Figure 8.
Fancd2 deficiency causes DEK accumulation in HSCs through the p38–ATF2 pathway. (A) Summary of the p-p38/p-ATR–ATF2–DEK axis. (B) Experimental schematic. Freshly sorted HSCs (CD48−CD150+LSK) from WT C57BL/6J mice were cultured in vitro with the treatment of DMSO, HU (100 µM), or the ATRi VE-821 (1 µM) for 24 h, followed by flow cytometry and qRT-PCR analysis. (C) The bar graph shows the MFI of p-ATR and p-p38 in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (D) The bar graphs show the MFI of p-ATF2 (Thr69/71) (left) and p-ATF2 (Ser490/498) (right) in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (E) qRT-PCR analysis and the bar graph shows the expression of the DEK gene in HSCs (n = 4 samples per group). (F) Experimental schematic. Freshly sorted HSCs (CD48−CD150+LSK) from Fancd2+/+ and Fancd2−/− mice were cultured in vitro with DMSO or HU (100 µM) for 24 h, followed by flow cytometry analysis. (G and H) The charts show the MFI of p-ATR and p-p38 (G); p-ATF2 (Thr69/71) and p-ATF2 (Ser490/498) (H) in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (I) Experimental schematic. HEK293T cells were transfected with the vector pGL3-DEK–promoter luciferase and a control vector phRL-SV40, along with the pMSCV (control) or ATF2 expression constructs. Next, HEK293T cells were treated with a range of doses of VE-821 (ATRi) and doramapimod (p38i) after transfection, followed by luciferase activity analysis. (J) The luciferase activity is presented as a heatmap with a relative value (n = 3 samples per condition). (K) Representative flow plots of p-ATF2 (Thr69/71) staining in HSCs. The right bar graph shows the MFI of p-ATF2 (Thr69/71) (n = 5 samples per group). Freshly sorted HSCs (CD48−CD150+LSK) from Fancd2+/+ and Fancd2−/− mice were cultured in vitro with DMSO, ATRi (1 µM), or p38i (0.5 µM) for 24 h. (L) The bar graph shows the MFI of dichlorofluorescein (DCF) in HSCs (n = 5 samples per group). Freshly sorted HSCs were cultured in vitro with DMSO, or HU (100 µM), or N-acetylcysteine (NAC) (5 mM) for 24 h, followed by staining of dichlorodihydrofluorescein diacetate (DCFH-DA) probe. (M) qRT-PCR analysis and the bar graph shows DEK expression in HSCs under indicated conditions (n = 4 samples per group). The data presented in panels C–M are representative of two independent experiments. The results shown in panels D, E, and K–M are shown as mean ± SD, and panel J are shown as heatmaps with mean values. Statistical analyses were performed using a two-tailed unpaired Student’s t test for panels C, G, and H and one-way ANOVA followed by Tukey’s post hoc test for panels D, E, and K–M. P values are presented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. Refer to the image caption for details.

Fancd2 deficiency causes DEK accumulation in HSCs through the p38–ATF2 pathway. (A) Summary of the p-p38/p-ATR–ATF2–DEK axis. (B) Experimental schematic. Freshly sorted HSCs (CD48−CD150+LSK) from WT C57BL/6J mice were cultured in vitro with the treatment of DMSO, HU (100 µM), or the ATRi VE-821 (1 µM) for 24 h, followed by flow cytometry and qRT-PCR analysis. (C) The bar graph shows the MFI of p-ATR and p-p38 in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (D) The bar graphs show the MFI of p-ATF2 (Thr69/71) (left) and p-ATF2 (Ser490/498) (right) in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (E) qRT-PCR analysis and the bar graph shows the expression of the DEK gene in HSCs (n = 4 samples per group). (F) Experimental schematic. Freshly sorted HSCs (CD48−CD150+LSK) from Fancd2+/+ and Fancd2−/− mice were cultured in vitro with DMSO or HU (100 µM) for 24 h, followed by flow cytometry analysis. (G and H) The charts show the MFI of p-ATR and p-p38 (G); p-ATF2 (Thr69/71) and p-ATF2 (Ser490/498) (H) in HSCs using flow cytometry staining and analysis (n = 5 samples per group). (I) Experimental schematic. HEK293T cells were transfected with the vector pGL3-DEK–promoter luciferase and a control vector phRL-SV40, along with the pMSCV (control) or ATF2 expression constructs. Next, HEK293T cells were treated with a range of doses of VE-821 (ATRi) and doramapimod (p38i) after transfection, followed by luciferase activity analysis. (J) The luciferase activity is presented as a heatmap with a relative value (n = 3 samples per condition). (K) Representative flow plots of p-ATF2 (Thr69/71) staining in HSCs. The right bar graph shows the MFI of p-ATF2 (Thr69/71) (n = 5 samples per group). Freshly sorted HSCs (CD48−CD150+LSK) from Fancd2+/+ and Fancd2−/− mice were cultured in vitro with DMSO, ATRi (1 µM), or p38i (0.5 µM) for 24 h. (L) The bar graph shows the MFI of dichlorofluorescein (DCF) in HSCs (n = 5 samples per group). Freshly sorted HSCs were cultured in vitro with DMSO, or HU (100 µM), or N-acetylcysteine (NAC) (5 mM) for 24 h, followed by staining of dichlorodihydrofluorescein diacetate (DCFH-DA) probe. (M) qRT-PCR analysis and the bar graph shows DEK expression in HSCs under indicated conditions (n = 4 samples per group). The data presented in panels C–M are representative of two independent experiments. The results shown in panels D, E, and K–M are shown as mean ± SD, and panel J are shown as heatmaps with mean values. Statistical analyses were performed using a two-tailed unpaired Student’s t test for panels C, G, and H and one-way ANOVA followed by Tukey’s post hoc test for panels D, E, and K–M. P values are presented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

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