Induction of Moderate DNA Damage Enhances Megakaryopoiesis and Platelet Production

Figures

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  Figure 1.. Low dose Niraparib treatment enhances platelet count in mice without affecting platelet function. A) Schematic outlining niraparib administration: Mice were injected intraperitoneally with niraparib or vehicle (DMSO) at the indicated times. Blood was drawn via tail vein venipuncture and complete blood counts (CBC) were measured using a Sysmex hematology analyzer. B) Platelet counts in vehicle or niraparib-treated mice (50 mg/kg); n=5 mice/group. C) Platelet counts in vehicle or niraparib-treated mice (25 mg/kg); n=5 mice/group. D) Mean platelet volume (MPV) in vehicle- or niraparib-treated mice (25 mg/kg); n=5 mice/group. E) Immature platelet fraction (IPF) in mice treated with niraparib (25 mg/kg); n=5 mice/group. Data in B) - E) shown as mean ± SD. Two-way ANOVA with Tukey’s correction for multiple comparisons. F) Frequency of reticulated, thiazole orange (TO)high platelets in whole blood of vehicle- or niraparib-treated mice (25 mg/kg); n=5 mice/group. Data as mean ± SD, unpaired, two-tailed student’s t-test. G) Spleen weights from vehicle- or niraparib-treated mice (25 mg/kg) after 3 or 11 days of treatment, as indicated; n=5 mice/group. Data as mean ± SD, unpaired, one-way…

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  Figure 2.. Low dose Niraparib treatment enhances megakaryopoiesis. A) HSPC counts in vehicle- or niraparib-treated mice; n = 5 mice/group. Data shown as mean ± SD, One-way ANOVA with Dunnett’s post hoc test. Gating strategy shown in Supplemental Figure 2. B) Representative images of femoral cryosections from vehicle or niraparib treated mice (CD41, green). Nuclei were counterstained using DAPI (blue). Scale bars: 1000 µm; inset scale bars: 200 µm. C) MK counts and D) area in vehicle- or niraparib-treated mice were quantified in femoral cryosections; n=4 mice/group. Data shown as mean ± SD, unpaired, two-tailed student’s t-test. E) MK/platelet reporter mice (vWF-GFP) were treated with 25 mg/kg niraparib or vehicle for 7 days and 2-photon intravital microscopy of the calvaria was performed. Representative images show MKs (cyan) and vasculature (magenta, labelled using Evans Blue). Scale bars: 20 µm. F) Live MKs were quantified by size and GFP signal; n=4 mice/group. Data shown as mean ± SD, unpaired, two-tailed student’s t-test.

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  Figure 3.. DNA damage dose dependently enhances megakaryopoiesis in vitro and in vivo. A) yH2AX positivity in indicated HSPC populations in vehicle- or niraparib-treated mice; n=5 mice/group. Data shown as mean ± SD, One-way ANOVA with Dunnett’s post hoc test. Gating strategy shown in Supplemental Figure 2. B) Representative widefield images of femoral cryosections derived from vehicle- or Niraparib-treated mice; (CD41; green), (γH2AX; purple). Scale bars: 20 µm. Quantification shows the percentage of femoral MKs that have either 0, 1–10, or >10 γH2AX foci in the nucleus in vehicle or niraparib treated mice; 100 MKs counted per femur, n=3 mice/group. Data shown as mean ± SD, multiple t-tests. C) Representative images of native MKs from vehicle- or Niraparib-treated mice (left); (green=SYBR Gold). Scale bars: 100 µm. MKs were isolated from the femurs of vehicle- or niraparib-treated mice. The tail DNA (%) of at least 120 MKs per femur were analyzed across three replicates (3 femurs) per condition; n=3 mice/group. Violin plots show distribution of all data points and median and interquartile range, unpaired, two-tailed student’s t-test. D) Ploidy distribution in native MKs from vehicle- or…