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. 2012 Aug 20;198(4):561–574. doi: 10.1083/jcb.201111136

Figure 1.

Figure 1.

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.