MKs up-regulate Tlr9 transcript expression during
pro-PLT production and express TLR9 in distinct granules that
partially colocalize with VAMP 8 at every stage in
thrombocytopoiesis. (A and B) Shown is a screen shot from
Integrated Genome Browser of reads distributing to the
Tlr9 (A) and Vamp8 (B) locus. The
black bars represent piled up sequencing reads aligning to the genomic
coordinates encoding the respective RNAs. More reads will align to more
highly expressed RNA regions, and the height of the black bars
correlates with RNA expression level. Below the read distributions are
the RefSeq annotations: thick horizontal lines represent exons, and thin
horizontal lines represent introns. Quantification was based on three
round MK and four pro-PLT–MK replicates and is expressed in reads
per kilobase of exon model per million mapped reads. (C) Intermediates
of PLT production from murine fetal liver cell cultures were spun down
onto poly-l-lysine–coated glass cover slides,
permeabilized with 0.5% Triton X-100 for 5 min, and probed for TLR9 and
either β1-tubulin or VAMP 8. Slides were examined by fluorescence
microscopy, and image fluorescence intensity is normalized to the round
MK fraction. Round MKs, pro-PLT–MKs, released pro-PLTs, and
individual PLTs revealed distinct, punctuate/granular localization of
TLR9 similar to that observed in whole-blood PLTs. β1-Tubulin
antibodies were used to delineate the cell periphery and denote the
different intermediate stages in PLT production. Insets (from top to
bottom) represent a PLT, pre-PLT, and barbell pro-PLT. (D) Background
fluorescence was subtracted, and image brightness/contrast was adjusted
linearly for each micrograph to resolve individual granules. TLR9 showed
significant colocalization with VAMP 8 in round MKs, along the pro-PLT
shafts of pro-PLT–MKs, and within released pro-PLTs and
individual PLTs. A Manders’ coefficient of 0.64 was calculated
for VAMP 8 colocalization with TLR9 throughout the entirety of MK cell
culture. Insets represent the magnified region outlined by the yellow
box for each image.