Figure 6.

Figure 6.

Accelerated differentiation by TMEM187 deficiency hastens cellular senescence and leads to being phagocytosed by macrophages. (A-B) Expression of apoptosis-related proteins (BCL2, BAX, Cleaved CASP3), detected by western blotting, in K562 cells before and after 7-day induction (A), quantified in (B). ACTB and GAPDH were used as internal controls. One-way ANOVA was used for significance. n = 3/group. ∗P < .05; ∗∗P < .01. (C) Flow cytometry with annexin V-PI staining for apoptotic analysis. (D) Sensitivity of K562 WT and KO cells to hypotonic solutions after 7-day induction, measured by lactate dehydrogenase (LDH) release. (E) β-Galactosidase activity assays, showing senescent cells indicated by arrows. (F-H) Phagocytosis assays in K562 WT and KO cells. A flowchart of the phagocytosis assays (F). In brief, viable K562 cells were stained with Hoechst 33342, following 7-day induction. After washing, the cells were coincubated with prestarved primary bone-marrow-derived macrophages for 6 hours to assess phagocytosis. The phagocytosed cells exhibiting Hoechst 33342-positive signals were imaged after thorough rinsing with 1× PBS (G) and quantified (H). t test was used for significance. n = 16/group. ∗∗∗∗P < .0001. (I) β-Galactosidase activity assays in CD34⁺ cells after 6-day induction by EPO addition.