Figure 4
Figure 4. RhoA inhibition restores proPLT formation and PLT release in MK infected with PKCε-specific shRNA. (A) Representative images of ppFMK in the absence or presence of RhoA pharmacological inhibitor. Cultures of cells treated with RhoA inhibitor presented increased quantities of proplatelets and released platelets. (B) Representative scatter plots of flow cytometric analyses of platelets released in culture. Fixed volumes of media from the different cultures were collected, labeled with CD41 and Calcein AM, and mixed with a fixed volume of calibration beads for absolute PLT count. Only cells CD41pos/Calcein AMpos were considered as preserved platelets (quadrants: a2, b2, c2, d2, e2, f2). The absolute count for each sample was reported inside the scatter plots. In the absence of RhoA inhibitor, the cells treated with PKCε-specific shRNA59 (c2) and shRNA60 (e2) released fewer platelets than did control (shRNACT; a2). In the presence of RhoA inhibitor, both control (shRNACT; b2) and PKCε-specific shRNA (shRNA59 and shRNA60; d2 and f2) samples released around 20-fold more platelets. (C) Analysis of PLT production. Data, from 4 replicates, are normalized for shRNACT (means ± 1 SD; **P < .01 ANOVA and Tukey tests). (D) Immunofluorescence analyses of proplatelets, labeled with specific antibodies against PKCε (green) and α/β-Tubulin (red), in the absence (a, c, e) or presence of RhoA inhibitor (b, d, f). In comparison with the control (shRNACT; a), proplatelets derived from PKCε-specific shRNAs infected MK (shRNA59 and shRNA60; c and e) were aberrant and lacking the typical spherical organization of PLT precursors. In the presence of the RhoA inhibitor, the shRNAs infected MK (shRNA59 and shRNA60; d and f) produced proplatelets with a morphology similar to the control (shRNACT; b). Samples were examined with microscope Axiovert 200 equipped with a 63× numerical aperture 1.4 oil immersion objective. Images were obtained using a CCD camera and analyzed using the MetaMorph image analysis software. Images were obtained at 22°C. PKCε was detected with a rabbit polyclonal antibody and a secondary goat anti-rabbit antibody, conjugated to an Alexa Fluor 488; α and β tubulin were detected with rmouse monoclonal antibodies and a secondary goat anti-mouse antibody, conjugated to an Alexa Fluor 568. (E) Analysis of PLT dimensions (means ± 1 SD; **P < .01 ANOVA and Tukey tests). For each group, several platelets were analyzed from different fields. In the absence of RhoA inhibitor the platelet counts were (1) shRNACT, 33 PLTs; (2) shRNA59, 18 PLTs; and (3) shRNA60, 23 PLTs. In the presence of RhoA inhibitor, they were (1) shRNACT, 19 PLTs; (2) shRNA59, 21 PLTs; and (3) shRNA60, 35 PLTs. The mean cell diameter was calculated with MetaMorph and Image J software. (F) Analysis of size distribution within PLT populations.

RhoA inhibition restores proPLT formation and PLT release in MK infected with PKCε-specific shRNA. (A) Representative images of ppFMK in the absence or presence of RhoA pharmacological inhibitor. Cultures of cells treated with RhoA inhibitor presented increased quantities of proplatelets and released platelets. (B) Representative scatter plots of flow cytometric analyses of platelets released in culture. Fixed volumes of media from the different cultures were collected, labeled with CD41 and Calcein AM, and mixed with a fixed volume of calibration beads for absolute PLT count. Only cells CD41pos/Calcein AMpos were considered as preserved platelets (quadrants: a2, b2, c2, d2, e2, f2). The absolute count for each sample was reported inside the scatter plots. In the absence of RhoA inhibitor, the cells treated with PKCε-specific shRNA59 (c2) and shRNA60 (e2) released fewer platelets than did control (shRNACT; a2). In the presence of RhoA inhibitor, both control (shRNACT; b2) and PKCε-specific shRNA (shRNA59 and shRNA60; d2 and f2) samples released around 20-fold more platelets. (C) Analysis of PLT production. Data, from 4 replicates, are normalized for shRNACT (means ± 1 SD; **P < .01 ANOVA and Tukey tests). (D) Immunofluorescence analyses of proplatelets, labeled with specific antibodies against PKCε (green) and α/β-Tubulin (red), in the absence (a, c, e) or presence of RhoA inhibitor (b, d, f). In comparison with the control (shRNACT; a), proplatelets derived from PKCε-specific shRNAs infected MK (shRNA59 and shRNA60; c and e) were aberrant and lacking the typical spherical organization of PLT precursors. In the presence of the RhoA inhibitor, the shRNAs infected MK (shRNA59 and shRNA60; d and f) produced proplatelets with a morphology similar to the control (shRNACT; b). Samples were examined with microscope Axiovert 200 equipped with a 63× numerical aperture 1.4 oil immersion objective. Images were obtained using a CCD camera and analyzed using the MetaMorph image analysis software. Images were obtained at 22°C. PKCε was detected with a rabbit polyclonal antibody and a secondary goat anti-rabbit antibody, conjugated to an Alexa Fluor 488; α and β tubulin were detected with rmouse monoclonal antibodies and a secondary goat anti-mouse antibody, conjugated to an Alexa Fluor 568. (E) Analysis of PLT dimensions (means ± 1 SD; **P < .01 ANOVA and Tukey tests). For each group, several platelets were analyzed from different fields. In the absence of RhoA inhibitor the platelet counts were (1) shRNACT, 33 PLTs; (2) shRNA59, 18 PLTs; and (3) shRNA60, 23 PLTs. In the presence of RhoA inhibitor, they were (1) shRNACT, 19 PLTs; (2) shRNA59, 21 PLTs; and (3) shRNA60, 35 PLTs. The mean cell diameter was calculated with MetaMorph and Image J software. (F) Analysis of size distribution within PLT populations.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal