Gem/HU causes replication stress in HSCs and induces the expression of IEGs. (A) Representative flow plots show the gating strategies for Lin⁻, HPCs, LSK, GMP, CMP, MEP, CD34⁻LSK, and HSCs (CD48⁻CD150⁺LSK or CD34⁻CD135⁻LSK). (B) HSCs (CD48⁻CD150⁺LSK) from WT C57BL/6J mice were cultured in vitro with Gem or HU for 48 h. The charts show the cell viability of HSCs with the treatment of a range of doses of Gem (left) or HU (right) using an MTT assay (n = 2 samples per group). (C) Representative flow plots of p-ATR staining in HSCs. The right bar graphs show the mean fluorescence intensity (MFI) of p-ATR in HSCs (n = 2 samples per group). (D) qRT-PCR analysis and the bar graph shows the expression of Egr1, Jun, Fos, and Rpl27 in HSCs under indicated conditions (related to Fig. 1 C) (n = 4 samples per group). (E) Representative volcano plot from ATAC-seq analysis between the DMSO and Gem/HU-treated HSCs. All peaks that were significantly changed (greater than twofold change, P < 0.05) appear as red dots. (F) Scatter plot of log₂ fold changes of genome-wide ATAC (x axis) and H3K27AC (y axis) signal. Each point represents one peak. The data presented in panels A–D are representative of two independent experiments. The results shown in panels B–D are shown as mean ± SD. Statistical analyses were performed using a two-tailed unpaired Student’s t test for panel D. P values are presented as follows: ***P < 0.001.