Figure 6.
Screening and applying of LDHA inhibitors in thrombocytopenia. (A) Coimmunoprecipitation (Co-IP) of Flag-LDHA and HA-eEF2 in HEK-293T treated with LDHA inhibitors. OXA = sodium oxamate 1 mM; FX11 = FX11 20 μM; STP = stiripentol 100 μM; GNE = (R)-GNE-140 20 μM; GSK = GSK 2837808A 20 nM; AZ-33 = AZ-33 20 μM. (B) The levels of protein synthesis were measured when FX11 (1 μM) and STP (10 μM) were added in the rabbit reticulocyte translation system with 2.5 μg LDHA and 2 mM NADH (n = 3). (C) Isolation of human MK (DAMI) ribosomes and detection of eEF2 content on the ribosomes under dimethyl sulfoxide (DMSO)/LDHA inhibitors treated for 24 hours. Ribosomal protein L4 (RPL4), ribosomal protein L28 (RPL28), ribosomal protein L35 (RPL35), and ribosomal protein S3A (RPS3A) were used to indicate the equal loading. (D) Megakaryocyte PPF assay in mice fetal liver MKs incubated with STP (100 μM). DMSO was used as control. Statistical analysis of the PPF-MK/total MK ratio is shown (n = 4). Scale bar, 100 μm. (E) Platelet counts of wild-type mice that were administered STP (200 mg/kg per 12 hours) or 10% DMSO through intraperitoneal injection (n = 3; 8 weeks old, male). (F) Platelet counts of LdhAf/f and LdhA−/− mice that were administered STP (200 mg/kg per 12 hours) through intraperitoneal injection (n = 4; 8 weeks old, male). (G-H) STP (200 mg/kg per 12 hours) or 10% DMSO was intraperitoneally injected in wild-type mice, and platelets were cleared by tail IV injection with anti-CD42b antibody (2 μg/g weight) at hour 24; platelet counts were monitored at different time points (n = 3; 8 weeks old, male). (I) Polyploidy (≥4N) assay of cord blood mononuclear cell (CBMC)-derived MKs treated with STP (10 μM; n =8 or 50 μM; n = 7), romiplostim (Romi; 100 ng/mL) (n = 6), and the combination of STP with Romi (n = 6) by using flow cytometry. (J) Detection of reticulated platelet counts from CBMC-derived MK culture supernatant that were incubated with STP (10 μM or 50 μM; n = 7), Romi (100 ng/mL; n = 5), and the combination of STP with Romi (n = 5) according to flow cytometry. (K) Schematic diagram of eEF2 release from LDH with NADH or LDHA inhibitors to regulate translation in maturation and differentiation of MKs. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control; ns, not significant.

Screening and applying of LDHA inhibitors in thrombocytopenia. (A) Coimmunoprecipitation (Co-IP) of Flag-LDHA and HA-eEF2 in HEK-293T treated with LDHA inhibitors. OXA = sodium oxamate 1 mM; FX11 = FX11 20 μM; STP = stiripentol 100 μM; GNE = (R)-GNE-140 20 μM; GSK = GSK 2837808A 20 nM; AZ-33 = AZ-33 20 μM. (B) The levels of protein synthesis were measured when FX11 (1 μM) and STP (10 μM) were added in the rabbit reticulocyte translation system with 2.5 μg LDHA and 2 mM NADH (n = 3). (C) Isolation of human MK (DAMI) ribosomes and detection of eEF2 content on the ribosomes under dimethyl sulfoxide (DMSO)/LDHA inhibitors treated for 24 hours. Ribosomal protein L4 (RPL4), ribosomal protein L28 (RPL28), ribosomal protein L35 (RPL35), and ribosomal protein S3A (RPS3A) were used to indicate the equal loading. (D) Megakaryocyte PPF assay in mice fetal liver MKs incubated with STP (100 μM). DMSO was used as control. Statistical analysis of the PPF-MK/total MK ratio is shown (n = 4). Scale bar, 100 μm. (E) Platelet counts of wild-type mice that were administered STP (200 mg/kg per 12 hours) or 10% DMSO through intraperitoneal injection (n = 3; 8 weeks old, male). (F) Platelet counts of LdhAf/f and LdhA−/− mice that were administered STP (200 mg/kg per 12 hours) through intraperitoneal injection (n = 4; 8 weeks old, male). (G-H) STP (200 mg/kg per 12 hours) or 10% DMSO was intraperitoneally injected in wild-type mice, and platelets were cleared by tail IV injection with anti-CD42b antibody (2 μg/g weight) at hour 24; platelet counts were monitored at different time points (n = 3; 8 weeks old, male). (I) Polyploidy (≥4N) assay of cord blood mononuclear cell (CBMC)-derived MKs treated with STP (10 μM; n =8 or 50 μM; n = 7), romiplostim (Romi; 100 ng/mL) (n = 6), and the combination of STP with Romi (n = 6) by using flow cytometry. (J) Detection of reticulated platelet counts from CBMC-derived MK culture supernatant that were incubated with STP (10 μM or 50 μM; n = 7), Romi (100 ng/mL; n = 5), and the combination of STP with Romi (n = 5) according to flow cytometry. (K) Schematic diagram of eEF2 release from LDH with NADH or LDHA inhibitors to regulate translation in maturation and differentiation of MKs. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; NC, negative control; ns, not significant.

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