Figure 5.
Igf2bp2 regulates mitochondrial activity in HSPCs of mice. (A-B) Freshly isolated, total CD150+ (high and low) HSCs (CD150+CD34−LSK) from wild-type males (range: 24-27 months) were virally infected with Igf2bp2 cDNA or an empty vector (control). After transduction (2.5 days), transduced cells (CD48−LSKs) were re-sorted for proteomics analysis (n = 5 replicates per group). (A) Ingenuity Pathway Analysis (IPA) on differentially expressed proteins, Fisher’s exact test with Benjamini-Hochberg–corrected P values. (B) The differentially expressed proteins related to protein synthesis (marked by blue asterisks) metabolism (marked by red asterisks). Volcano plots on differentially expressed proteins related to protein synthesis (left, marked by blue asterisks in panel A) and metabolism (right, marked by red asterisks in panel A) in Igf2bp2-overexpressing cells compared with control cells. Relative quantification was performed in Spectronaut for each pairwise comparison using the replicate samples from each condition. (C-D) Freshly isolated LSK (lineage−cKit+Sca1+) from young mice (range: 3-6 months) and total CD150+ (high and low) HSCs (CD150+CD34−LSK) and multipotent progenitors (CD34+LSKs = MPPs) from old mice (range: 22-26 months) were used for respirometry analysis of Igf2bp2−/− vs Igf2bp2+/+ mice. (C) Quantification of oxygen consumption rates (OCRs) for basal respiration, ATP-linked respiration and maximal respiration of cells of the indicated genotype and age (young LSK cells, 9-10 mice per genotype, 2 independent experiments; old HSCs, 6 to 8 mice per genotype, 2 independent experiments; and old MPPs, 9 to 10 mice per genotype, 3 independent experiments). Linear modeling and analysis of variance (ANOVA; using "program package R v3.6.3, function aov()") on genotype and all respirometric parameters revealed a significant reduction of the OCR in LSK cells of young mice (P = .0002) but not in HSCs (P = .0592) or MPPs (P = .0819) of old mice. (D) Histogram of the ATP production rate. Young LSKs (left): 11 to 13 mice per genotype, 4 independent experiments; old HSCs (right), 8 to 11 mice per genotype, 3 independent experiments. Statistical analysis was performed with Welch’s t test. (E-F) Transduced HSCs were cultured with dimethyl sulfoxide (DMSO; control; circles) or inhibitors of PI3K (squares) or rapamycin (triangles) starting 12 hours after transduction. (E) The mitochondrial potential of transduced CD48− LSK cells was determined 2 days after culture initiation by MitoRed fluorescence-activated cell sorting (FACS) analysis. The mean fluorescence intensity of MitoRed was normalized to DMSO treated, vector-transduced cells set to 1. A total of 11 mice per group in 3 independent experiments. (F) The absolute number of DAPI−GFP+CD11b+ cells in the indicated groups was analyzed 7 days after culture initiation by FACS (n = 7 mice per group in 3 independent experiments). (E-F) Statistical analysis by 2-way ANOVA on log-transformed data followed by pairwise t tests with Sidak’s correction for multiple comparisons. All data are expressed as the mean ± SD; ns, nonsignificant.

Igf2bp2 regulates mitochondrial activity in HSPCs of mice. (A-B) Freshly isolated, total CD150+ (high and low) HSCs (CD150+CD34−LSK) from wild-type males (range: 24-27 months) were virally infected with Igf2bp2 cDNA or an empty vector (control). After transduction (2.5 days), transduced cells (CD48−LSKs) were re-sorted for proteomics analysis (n = 5 replicates per group). (A) Ingenuity Pathway Analysis (IPA) on differentially expressed proteins, Fisher’s exact test with Benjamini-Hochberg–corrected P values. (B) The differentially expressed proteins related to protein synthesis (marked by blue asterisks) metabolism (marked by red asterisks). Volcano plots on differentially expressed proteins related to protein synthesis (left, marked by blue asterisks in panel A) and metabolism (right, marked by red asterisks in panel A) in Igf2bp2-overexpressing cells compared with control cells. Relative quantification was performed in Spectronaut for each pairwise comparison using the replicate samples from each condition. (C-D) Freshly isolated LSK (lineage−cKit+Sca1+) from young mice (range: 3-6 months) and total CD150+ (high and low) HSCs (CD150+CD34−LSK) and multipotent progenitors (CD34+LSKs = MPPs) from old mice (range: 22-26 months) were used for respirometry analysis of Igf2bp2−/− vs Igf2bp2+/+ mice. (C) Quantification of oxygen consumption rates (OCRs) for basal respiration, ATP-linked respiration and maximal respiration of cells of the indicated genotype and age (young LSK cells, 9-10 mice per genotype, 2 independent experiments; old HSCs, 6 to 8 mice per genotype, 2 independent experiments; and old MPPs, 9 to 10 mice per genotype, 3 independent experiments). Linear modeling and analysis of variance (ANOVA; using "program package R v3.6.3, function aov()") on genotype and all respirometric parameters revealed a significant reduction of the OCR in LSK cells of young mice (P = .0002) but not in HSCs (P = .0592) or MPPs (P = .0819) of old mice. (D) Histogram of the ATP production rate. Young LSKs (left): 11 to 13 mice per genotype, 4 independent experiments; old HSCs (right), 8 to 11 mice per genotype, 3 independent experiments. Statistical analysis was performed with Welch’s t test. (E-F) Transduced HSCs were cultured with dimethyl sulfoxide (DMSO; control; circles) or inhibitors of PI3K (squares) or rapamycin (triangles) starting 12 hours after transduction. (E) The mitochondrial potential of transduced CD48− LSK cells was determined 2 days after culture initiation by MitoRed fluorescence-activated cell sorting (FACS) analysis. The mean fluorescence intensity of MitoRed was normalized to DMSO treated, vector-transduced cells set to 1. A total of 11 mice per group in 3 independent experiments. (F) The absolute number of DAPI−GFP+CD11b+ cells in the indicated groups was analyzed 7 days after culture initiation by FACS (n = 7 mice per group in 3 independent experiments). (E-F) Statistical analysis by 2-way ANOVA on log-transformed data followed by pairwise t tests with Sidak’s correction for multiple comparisons. All data are expressed as the mean ± SD; ns, nonsignificant.

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