Trsp KO modulates HSCs’ state and... | American Society of Hematology

$Trsp KO modulates HSCs’ state and elicits NRF2 activation across HSPCs. (A) Frequency of LSK (Lin–c-Kit+Sca1+), LT-HSCs (CD48–CD150+ LSK), and ST-HSCs (CD48–CD150– LSK), megakaryocytic and erythroid-biased MPP2 (CD135–CD150+CD48+ LSK), myeloid-biased MPP3 (CD135–CD150–CD48+ LSK), lymphoid-biased MPP4 (CD135+ LSK), LK (Lin–c-Kit+Sca1–), common myeloid progenitors (CMPs; CD34+FcγR– LK), granulocyte-monocyte progenitors (GMPs; CD34+FcγR+ LK), megakaryocyte-erythrocyte progenitors (MEPs; CD34–FcγR– LK), and CLPs (Lin–c-KitintSca1intCD127+CD135+) in the BM of each group (LSK, LT-HSCs, ST-HSCs, LK, CMPs, GMPs, and MEPs, n = 5 per group; MPP2, MPP3, MPP4, and CLP, n = 3 per group). P values were calculated by a 2-sided Student t test. (B) Representative flow cytometric profiles and percentages of bromodeoxyuridine-positive (BrdU+; S phase), DAPI+ (4N) BrdU– (G2/M phase), and DAPI+ (2N) BrdU– (G0/G1 phase) LT-HSCs in the BM (control group, n = 4 mice; Trsp KO group, n = 3 mice). P values were calculated by a 2-sided Student t test. (C) Volcano plot illustrating differential mRNA expression in Trsp KO Lin–c-Kit+ cells, with selenoproteins highlighted in blue and NRF2 pathway genes highlighted in red. Control mice were pI-pC–treated Trspfl/fl mice, whereas Trsp KO mice represent Trspfl/fl: Mx1-Cre mice (n = 4 per group). (D) GSEA enrichment plot for upregulated and downregulated genes in RNA-seq of Trsp KO vs control in Lin–c-Kit+ cells (n = 4 per group). (E) Identification of distinct hematopoietic clusters in control and KO Lin–c-Kit+ cells base on uniform manifold approximation and projection analysis from scRNA-seq. The estimated fractions of stem, progenitor, and mature cells are labeled and highlighted (n = 1 per group). (F) Violin plots depicting mRNA expression of selenoptoteins (Gpx1, Selh, and Sepw1) in the indicated clusters (HSC, MPP2, MPP3, and MPP4). Box plot and kernel density plot of log2 expression values are shown. P values were reanalyzed using Loupe browser. In the box-and-whisker plots, the 0th, 25th, 50th, 75th, and 100th percentiles and mean (dashed lines) are shown. ∗P < .05; ∗∗∗∗P < .0001. DAPI, 4',6-diamidino-2-phenylindole; FDR, false discovery rate; NES, normalized enrichment score; ns, not significant.$

Trsp KO modulates HSCs’ state and elicits NRF2 activation across HSPCs. (A) Frequency of LSK (Lin^–c-Kit⁺Sca1⁺), LT-HSCs (CD48^–CD150⁺ LSK), and ST-HSCs (CD48^–CD150^– LSK), megakaryocytic and erythroid-biased MPP2 (CD135^–CD150⁺CD48⁺ LSK), myeloid-biased MPP3 (CD135^–CD150^–CD48⁺ LSK), lymphoid-biased MPP4 (CD135⁺ LSK), LK (Lin^–c-Kit⁺Sca1^–), common myeloid progenitors (CMPs; CD34⁺FcγR^– LK), granulocyte-monocyte progenitors (GMPs; CD34⁺FcγR⁺ LK), megakaryocyte-erythrocyte progenitors (MEPs; CD34^–FcγR^– LK), and CLPs (Lin^–c-Kit^intSca1^intCD127⁺CD135⁺) in the BM of each group (LSK, LT-HSCs, ST-HSCs, LK, CMPs, GMPs, and MEPs, n = 5 per group; MPP2, MPP3, MPP4, and CLP, n = 3 per group). P values were calculated by a 2-sided Student t test. (B) Representative flow cytometric profiles and percentages of bromodeoxyuridine-positive (BrdU⁺; S phase), DAPI⁺ (4N) BrdU^– (G2/M phase), and DAPI⁺ (2N) BrdU^– (G0/G1 phase) LT-HSCs in the BM (control group, n = 4 mice; Trsp KO group, n = 3 mice). P values were calculated by a 2-sided Student t test. (C) Volcano plot illustrating differential mRNA expression in Trsp KO Lin^–c-Kit⁺ cells, with selenoproteins highlighted in blue and NRF2 pathway genes highlighted in red. Control mice were pI-pC–treated Trsp^fl/fl mice, whereas Trsp KO mice represent Trsp^fl/fl: Mx1-Cre mice (n = 4 per group). (D) GSEA enrichment plot for upregulated and downregulated genes in RNA-seq of Trsp KO vs control in Lin^–c-Kit⁺ cells (n = 4 per group). (E) Identification of distinct hematopoietic clusters in control and KO Lin^–c-Kit⁺ cells base on uniform manifold approximation and projection analysis from scRNA-seq. The estimated fractions of stem, progenitor, and mature cells are labeled and highlighted (n = 1 per group). (F) Violin plots depicting mRNA expression of selenoptoteins (Gpx1, Selh, and Sepw1) in the indicated clusters (HSC, MPP2, MPP3, and MPP4). Box plot and kernel density plot of log₂ expression values are shown. P values were reanalyzed using Loupe browser. In the box-and-whisker plots, the 0th, 25th, 50th, 75th, and 100th percentiles and mean (dashed lines) are shown. ∗P < .05; ∗∗∗∗P < .0001. DAPI, 4',6-diamidino-2-phenylindole; FDR, false discovery rate; NES, normalized enrichment score; ns, not significant.

This Feature Is Available To Subscribers Only