Figure 7
Figure 7. Molecular mechanisms of necdin in regulating the response of HSCs to irradiation. (A) Transcript profiling of LSK cells from mice reconstituted with wild-type (WT) and necdin-null (Ndn null) fetal liver cells after a sublethal dose of irradiation (6.5 Gy) were analyzed by Affymetrix oligonucleotide array. Genes that are differentially expressed between wild-type and necdin-null HSPCs are shown. (B) The relative mRNA expression level of Gas2L3 in LSK cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio (± SD) of transcript levels relative to that of wild-type cells (n = 2). (C) Effect of down-regulating Gas2L3 expression on the HSPC response to irradiation. Wild-type or necdin-null LSK cells were nucleofected with control or Gas2L3-directed siRNAs. Twenty-four hours after nucleofection, the cells, which showed efficient Gas2L3 knockdown in LSK cells by quantitative PCR (left panel), were irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P < .0001 by 1-way ANOVA, n = 6, right panel). Significant differences were observed between control/wild-type and Gas2L3 knockdown/wild-type, control/wild-type and control/necdin-null, control/wild-type and Gas2L3 knockdown/necdin-null, Gas2L3 knockdown/wild-type and control/necdin-null, and control/necdin-null and Gas2L3 knockdown/necdin-null. (D) Effect of Gas2L3 overexpression on the HSPC response to irradiation. Gas2L3 overexpression in LSK cells was confirmed by quantitative PCR (n = 2, left panel); the cells were then irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P = .01, n = 5, right panel). (E) Green fluorescent protein–positive HSCs (Lin−Sca-1+c-Kit+CD48−CD150+) from the BM of the mice repopulated with wild-type control, wild-type Gas2L3 knocked-down 1 (KD1), wild-type Gas2L3 KD2, necdin-null control, necdin-null Gas2L3 KD1, or necdin-null Gas2L3 KD2 fetal liver cells were assessed for apoptosis 12 hours after a dose of total-body irradiation (6.5 Gy) using DAPI and annexin V staining. Data shown are the mean percentage ± SD of the annexin V+/DAPI− population within HSCs (P < .0001 by 1-way ANOVA). Significant differences were observed between wild-type control and wild-type Gas2L3 KD1, wild-type control and wild-type Gas2L3 KD2, wild-type control and necdin-null control, wild-type control and necdin-null Gas2L3 KD1, wild-type control and necdin-null Gas2L3 KD2, wild-type Gas2L3 KD1 and necdin-null control, wild-type Gas2L3 KD2 and necdin-null control, necdin-null control and necdin-null Gas2L3 KD1, and necdin-null control and necdin-null Gas2L3 KD2. (F) The relative mRNA expression levels of Gas2L3 in CD48−CD150+LSK cells, CD34−LSK cells, and CD34+LSK cells isolated from mice before and after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio of transcript levels relative to the wild-type cells (± SD; n = 3).

Molecular mechanisms of necdin in regulating the response of HSCs to irradiation. (A) Transcript profiling of LSK cells from mice reconstituted with wild-type (WT) and necdin-null (Ndn null) fetal liver cells after a sublethal dose of irradiation (6.5 Gy) were analyzed by Affymetrix oligonucleotide array. Genes that are differentially expressed between wild-type and necdin-null HSPCs are shown. (B) The relative mRNA expression level of Gas2L3 in LSK cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio (± SD) of transcript levels relative to that of wild-type cells (n = 2). (C) Effect of down-regulating Gas2L3 expression on the HSPC response to irradiation. Wild-type or necdin-null LSK cells were nucleofected with control or Gas2L3-directed siRNAs. Twenty-four hours after nucleofection, the cells, which showed efficient Gas2L3 knockdown in LSK cells by quantitative PCR (left panel), were irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P < .0001 by 1-way ANOVA, n = 6, right panel). Significant differences were observed between control/wild-type and Gas2L3 knockdown/wild-type, control/wild-type and control/necdin-null, control/wild-type and Gas2L3 knockdown/necdin-null, Gas2L3 knockdown/wild-type and control/necdin-null, and control/necdin-null and Gas2L3 knockdown/necdin-null. (D) Effect of Gas2L3 overexpression on the HSPC response to irradiation. Gas2L3 overexpression in LSK cells was confirmed by quantitative PCR (n = 2, left panel); the cells were then irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P = .01, n = 5, right panel). (E) Green fluorescent protein–positive HSCs (Lin−Sca-1+c-Kit+CD48−CD150+) from the BM of the mice repopulated with wild-type control, wild-type Gas2L3 knocked-down 1 (KD1), wild-type Gas2L3 KD2, necdin-null control, necdin-null Gas2L3 KD1, or necdin-null Gas2L3 KD2 fetal liver cells were assessed for apoptosis 12 hours after a dose of total-body irradiation (6.5 Gy) using DAPI and annexin V staining. Data shown are the mean percentage ± SD of the annexin V+/DAPI− population within HSCs (P < .0001 by 1-way ANOVA). Significant differences were observed between wild-type control and wild-type Gas2L3 KD1, wild-type control and wild-type Gas2L3 KD2, wild-type control and necdin-null control, wild-type control and necdin-null Gas2L3 KD1, wild-type control and necdin-null Gas2L3 KD2, wild-type Gas2L3 KD1 and necdin-null control, wild-type Gas2L3 KD2 and necdin-null control, necdin-null control and necdin-null Gas2L3 KD1, and necdin-null control and necdin-null Gas2L3 KD2. (F) The relative mRNA expression levels of Gas2L3 in CD48−CD150+LSK cells, CD34−LSK cells, and CD34+LSK cells isolated from mice before and after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio of transcript levels relative to the wild-type cells (± SD; n = 3).

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