Figure 6.

Ripk3 signaling induces protein synthesis and cellular senescence in HSCs by stimulating PDC-mediated OXPHOS and attenuating mitochondrial ISR

(A and B) The basal activity of eIF2a-Atf4 signaling in WT, Ripk3^−/−, Mlkl^−/−, and Tnfr^−/− HSCs was examined by flow cytometry for p-eIF2a levels (mean fluorescence intensity, MFI) (A) and qRT-PCR for the expression of Atf4 target genes (B).

(C–J) WT, Ripk3^−/−, Mlkl^−/−, and Tnfr^−/− mice were irradiated with X-rays, 1.75 Gy weekly × 4. LSK cells were collected from mouse BM 1 month after the final IR. The activity of eIF2a-Atf4 signaling was examined by flow cytometry for p-eIF2a levels (C), western blotting for ATF4 expression (D), and qRT-PCR for the expression of Atf4 target genes (E). The activity of Perk-ER stress signaling was examined by p-Perk levels (F). The activity of OMA1 was examined by western blotting for OPA cleavage (G); OXPHOS was examined by OCR (H); and mitochondrial mass and mtROS were examined by MitoTracker green staining (I) and MitoSOX red staining (J). Data in (A), (C), (F), (I), and (J) show one of the three biological triplicate experiments. ^∗∗p < 0.01 compared with LK in (A) or WT and Mlkl^−/− mice.