(a,b) Characterization of the purified platelet preparations from starting platelet-rich plasma (PRP) by reverse transcriptase-polymerase chain reaction (RT-PCR) (a) and hemocytometer counting (b). (c) Bioanalyzer assessment of RNA samples prepared from purified platelets (left) or megakaryocytes (right). (d) Platelet miRNA profiling analysis. MiRNA probes with signals above the detection threshold are shown in order of increasing relative fluorescence unit (RFI). (e) Northern blot validation of 4 selected miRNAs shown in order of decreasing micro-array RFI. A 10-nt RNA ladder was used as a size marker. cDNA, complementary DNA; GP, glycoprotein; Leuk., leukocyte; nt, nucleotide.

(a) Detection of the protein components of the miRNA-guided RNA silencing machinery in megakaryocytes (left) or platelets (right) by Western blot¹⁰. β-Actin is used as a control. (b) Assessment of Dicer processing activity in protein extracts and Dicer immunoprecipitates (IP) prepared from purified platelets upon incubation with ³²P-labeled human let-7a-3 pre-miRNA³⁰. (c) Extracts from purified platelets were separated by gel filtration on a Superose 6 column and the fractions analyzed by Western blot using anti-Dicer antibody, as described^6,10. Selected (odd) fractions were tested for Dicer activity as in b. (d) Immunoblot analysis of TRBP2 IP derived from platelet extracts by using an anti-Dicer antibody. (e) Dicer processing activity assays on IP derived from platelet extracts using anti-TRBP2, control IgG or anti-Dicer antibodies. S10, 10,000 g supernatant fraction.

(a, b) In vitro RNA-induced silencing complex (RISC) activity assays. (a) Protein extracts were incubated in the presence of a ³²P-labeled RNA sensor bearing a binding site complementary to miR-223 without or with proteinase K (Prot. K; 1 mg ml⁻¹) (center-right panel) or EDTA (5 mM) (right panel). (b) Same as in a (left panel), but using sensor RNAs bearing a binding site complementary to let-7c (left panel), miR-19a (center panel) or miR-199a-3p (right panel). (c) Same as in Figure 2c, except using anti-Ago2 antibody for immunoblotting. (d) Pooled fractions were probed for miR-223 (upper panel) and let-7c (lower panel) content by Northern blot. (e) Selected (even) fractions were tested for RISC activity as in a. (f) Ago2 immunoprecipitates (IP) were analyzed by Northern blot for the presence of miR-223. −, indicates IP using control IgG. (g) Ago2 IP were analyzed for RISC activity. (h,i) Same as in a, except in the presence of an antisense RNA to miR-223 (+) or Rluc (−) (h), or upon disrupting pairing of either the miR-223 cleavage site (lane 2) or seed region (lane 3) on the sensor transcript (i). * Indicates the expected cleavage products (38–40 nt). Temp., temperature.

(a) Base pairing of the miR-223 seed region to its predicted binding site, wild-type (WT) or mutated (Mut), in the 3′ untranslated region (3′UTR) of P2Y₁₂ messenger RNA (mRNA). (b) Detection of P2Y₁₂ protein (left) and mRNA (right) in extracts of platelets and megakaryocytes by Western blot and RT-PCR, respectively. (c) Functional validation of the predicted binding site for miR-223 present in P2Y₁₂ mRNA 3′UTR by reporter gene assays performed in HEK293 cells (left and center panels) or megakaryocytes (right panel). Results are expressed as mean ± standard error of the mean (s.e.m.) (n = 5 to 7 experiments, in duplicate). * p<0.05; **p<0.001; *** p<0.0001 (two-tailed, unpaired Student’s t-test). (d) Detection of P2Y₁₂ mRNA in Ago2 immunoprecipitates (IP) derived from either megakaryocyte or platelet extracts by RT-PCR. (e) Proposed model for platelet inheritance of a functional miRNA pathway, devoid of its initiation step, from megakaryocyte precursor cells. BS, binding site; miRNP, miRNA-containing ribonucleoprotein complex; RISC, RNA-induced silencing complex.