Figure 6.
LKB1 shRNA interference offset rescuing effect of decitabine-treated MDSC in a murine model of ITP. (A) A schematic diagram denoting different interventions for ITP mice. KO, knockout. (B) The dotted lines represent the platelet counts of SCID mice.The star indicates significant differences between groups emerged on day 28. On days 28 and 35, platelet counts were significantly higher in NC + Dec group. Significance among groups was determined by 2-way ANOVA (multiple comparisons on day 28: ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC+Dec vs LKB1+Dec = .0312; day 35: ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC shRNA vs NC+Dec = .0198, ∗∗PNC+Dec vs LKB1+Dec = .0033). (C) Immunofluorescence images of femurs stained on day 35 with fluorescein isothiocyanate (green; MDSCs), cy3 (red; LKB1), and 4′,6-diamidino-2-phenylindole (DNA). Representative staining of nuclei, Ly-6C, LKB1, and merged spleen images. Ten areas of 80 × 80 μm were randomly selected from the 6G/Ly-6C+ cell-rich areas of the film. MDSCs were counted per merged view, and MDSC-positive cell LKB1 expression was estimated from the mean fluorescence intensity, using ImageJ. Five trials were conducted for each group. Original magnification, scale bars: 20 μm. LKB1 fluorescence intensity (reflecting LKB1 expression) was significantly higher in the NC + Dec group than in Ctrl and NC shRNA group, and there was no statistically significant difference between the LKB1 shRNA group and LKB1 + Dec group (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC shRNA vs NC+Dec = .0349, PLKB1 shRNA vs LKB1+Dec = .9713). (D) ATP production was significantly higher in the NC + Dec group than Ctrl and NC shRNA group; however, there was no statistically significant difference between the LKB1 shRNA group and the LKB1 + Dec group (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0002, ∗PNC shRNA vs NC+Dec = .0152, PLKB1 shRNA vs LKB1+Dec = .9681). (E) MDSCs were sorted from bone marrow for OCR assessment. OCR was significantly higher in the NC + Dec group than in Ctrl and NC shRNA group. (F) basal respiration (unpaired t test, ∗∗PCtrl vs NC+Dec = .0010, ∗PNC shRNA vs NC+Dec = .0370, PLKB1 shRNA vs LKB1+Dec = .6933), ATP production (unpaired t test, ∗∗PCtrl vs NC+Dec = .0018, ∗PNC shRNA vs NC+Dec = .0355, PLKB1 shRNA vs LKB1+Dec = .9938), and maximal respiration (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0003, ∗∗PNC shRNA vs NC+Dec = .0098, PLKB1 shRNA vs LKB1+Dec = .8724). (G-I) ACADM and PGC1β mRNA expression were significantly higher in the NC + Dec group than in the Ctrl and NC shRNA group (Unpaired t test. ACADM: ∗∗∗P Ctrlvs. NC+Dec=0.0005, ∗P NC shRNAvs. NC+Dec=0.0331; PGC1β: ∗∗∗P Ctrlvs. NC+Dec=0.0008, ∗P NC shRNAvs. NC+Dec=0.0390). HADHA mRNA expression was significantly higher in the NC + Dec group than in the Ctrl group and was not statistically significantly different from that of NC shRNA (∗P Ctrlvs. NC+Dec=0.0212, P NC shRNAvs. NC+Dec=0.6881). The mRNA expressions of ACADM, PGC1β, and HADHA showed no significant difference between LKB1 shRNA and LKB1 + Dec.

LKB1 shRNA interference offset rescuing effect of decitabine-treated MDSC in a murine model of ITP. (A) A schematic diagram denoting different interventions for ITP mice. KO, knockout. (B) The dotted lines represent the platelet counts of SCID mice.The star indicates significant differences between groups emerged on day 28. On days 28 and 35, platelet counts were significantly higher in NC + Dec group. Significance among groups was determined by 2-way ANOVA (multiple comparisons on day 28: ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC+Dec vs LKB1+Dec = .0312; day 35: ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC shRNA vs NC+Dec = .0198, ∗∗PNC+Dec vs LKB1+Dec = .0033). (C) Immunofluorescence images of femurs stained on day 35 with fluorescein isothiocyanate (green; MDSCs), cy3 (red; LKB1), and 4′,6-diamidino-2-phenylindole (DNA). Representative staining of nuclei, Ly-6C, LKB1, and merged spleen images. Ten areas of 80 × 80 μm were randomly selected from the 6G/Ly-6C+ cell-rich areas of the film. MDSCs were counted per merged view, and MDSC-positive cell LKB1 expression was estimated from the mean fluorescence intensity, using ImageJ. Five trials were conducted for each group. Original magnification, scale bars: 20 μm. LKB1 fluorescence intensity (reflecting LKB1 expression) was significantly higher in the NC + Dec group than in Ctrl and NC shRNA group, and there was no statistically significant difference between the LKB1 shRNA group and LKB1 + Dec group (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0005, ∗PNC shRNA vs NC+Dec = .0349, PLKB1 shRNA vs LKB1+Dec = .9713). (D) ATP production was significantly higher in the NC + Dec group than Ctrl and NC shRNA group; however, there was no statistically significant difference between the LKB1 shRNA group and the LKB1 + Dec group (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0002, ∗PNC shRNA vs NC+Dec = .0152, PLKB1 shRNA vs LKB1+Dec = .9681). (E) MDSCs were sorted from bone marrow for OCR assessment. OCR was significantly higher in the NC + Dec group than in Ctrl and NC shRNA group. (F) basal respiration (unpaired t test, ∗∗PCtrl vs NC+Dec = .0010, ∗PNC shRNA vs NC+Dec = .0370, PLKB1 shRNA vs LKB1+Dec = .6933), ATP production (unpaired t test, ∗∗PCtrl vs NC+Dec = .0018, ∗PNC shRNA vs NC+Dec = .0355, PLKB1 shRNA vs LKB1+Dec = .9938), and maximal respiration (unpaired t test, ∗∗∗PCtrl vs NC+Dec = .0003, ∗∗PNC shRNA vs NC+Dec = .0098, PLKB1 shRNA vs LKB1+Dec = .8724). (G-I) ACADM and PGC1β mRNA expression were significantly higher in the NC + Dec group than in the Ctrl and NC shRNA group (Unpaired t test. ACADM: ∗∗∗P Ctrlvs. NC+Dec=0.0005, ∗P NC shRNAvs. NC+Dec=0.0331; PGC1β: ∗∗∗P Ctrlvs. NC+Dec=0.0008, ∗P NC shRNAvs. NC+Dec=0.0390). HADHA mRNA expression was significantly higher in the NC + Dec group than in the Ctrl group and was not statistically significantly different from that of NC shRNA (∗P Ctrlvs. NC+Dec=0.0212, P NC shRNAvs. NC+Dec=0.6881). The mRNA expressions of ACADM, PGC1β, and HADHA showed no significant difference between LKB1 shRNA and LKB1 + Dec.

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