1. Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis
Camelia Iancu-Rubin, Goar Mosoyan, Kelli Glenn, Ronald E. Gordon, Gwen L. Nichols, Ronald Hoffman 
Experimental Hematology 
Volume 42, Issue 2, Pages e5 (February 2014)
DOI: /j.exphem
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

2. Figure 1
Effects of RG7112 on platelet numbers. (A) Four groups of 18 male Wistar rats each were treated with vehicle (Gr 1) or with 25, 50 and 100 mg/Kg/day RG7112 (grade 2, 3, and 4, respectively) for 10 consecutive days. The platelet counts of individual animals were evaluated on the days indicated on the x axis. Each time point represents the mean value ± SD of platelet counts recorded for each group (n = 18 animals per group). (B) Four groups of five male cynomongolus monkeys each were treated with vehicle (grade 1) or with 5, 10, and 20 mg/kg/day RG7112 (grade 2, 3, and 4, respectively) for 10 consecutive days. Platelets were enumerated in the peripheral blood of individual animals on the days indicated on the x axis. Each time point represents the mean value ± SD of platelet numbers recorded for each group (n = 5 animals per group).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

3. Figure 2
Effects of RG7112 on proplatelets formation and platelet production. (A) Quantification and microscopic visualization of proplatelets generated by MK in untreated cultures (Control) and in cultures treated with RG7112. Representative microphotographs show the presence of MK bearing long proplatelets with nascent platelet buds at their ends in control MK cultures (arrows in a) and of atypical, short cytoplasmic extensions in RG7112-treated MK cultures (arrows in b). MK-bearing proplatelets were enumerated in 50 high-power fields, and the average number ± SD is indicated for each condition. Microphotographs were obtained using an inverted Olympus 1X71 microscope with a 10X/0.30 objective. Scale bars: 50 μm in a, 20 μm in b. (B) Representative dot plot analyses used to evaluate and quantify the fraction of culture-derived platelets. Top diagrams represent analyses of freshly isolated peripheral blood platelets (PB-Platelets) that were used as control to set up an analytical gate for the size (FSC, x axis) and side scatter (SSC, y axis) properties: the red-colored events in gate P1 designate platelets (top left diagram). CD41 expression (x axis) and thiazole orange (y axis) labeling of PB-platelets gated in P1 are shown in the top right diagram. Identical analyses were applied to MK cultures, as illustrated by representative dot plots diagrams of Control, RG7112i-treated, and RG7112-treated cultures. (C) Quantification of platelets generated by untreated MK (Control) and by MK treated with 5 μmol/L of inactive drug (RG7112i) or 5 μmol/L active drug (RG7112). The columns represent the percentage of CD41+/TO+ platelet-sized cells ± SD detected in MK cultures derived from BM-CD34+ from at least three different healthy donors analyzed independently by flow cytometry as shown in (B).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

4. Figure 3
Effects of RG7112 on apoptosis. (A) Assessment of apoptosis in control cultures and in cultures treated with 1 and 5 μmol/L of RG7112i or RG7112 during the initial 7 days of culture. The results represent the percentage of apoptotic cells (i.e., percent of annexin V positive quantified in three different experiments on days 4 and 7 of incubation). Representative histograms of annexin V expression (B) and Caspase 3 activation (C) by control MK cultures and by cultures treated with 5 μmol/L RG7112i or RG7112 for 7 days.
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

5. Figure 4
Effects of RG7112 during the early stages of megakaryocytopoiesis. (A) Quantification of viable cells generated in liquid cultures initiated with 1.25 × 105 BM-CD34+ cells cultured for 7 days in serum-free media supplemented with stem cell factor and TPO in the absence (Control) or presence of 1 μmol/L and 5 μmol/L RG7112i or RG7112. (B) Quantification of CD61+ MK generated in control cultures and in cultures treated with 1 and 5 μmol/L RG7112i or RG7112 during the initial 7 days of culture. The results represent the percentage of CD61+ cells ± SD detected in four independent experiments using an analytical gate that was set to include only viable cells (i.e., cells that were negative for 7-AAD staining). (C) Evaluation of RG7112i or RG7112 treatment on CFU-MK formation by BM-CD34+ cells. CD34+ cells from at least three different donors were untreated or treated with 1 or 5 μmol/L of the inactive or active RG7112 in liquid culture in independent experiments. After 24 hours, an equal number of cells (0.6 × 103) were plated in duplicate in MegaCult collagen-based semisolid media and allowed to form colonies. After 14–16 days of incubation, the number of CFU-MKs formed was enumerated, and the numbers obtained in drug-treated cultures were normalized to the number of colonies that were formed in the control cultures (100% efficiency).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

6. Figure 5
Evaluation of RG7112 on MK maturation. (A) Representative flow cytometric analyses of CD41 and CD42b expression by control MK cultures and MK cultures treated with 5 μmol/L RG7112i or RG7112 for 14 days. Gate P1 indicates less mature CD41+/CD42b–/low MK and P2 indicates fully mature CD41+/CD42b+/high MK. (B) Representative Wright-Giemsa stained cytospin preparations of cells from control cultures and from cultures incubated for 14 days in the presence of 5 μmol/L RG7112i or RG7112. Large, multilobulated MKs are present in control cultures and in RG7112i-treated cultures (arrows in a and b), whereas smaller hypolobulated MKs are present in RG7112-treated cultures (arrows in c). Microphotographs were obtained using an Olympus BX40 microscope with a dry 40X/0.75 objective. Scale bars, 20 μm.
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

7. Figure 6
Evaluation of RG7112 on MK polyploidization. (A) Quantification of MK ploidy detected after 14 days incubation in control cultures and in cultures generated in the presence of 1 and 5 μmol/L RG7112i or RG7112. The numbers represent the percentage of MKs with DNA content greater than 4N as determined by flow cytometric analysis of cells labeled with propidium iodide using an analytical gate that included CD61+ MK only. (B) Western blot analyses of immunomagnetically purified CD61+ MKs that were untreated (−) or treated with 5 μmol/L RG7112 (+) for 48 hours (D9) or 72 hours (D11) exclusively during MK maturation and polyploidization stages (i.e., the last 7 days of culture). After blotting, the membranes were incubated with MDM2 and p21 antibodies. Coomassie blue staining was included as protein-loading control. (C) Representative dot-plot histograms of BrdU-labeled MK generated in untreated cultures (Control) and in cultures exposed to 5 μmol/L of RG7112i or RG7112 for 48 hours during MK polyploidization. BrdU incorporation, indicating active DNA synthesis, is represented on the y axis, whereas DNA content as evaluated by 7-AAD staining is represented on the x axis. The fraction of MK with greater than 4N DNA content represents a population of MKs that is BrdU negative (P1) and a population that is BrdU positive (P2). The cells within P2 denote DNA synthesis by MK with DNA content of 4N and greater. P2 population of MK was absent in the cultures treated with RG7112.
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

8. Supplementary Figure E1
Effects of RG7112 on hemoglobin (HGB) and white blood cell (WBC) counts. Four groups of 18 male Wistar rats each were treated with vehicle (grade 1) or with 25, 50, and 100 mg/kg/day RG7112 (grades 2, 3, and 4, respectively) as described in the Methods section. Hemoglobin levels (A) and WBC counts (B) of individual animals were evaluated on the days indicated on the x axis. Each time point represents the mean value ± SD recorded for each group (n = 18 animals per group). Four groups of five male cynomolgus monkeys each were treated with vehicle (grade 1) or with 5, 10, and 20 mg/kg/day RG7112 (grades 2, 3, and 4, respectively). Hemoglobin levels (C) and WBC counts (D) of individual animals were evaluated on the days indicated on the x axis. Each time point represents the mean value ± SD recorded for each group (n = 5 animals per group).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

9. Supplementary Figure E2
Effects of RG7112 on platelet production. Representative flow cytometric analyses of platelets generated in cultures treated with 5 μM of inactive drug (RG7112i) or with 5 mM RG7112. The panels on the left show dot plot analyses of platelets generated in cultures treated with RG7112i or RG7112 for the entire 14-day culture period (Continuous) or in cultures from which the drugs were withdrawn during the maturation step, i.e. the last 7 days of culture (Withdrawn). The cells were gated based on the side scatter and forward scatter properties of peripheral blood platelets that were used as control then analyzed for CD41 expression and thiazole orange (TO) staining as illustrated in Figure 2 in the main manuscript. The oval gate indicates platelet-sized CD41+/TO+ cells.
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

10. Supplementary Figure E3
Effects of RG7112 on burst-forming erythroid (BFU-E) and colony-forming unit granulocyte-monocyte (CFU-GM) colony formation. CD34+ cells from at least three different donors were untreated or treated with 1 or 5 μmol/L of the inactive (RG7112i) or active RG7112 in liquid culture in independent experiments. After 24 hours, an equal number of cells (0.5 × 103) were plated in duplicate in methylcellulose semisolid media supplemented with 50 ng/mL interleukin 3, 50 ng/mL SCF, and 2 U EPO in 35 mm tissue culture dishes and allowed to form colonies. After 14–16 days of incubation, the number of BFU-E (A) and CFU-GM (B) colonies were enumerated, and the numbers obtained in drug-treated cultures were normalized to the number of colonies formed in the control cultures (100% efficiency).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

11. Supplementary Figure E4
Transmission electron microscopy (TEM) analysis of mature MK. MK cultures were generated from human CD34+ cells cultured in MK differentiation media in the presence or absence of 5 μmol/L RG7112 as described in the Methods section. After 13 days of incubation, large mature MKs were isolated by bovine serum albumin density gradient, fixed and processed for transmission electron microscopy (TEM). (A) Representative TEM microphotographs of untreated MK (a, b, and the corresponding insets) show polylobulated nuclei (N), characteristic demarcation membranes system (DMS), numerous granules (G) and an open canalicular system (OCS). Abundant DMS that fills the cytoplasm and delimits platelet-like territories (PTL-t) are observed in the MK illustrated in b. (B) Representative TEM microphotographs of MK treated with RG7112 (c, d, and the corresponding insets) show atypical DMS (arrow heads in c and corresponding inset indicate enlarged DMS), ultrastructural features of early apoptosis such as abundant vacuolization (V) in which many vacuoles resemble autophagosomes containing partially degraded material and multiple membranes (asterisks). Chromatin condensation to the margins of the nucleus (N) is also observed (arrows). Of note, the cytoplasm of RG7112-treated MK lacks characteristic granules. Scale bar: 10 mm (A) and 5 mm (B); TEM magnification: 2K in (A) and 1.5K in (B).
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

12. Supplementary Figure E5
Effects of RG7112 on MK polyploidization. Representative DNA histograms obtained by flow cytometric analysis of propidium iodide-stained CD61+ MK generated in the presence of 5 μmol/L RG7112i or RG7112. The panels on the left show DNA content of MK treated with either RG7112i or RG7112 for the entire culture period (i.e., 14 days; continuous), and the panels on the right show DNA content of MK generated in cultures from which the drugs were removed during the maturation step (i.e., the last 7 days of culture; withdrawn). Gate P1 includes the population of MK with greater than 4N DNA content, and the arrows indicate different ploidy classes (e.g., 8N, 16N, 32N, 64N). Note that the absence of ploidy peaks on the histograms representing MK exposed continuously to RG7112 and partial reversal of ploidy peaks on the histograms representing MK cultures from which RG7112 was withdrawn during the maturation step.
Experimental Hematology  , e5DOI: ( /j.exphem )
Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions