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. 2023 Jan 31;12:e79196. doi: 10.7554/eLife.79196

Figure 5. Aging triggers RPM loss via mechanisms that resemble ferroptosis and involve proteotoxicity.

(A) Lipid peroxidation was determined in RPMs derived from mice at the indicated age using the Lipid Peroxidation Assay Kit with flow cytometry. (B) Lipid peroxidation was determined in RPMs derived from young, aged, and aged IR mice using the Lipid Peroxidation Assay Kit with flow cytometry. (C) Lipid peroxidation was determined in RPMs derived from dextran- and iron-dextran-injected mice (8 hr post-injection) using the Lipid Peroxidation Assay Kit with flow cytometry. (D) The percentage of RPMs from CD45+ live cells present in the spleen of dextran- and iron dextran-injected mice (8 hr post-injection) was assessed by flow cytometry. (E) Mitochondrial mass and (F) mitochondrial activity were determined in RPMs derived from young, aged, and aged IR mice using MitoTracker Green and TMRE probes, respectively, with flow cytometry. (G) Ultrastructural analyses of mitochondrial morphology in spleen red pulp sections obtained from young, aged, and aged IR mice. Yellow arrows indicate mitochondria. (H) Protein aggregation and (I) cell viability in cultured iRPMs were determined using PROTEOSTAT Aggresome detection kit and a fluorescent Aqua Live/Dead probe, respectively, with flow cytometry. Cells were treated with FAC (150 µM, 24 hr), PR73 mini-hepcidin (2 µg/mL, 24 hr), or exposed to heat shock (HS) stress (42 °C, 4 hr) as indicated. (J) Lipid peroxidation and (K) cell viability of cultured iRPMs were determined using the Lipid Peroxidation Assay Kit and fluorescent Aqua Live/Dead probe, respectively, with flow cytometry. Cells were treated with FAC (150 µM, 24 hr), Liproxstatin-1 (Lip-1; 2 µM, 25 hr) or exposed to heat shock (HS) stress (42 °C, 4 hr) as indicated. Each dot represents one mouse or independent cell-based experiment. Data are represented as mean ± SEM. Welch’s unpaired t-test determined statistical significance between the two groups; statistical significance among the three or more groups was determined by One-Way ANOVA test with Dunnett’s or Tukey’s Multiple Comparison test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Figure 5—source data 1. Related to Figure 5A–F and H–K.

Figure 5.

Figure 5—figure supplement 1. Mitochondrial oxidative stress and ATP levels in young, aged and aged IR RPMs.

Figure 5—figure supplement 1.

(A) The mitochondria-associated ROS levels in young, aged and aged IR RPMs were determined using the MitoSOX Red probe with flow cytometry. (B) Levels of ATP in FACS-sorted RPMs isolated from young, aged, and aged IR mice were measured using ATP Fluorometric Assay Kit. Each dot represents one mouse. Data are represented as mean ± SEM. Statistical significance among the three groups was determined by the One-Way ANOVA test with Tukey’s Multiple Comparison test. ns p>0.05, *p<0.05.
Figure 5—figure supplement 1—source data 1. Related to Figure 5—figure supplement 1A–B.
Figure 5—figure supplement 2. Apoptotic cell death was detected using In Situ Cell Death Detection Kit in spleen sections of young, aged and aged IR mice spleens.

Figure 5—figure supplement 2.

The red pulp (RP) and white pulp (WP) are indicated. Yellow arrows indicate apoptotic cell death. The micrographs are representative of two or three mice per group.