Loss of Sphk2 has no effect on positioning, motility, or size of MKs or PP formation. (A) Representative in vivo images of YFP⁺ MKs (green) and vasculature (red) in mouse BM. Images were captured through a ×20 water immersion objective lens with NA = 0.95 using a BioTech TriM Scope system. WT × CD41-YFP^ki/+ or Sphk2^−/− × CD41-YFP^ki/+ naïve (nontransplanted) mice (upper row); WT × CD41-YFP^ki/+ or Sphk2^−/− × CD41-YFP^ki/+ BM chimeric mice (lower row). (B) Instantaneous lateral (x-y) velocity of MKs. Data were pooled from 3 mice per group. (C) Distance of MKs from BM sinusoids. Red lines, medians. Data were pooled from 3 mice in each group. (D) Surface area of MKs in femoral BM. Red lines, medians. Data were pooled from 3 mice per group. (E) The percentage of MKs displaying PP formation (PPF) (3 independent experiments performed in triplicate). (F) Intrasinusoidal PP formation in WT × CD41-YFP^ki/+ or Sphk2^−/− × CD41-YFP^ki/+ naïve (nontransplanted) mice. MKs displaying intrasinusoidal PPF in vivo are presented as the percentage of all MKs carrying PPs (50-76 MKs per group, 3 independent experiments per genotype). (G) Intrasinusoidal PP formation in WT × CD41-YFP^ki/+ or Sphk2^−/− × CD41-YFP^ki/+ BM chimeras mice. MKs displaying intrasinusoidal PPF in vivo represented as the percentage of all MKs displaying PPs (49 MKs per group, 3 independent experiments per genotypes). (H) Representative MP-IVM images of MKs with YFP⁺ PPs. Images were captured through a ×20 water immersion objective lens with NA = 0.95 using a BioTech TriM Scope system. Green, MKs and PP; red, sinusoids; arrowheads, intrasinusoidal YFP⁺ PPs. The dashed line highlights the sinusoids. All scale bars represent 20 µm. All error bars represent SEM.