Experimental validation of trackSeq candidate Itgb4. (A) Identifying CC-independent expression differences. Histogram of absolute transcriptome-inferred pseudo time (ie, CC) difference between paired daughters. Cutoff at <0.2 excludes 63 pairs. (B) Venn diagram of top 500 trackSeq candidates including or excluding sister pairs with CC differences. Gene set size and pathway/process enrichments from STRING are shown. (C) Itgb4 is a central node of the ECM-receptor interaction pathway indicating a CC-independent putative niche retention mechanism regulated by ACD. A total of 206 CC-independent trackSeq candidates mapped to STRING database, with unconnected nodes omitted. Yellow nodes = candidates of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ECM-receptor interaction. (D) Itgb4 is a weakly expressed LysoLow trackSeq candidate. Plot as in Figure 2B; top 500 trackSeq candidate genes in blue. (E) Higher ITGB4 protein expression in ACD LysoLow than in LysoHigh HSC daughters. ITGB4 detection by proximity ligation assay immunostaining. Eighteen ACD (lysosome ratio >1.6×) pairs from 2 replicates. P value from paired t test. Whiskers = minimum–maximum. Example ACD HSC daughter pair after PLA (top). Scale bar = 5 μm. (F) Surface expression of ITGB4 and dimerization partner ITGA6 quantified by fluorescence-activated cell sorting. Their expression decreases during HSC differentiation (middle). ITGB4 expression varies in HSCs (right). (G) ITGB4-high HSCs are more adherent to LAMININ than ITGB4-low HSCs. Mean HSC displacement per time quantified by time-lapse imaging. t test. 53/48 ITGB4-low/high HSCs from 3 replicates. (H) Higher LAMININ adhesion of LysoLow (higher Itgb4 expression) than LysoHigh HSC daughters. Quantification of ACD (lysosome ratio >1.6×) HSC daughter displacement on LAMININ 511 (bound by ITGB4) or 421 (not bound by ITGB4). 32/33 pairs for LAMININ 511/421. Paired t tests from 3 replicates; error bars = minimum–maximum. GO, gene ontology.