Page 330 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
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BCD
Figure 3. Molecular qualities of proplatelet-promoting factor. (A) Table showing molecular treatments done to S100 derived from proplatelet-producing megakaryocytes (MK), and corresponding percentage of MK making proplatelets after 1 hour (hr) when treated S100 was microinjected into round MK (indicated in red). UV irradiation was carried out at 254 nm. S100 was irradiated on ice and placed beneath GE germicidal lamps (emax = 254 nm) at approximately 5 cm. A Spectroline UV dosimeter was used to measure the delivered doses, calculated at 7.2 + 1.2 J/m2/sec. The calculated dose on the S100 was 10,000 J/m2. For high-speed centrifugation, S100 was centrifuged at 18,000 g for 90 minutes (min) in a Thermo Scientific Sorvall Lynx 4000 centrifuge. For size separation, 100 μL of S100 was placed in a Millipore filter with a molecular weight cut off at 5 kDa and centrifuged for 2 hr. The lower layer was collected, and upper layer reconstituted to the original volume in 10 mM KH2HPO4 (pH 6.8) buffer. Proteinase K, trypsin, PMSF, sodium dodecyl sulfate, puromycin, Triton X-100, Nonidet P-40, octyl glucoside, and Tween-20 were all obtained from Sigma. Proteinase K digestion was carried out at 100 μg/mL for 1 hr at 37°C. PhMeSO2F (PMSF) was used at 0.1 mM to inhib- it proteinase K. Digestion with RNase A or DNase I was carried out at 50 μg/mL for 1 hr. Trypsin was used at 100 μg/mL for 1 hr. For phenol extraction, prior to extraction with phenol, cytosol was suspended in 30 mM Tris-acetate, pH 8.3. Equal volumes of cold phenol were used to extract the samples at 4°C. Phase separation was accomplished by centrifugation at 4,000 g for 10 min at 4°C. The aqueous phase was recovered. N=3 biological replicates. Data are presented as mean + SD. (B) Puromycin treatment (250 μg/mL) caused a block in proplatelet formation10 (4.6+1.7% proplatelet production). Representative images of 8-hr time-point after puromycin treatment (original magnification 40x). (C) S100 from proplatelet-producing MK was microinjected into puromy- cin-treated MK, which triggered proplatelet production. Representative images of 1-hr time-point (original magnification 40x) after injection of S100 obtained from proplatelet-producing MK (71.6+8.2% proplatelet initiation). Scale bars = 20 μm. (D) Pro- platelet production in the presence of puromycin and after microinjection with S100 from proplatelet-producing megakaryocytes. N=40 cells per condition per replicate. N=3 biological replicates. Data are presented as mean + SD. ****P=0.0001. PPF: proplate- let-promoting factor.
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