Fig. 5

Further investigation of HES1 and CD9 in BM. a Volcano plots highlighting significant differences in gene expression between ITP and HC in HSC (left), MkP1 (middle), and MkP2 (right). Red points represent significantly upregulated genes, blue points represent significantly downregulated genes, and gray points represent non-DEGs. Genes with an adjusted p value < 0.05, log-transformed fold change absolute value >0.25, and minimum percentage >0.25 were considered as differentially expressed genes. b Violin plot representing the expression levels of HES1 and CD9 in HSC, MkP1, and MkP2. ***adjusted p value < 0.001. ns, not significant. c Flowchart. Related to Fig. 5d–j. d Expression of HES1 and CD9 in Lin⁻CD34⁺CD45RA⁻ HSPCs of ITP and HC samples. e Box plots showing the proportion of CD9 (left) and HES1 (right) in Lin⁻CD34⁺ CD45RA⁻ HSPCs of ITP (n = 27) and HC (n = 12) samples. ***p <0.001 by two-tailed Student’s t test. f Expression of CD41 and CD235a in cultures of CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs on the indicated days. Cells varied in size and granularity at different culture time-points, thus the voltages for flow cytometric analysis were adjusted accordingly, resulting in inconsistent thresholds. g Proportion of CD41⁺CD235a⁻ and CD235a⁺CD41⁻ cells on days 22, 26, and 28 after culturing flow-sorted CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs. Error bars, mean ± S.E. Data were subjected to variable transformation (arcsine square root transformed) and analyzed using one-way ANOVA. ITP, n = 5; HC, n = 4. ***p < 0.001. h Representative immunofluorescence microscopy images (left panel) showing the morphology of megakaryocytes after culture. Scale bar, 10 μm. Representative light microscopy images of Wright–Giemsa-stained cytospins (right panel) showing the morphology of megakaryocytes after culture. i The expression of CD41a, and CD61 in cultures of CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs on day 28. j Box plots showing the proportion of CD41a⁺CD61⁺ cells in cultures of CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs flow^-sorted from ITP and HC BM samples. Data were subjected to variable transformation (arcsine square root transformed) and analyzed using one-way ANOVA with Scheffe’s post hoc test. ITP, n = 5; HC, n = 4. ***p < 0.001. ns, not significant