Syndecan-2 regulates HSC quiescence through Cdkn1c. Representative cell cycle analysis of shSdc2 Clone #1–treated (A) and shSdc2 Clone #2–treated (B) and shCtrl-treated Sdc2⁺34^–KSL cells. (C) Percentages of 34^–KSL cells in G₀, G₁, and G₂/S/M phases in each group (n = 4-5 replicates per group). (D) Quantitative reverse transcription polymerase chain reaction analysis for the expression of Cdkn1c in Sdc2⁺ and Sdc2^– BM 34^–KSL cells treated with shCtrl, shSdc2, shCdkn1c, or shSdc2 and shCdkn1c (n = 3-12 replicates per group). (E) Quantitative reverse transcription polymerase chain reaction analysis for the expression of Cdkn1a, Cdkn1b, and p16 in Sdc2⁺ and Sdc2^– BM 34^–KSL cells treated with shCtrl, shSdc2, shCdkn1c, or shSdc2 and shCdkn1c (n = 3-12 replicates per group). (F) Numbers of CFU-GEMMs from 250 Sdc2⁺ HSCs treated with shCtrl, shSdc2, shCdkn1c, or shSdc2 + shCdkn1c. (G) Percentages of Sdc2⁺ HSCs in G₀ after treatment with shSdc2 or shCtrl and 5 µM SIS3 or vehicle for 24 hours (n = 3-6 replicates per group). (H) Percentages of CD150⁺CD48^–34^–KSL LT-HSCs in G₀, G₁, and G₂/S/M phases from Ki-67/7-AAD analysis (n = 3-4 replicates per group). (I) At left, representative flow cytometric analysis showing phospho-SMAD3 expression in 34^–KSL, 34^–KSL Sdc2^–, and 34^–KSL Sdc2⁺ HSCs as baseline and after TGFβ stimulation for 30 minutes; at right, quantification of the percent phospho-SMAD3 cells within each population. (J) Quantification of CFU-GEMMs from 100 Sdc2⁺ HSCs or Sdc2^– HSCs treated with 5 µM SIS3 or vehicle (n = 5 mice, pooled for sort; n = 4 replicates per group). (K) Percent 34^–KSL cells (percentage of live cells) after SIS3 treatment of Sdc2⁺ and Sdc2^– HSCs for 7 days in TSF media (n = 3-6 replicates per group). (L) Volcano plots depicting differentially expressed genes in Sdc2⁺ HSCs vs Sdc2^– HSCs. Differentially expressed genes were detected with parameters of P < .05, and fold change (FC) >2 or less than −2. (M) Heat map depicting Ingenuity Pathway Analysis comparing Sdc2⁺ HSCs and Sdc2^– HSCs (n = 10 mice pooled to sort each population; n = 3 replicates per population; comparisons made between individual samples). (N) Heat map depicting selected differentially expressed genes within Hematological System Development, and (O) Cell Growth and Proliferation genes from Ingenuity Pathway Analysis of 34^–KSL HSCs, Sdc2^– HSCs, and Sdc2⁺ HSCs (n = 10 mice pooled to sort each population; n = 3 replicates per population). Error bars = standard error of the mean; statistics denote 2-way analysis of variance followed by unpaired Student t test. *P < .05, **P < .01, ***P < .001, ****P < .0001. FDR, false discovery rate; ns, not significant; SSC-A, side scatter area.