Loss of CD99 leads to increased protein synthesis and impaired proteostasis in HSCs. (A) In vivo OP-puro incorporation in HSCs. Representative histogram with MFI indicated for CD99 Gt/Gt and WT mice injected with OP-puro and PBS control (left). In vivo protein synthesis in CD99 Gt/Gt and WT HSCs (n = 9 and 8, respectively), normalized to the mean in WT HSCs (right). (B) Schematic for treatment of CD99 Gt/Gt and WT mice with Cy (4 mg) followed by 2 daily doses of G-CSF (5 μg). (C) CD99 cell surface protein expression on WT HSCs following Cy/G-CSF treatment. Representative histogram (left). CD99 MFI for Veh and Cy/G-CSF–treated mice (n = 3 and 6, respectively). (D) Representative gating based on DNA content to identify 2N (G0/G1) and >2N (S/G2/M) cells. (E) In vivo protein synthesis rates in CD99 Gt/Gt and WT HSCs (n = 12 per genotype) after Cy/G-CSF treatment relative to the mean in WT HSCs for cells in G0/G1 (left) and S/G2/M (right). (F) Schematic for ex vivo culture of HSCs followed by OP-puro analysis. (G) Protein synthesis in ex vivo cultured CD99 Gt/Gt and WT HSCs (n = 12 per genotype), normalized to the mean in WT HSCs for total cells (left), as well as those in G0/G1 (middle) and S/G2/M (right). (H) Schematic for treatment of CD99 Gt/Gt and WT mice with 3 daily doses of bortezomib (1 mg/kg) followed by western blot analysis of HSPCs. (I-M) Western blots examining ubiquitylated protein (I), p-eIF2α, eIF2α, p-S6, S6, and β-actin (K) in 3 × 104 sorted HSC/MPPs, CMPs, GMPs, and MEPs from CD99 Gt/Gt and WT mice treated with bortezomib. Quantification of western blots performed on independent mice for ubiquitylated protein (n = 3 per genotype) (J), p-eIF2α (n = 4 per genotype) (L), and S6 (n = 5 per genotype) (M), with measurements normalized to WT HSCs. Statistical significance was assessed using 2-tailed Student t tests (∗P < .05; ∗∗∗P < .005; +P < .0005). P values for selected nonsignificant trends are also shown; data are represented as mean ± standard deviation. Max, maximum; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; Veh, vehicle.
Figure 4.

Loss of CD99 leads to increased protein synthesis and impaired proteostasis in HSCs. (A) In vivo OP-puro incorporation in HSCs. Representative histogram with MFI indicated for CD99 Gt/Gt and WT mice injected with OP-puro and PBS control (left). In vivo protein synthesis in CD99 Gt/Gt and WT HSCs (n = 9 and 8, respectively), normalized to the mean in WT HSCs (right). (B) Schematic for treatment of CD99 Gt/Gt and WT mice with Cy (4 mg) followed by 2 daily doses of G-CSF (5 μg). (C) CD99 cell surface protein expression on WT HSCs following Cy/G-CSF treatment. Representative histogram (left). CD99 MFI for Veh and Cy/G-CSF–treated mice (n = 3 and 6, respectively). (D) Representative gating based on DNA content to identify 2N (G0/G1) and >2N (S/G2/M) cells. (E) In vivo protein synthesis rates in CD99 Gt/Gt and WT HSCs (n = 12 per genotype) after Cy/G-CSF treatment relative to the mean in WT HSCs for cells in G0/G1 (left) and S/G2/M (right). (F) Schematic for ex vivo culture of HSCs followed by OP-puro analysis. (G) Protein synthesis in ex vivo cultured CD99 Gt/Gt and WT HSCs (n = 12 per genotype), normalized to the mean in WT HSCs for total cells (left), as well as those in G0/G1 (middle) and S/G2/M (right). (H) Schematic for treatment of CD99 Gt/Gt and WT mice with 3 daily doses of bortezomib (1 mg/kg) followed by western blot analysis of HSPCs. (I-M) Western blots examining ubiquitylated protein (I), p-eIF2α, eIF2α, p-S6, S6, and β-actin (K) in 3 × 104 sorted HSC/MPPs, CMPs, GMPs, and MEPs from CD99 Gt/Gt and WT mice treated with bortezomib. Quantification of western blots performed on independent mice for ubiquitylated protein (n = 3 per genotype) (J), p-eIF2α (n = 4 per genotype) (L), and S6 (n = 5 per genotype) (M), with measurements normalized to WT HSCs. Statistical significance was assessed using 2-tailed Student t tests (∗P < .05; ∗∗∗P < .005; +P < .0005). P values for selected nonsignificant trends are also shown; data are represented as mean ± standard deviation. Max, maximum; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; Veh, vehicle.

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