Figure 4.
Ultrastructures of DGs in MEG-01 cells and platelets with deficiencies in dysbindin or TMEM163. (A) Thin-section TEM pictures of DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells after induction with 100 nM TPA for 4 days. Red arrows represent round membranous organelles resembling EEs with reduced intraluminal contents, yellow arrows represent round membranous organelles resembling LEs with increased intraluminal contents, and blue arrows represent MVB circular membranous organelles. Scale bars, 1 µm. (B) The average number of vacuolar compartment (red arrows shown in panel A) per unit area (µm2) in DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells are significantly higher than that in control (WT) cells (WT, 0.027 ± 0.002, n = 47; D1, 0.037 ± 0.003, n = 48; D24, 0.036 ± 0.002, n = 52; T20, 0.042 ± 0.003, n = 55; T87, 0.044 ± 0.004, n = 40). (C) The average size of the vacuolar compartment (µm2) in DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells is significantly larger than that in WT cells (WT, 1.343 ± 0.080, n = 158; D1, 3.637 ± 0.234, n = 299; D24, 3.736 ± 0.302, n = 273; T20, 2.791 ± 0.145, n = 382; T87, 3.890 ± 0.245, n = 304). (D-E) TEM pictures of whole-mount platelets of the B6 (WT, C57BL/6J), sdy mutant, and Tmem163-KO mice. Arrows show platelet DGs. Lighter-intensity dots or smaller dots (diameter <100 nm) are not counted as DGs. Scale bars, 0.5 μm. DGs are absent in sdy (0.029 ± 0.020, n = 69) and Tmem163-KO (0.095 ± 0.020, n = 222) mice compared with B6 (7.069 ± 0.451, n = 58). (F-G) Washed platelets were lysed by 100 µL ice-cold lysis buffer, and the lysates used for further enzyme-linked immunosorbent assay were analyzed spectrophotometrically at 450 nm. The content of platelet ADP (sdy vs Tmem163-KO, 75.71% vs 79.77%, n = 4, 13, respectively) (F) and serotonin (sdy vs Tmem163-KO, 33.01% vs 79.51%, n = 6, 10, respectively) (G) of sdy, Tmem163-KO mice were calculated and plotted relative to their control littermates (sdy+/− or B6). (H) Platelets were incubated with 1.7 μM mepacrine at 37°C for 30 minutes and then analyzed by flow cytometry. The mepacrine uptake (green) fluorescence of sdy and Tmem163-KO was expressed as the percentage (mean ± SEM) of the mean normalized value for control mice (sdy vs Tmeme163-KO, 85.41% vs 89.29%, n = 9, 8, respectively). *P < .05; **P < .01; ***P < .001.

Ultrastructures of DGs in MEG-01 cells and platelets with deficiencies in dysbindin or TMEM163. (A) Thin-section TEM pictures of DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells after induction with 100 nM TPA for 4 days. Red arrows represent round membranous organelles resembling EEs with reduced intraluminal contents, yellow arrows represent round membranous organelles resembling LEs with increased intraluminal contents, and blue arrows represent MVB circular membranous organelles. Scale bars, 1 µm. (B) The average number of vacuolar compartment (red arrows shown in panel A) per unit area (µm2) in DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells are significantly higher than that in control (WT) cells (WT, 0.027 ± 0.002, n = 47; D1, 0.037 ± 0.003, n = 48; D24, 0.036 ± 0.002, n = 52; T20, 0.042 ± 0.003, n = 55; T87, 0.044 ± 0.004, n = 40). (C) The average size of the vacuolar compartment (µm2) in DTNBP1-KO (D1 and D24) and TMEM163-KO (T20 and T87) MEG-01 cells is significantly larger than that in WT cells (WT, 1.343 ± 0.080, n = 158; D1, 3.637 ± 0.234, n = 299; D24, 3.736 ± 0.302, n = 273; T20, 2.791 ± 0.145, n = 382; T87, 3.890 ± 0.245, n = 304). (D-E) TEM pictures of whole-mount platelets of the B6 (WT, C57BL/6J), sdy mutant, and Tmem163-KO mice. Arrows show platelet DGs. Lighter-intensity dots or smaller dots (diameter <100 nm) are not counted as DGs. Scale bars, 0.5 μm. DGs are absent in sdy (0.029 ± 0.020, n = 69) and Tmem163-KO (0.095 ± 0.020, n = 222) mice compared with B6 (7.069 ± 0.451, n = 58). (F-G) Washed platelets were lysed by 100 µL ice-cold lysis buffer, and the lysates used for further enzyme-linked immunosorbent assay were analyzed spectrophotometrically at 450 nm. The content of platelet ADP (sdy vs Tmem163-KO, 75.71% vs 79.77%, n = 4, 13, respectively) (F) and serotonin (sdy vs Tmem163-KO, 33.01% vs 79.51%, n = 6, 10, respectively) (G) of sdy, Tmem163-KO mice were calculated and plotted relative to their control littermates (sdy+/− or B6). (H) Platelets were incubated with 1.7 μM mepacrine at 37°C for 30 minutes and then analyzed by flow cytometry. The mepacrine uptake (green) fluorescence of sdy and Tmem163-KO was expressed as the percentage (mean ± SEM) of the mean normalized value for control mice (sdy vs Tmeme163-KO, 85.41% vs 89.29%, n = 9, 8, respectively). *P < .05; **P < .01; ***P < .001.

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