTX6058 (HY-139400), pomalidomide (HY-10984) and lenalidomide (HY-A0003) were purchased from MedChemExpress.

Techniques: Expressing, ChIP-qPCR, Genome Wide, DNA Methylation Assay, Methylation