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. 2022 Jun 20;24(7):1038–1048. doi: 10.1038/s41556-022-00931-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, Experimental design of GPRC5C OE in mPB and CB. Internal ribosomal entry site (ires). b, FACS validation of GPRC5C OE at protein level. n = 3 experiments. c, FACS-based cell cycle analysis of GPRC5C OE and control. n = 6 experiments. d, SiC division assay of GPRC5C OE and control quantified after 48 h in vitro culture. n = 4 experiments. e, qPCR of CDK6 expression. Normalized to housekeeping gene GAPDH and control. n = 6 experiments for GPRC5C OE and n = 12 experiments for control. f, Surface expression of CD34⁺EPCR⁺ in GPRC5C OE and control. n = 6 experiments. g, Surface expression of CD34⁺CD90⁺ in GPRC5C OE and control. n = 4 experiments. h, Volcano plot depicting DEGs between GPRC5C OE and control. Key genes associated with dormancy and stemness are highlighted. BH-adjusted P values after Wald test. log₂FC ≥0.5; adjusted P < 0.1. i, Smoothed pseudo-temporal expression profile of the GPRC5C-OE signature in LT-HSCs. Smoothed profiles were computed by local regression. The y axis indicates normalized expression. j, GSEA of KEGG pathways in GPRC5C OE compared with control. Performed with BH-adjusted P values after adaptive multi-level splitting Monte Carlo approach. k, FACS measurement of OP-Puro MFI in GPRC5C OE and control. n = 5 experiments. l, FACS measurement of MitoTracker Red MFI in GPRC5C OE and control. n = 7 experiments. m, Transplantation of GPRC5C OE and control in CB cells from two biological replicates. Engraftment was measured from BM and calculated by the ratio of the total percentage GFP⁺hCD45⁺ to the percentage input GFP post-transplantation and normalized to control. n = 5 control and n = 6 GPR-OE biological replicates. n, Lineage distribution of GPRC5C OE and control in CB cells. Distribution of myeloid (CD33), B-lymphoid (CD19) and immature surface phenotype (CD34) are presented. n = 5 control and n = 6 GPR-OE biological replicates. o, Secondary transplantation of 150,000 GFP⁺hCD45⁺ cells. n = 4 biological replicates. All data presented as mean ± s.d. Statistical significance was determined using two-tailed t-test (b, e, f, g, l, m, n and k) or two-way ANOVA (c and d). n indicates number of replicates. For all experiments, at least two independent experiments were performed. At least two human mPB or CB donors were used. Source numerical data are available in source data.

Source data