Fig 2: ELISA analyses of cerebrocortical EPO and BDNF. Cerebrocortical EPO (A) and BDNF (B) contents are significantly greater in the AD+IHT than pre-intervention 3xTg-AD mice. For EPO and BDNF contents, treatment factors are P = 0.008 and P = 0.001, respectively, and time factors are P = 0.047 and P = 0.002, respectively. *P < 0.05 for comparisons indicated by horizontal lines. Individual data points and group mean ± standard error of the mean are shown.

Fig 3: Hypoactivation of JAK-STAT signaling underlies the comprised erythroid progenitor production in Fundc1-/- mice during stress erythropoiesis.(A) Volcano Plot illustrates the differentially expressed genes (DEGs) of FACS-purified erythroid progenitors (R2 populations) in WT (n = 3) and Fundc1-/- (n = 3) spleen at 48 hr after PHZ treatment. Top 10 genes in each group were labeled. (B) Top 5 KEGG functional enrichment pathways analysis of downregulated DEGs in Fundc1-/- R2 cells. (C) GSEA showing the enrichment of the JAK-STAT signaling pathway in splenic R2 cells from WT and Fundc1-/- mice. Normalized enrichment score (NES) and false discovery rate (FDR) are shown. (D) The box plot depicts the quantitative analysis of the genes in JAK-STAT signaling pathway in splenic R2 cells of WT and Fundc1-/- mice. The boxes represent the median and quartile of the sum of log2 transformed (FPKM +1). p-value was determined by using paired Wilcoxon signed-rank Test. (E) The expression of key components in the JAK-STAT pathway in splenic R2 cells from WT and Fundc1-/- mice. (F) Western blotting of STAT3, STAT5 and phosphorylated STAT3 and STAT5 in FACS-sorted R2 populations in spleen of WT and Fundc1-/- mice after 48 hr of PBS or PHZ treatment, respectively. ACTIN was used as a loading control. n = 3. (G) The quantification of the signal intensity of phosphorylated STAT3 and STAT5 out of the total STAT3 and STAT5 in (F). (H) The expression of Epor mRNA in the spleen of WT and Fundc1-/- mice after 48 hr of PBS or PHZ treatment. (I) The serum EPO concentration in Fundc1-/- and WT mice after 48 hr of PBS or PHZ treatment. (J) Schematic illustrating the administration of EPO to PHZ-Fundc1-/- and PHZ-WT mice. (K) RBC number (upper) and HGB level (bottom) after EPO supplement in PHZ-Fundc1-/- and PHZ-WT mice. (L) Erythroid differentiation in spleen of PHZ-Fundc1-/- and PHZ-WT after EPO administration. The left panel shows representative FACS plots and right bar graph shows the statistical quantification of the percentage of the R2 compartment. For each experiment (E–L), n = 3–5 mice for each group. Individual mice are represented by symbols. Data shown are representative of at least three independent experiments. Similar results were found in each experiment. All data are mean ± SEM; *: p<0.05; ns: no statistical significance. Statistical significance was analyzed by using the two-tailed unpaired Student’s t-test unless stated otherwise. Figure 2—source data 1.The JAK-STAT signaling in WT and Fundc1-/- mice during stress erythropoiesis.

Fig 4: Impaired mitophagy triggers inflammation and reduced Epo expression in REPs.(A) Representative immunofluorescence images (left) of PDGFRß and a-SMA co-staining in renal tissue sections from WT and Fundc1-/- mice after 48 hr of PBS or PHZ treatment, respectively. The right bar graph shows the percentage of a-SMA positive fibrotic cells out of PDGFRß labeled REPs. For quantification, approximately 10 fields for each mouse (200×) were randomly selected to evaluate the frequency of a-SMA+ cells out of PDGFRß+ cells. Scale bars, 30 µm. (B) The schematic diagram showing the procedures of isolating REPs for flow cytometry assay. (C) The representative FACS plots showing the sorting strategy for PDGFRß+ REPs. (D) Epo mRNA expression in PDGFRß- and PDGFRß+ cells by qRT-PCR. (E) Western blotting showing protein levels of mitochondrial membrane proteins (TIMM23, COXIV, and TOMM20) and autophagic proteins LC3 in FACS-sorted PDGFRß+ REPs from WT and Fundc1-/- mice under the treatment of hypoxia (1% O2). (F) The quantification of the signal intensity of indicated mitochondrial membrane proteins (TIMM23, COXIV and TOMM20) and ratio of LC3II/I from (E). (G) Mitochondrial mass examined by using MitoTracker Green in FACS-sorted PDGFRß+ REPs in WT and Fundc1-/- mice with or without PHZ treatment. (H) Flow cytometry analysis of mitophagy in REPs of mt-Keima/WT and mt-Keima/Fundc1-/- mice after 48 hr of PBS or PHZ treatment. (I) mtDNA copy number in FACS-purified REPs in WT and Fundc1-/- mice with or without PHZ treatment. (J, K) Mitochondrial membrane potential detected by using TMRM (J) or ROS level (K) measured by using MitoSOX in FACS-purified REPs of WT or Fundc1-/- mice after 48 hr of PBS or PHZ treatment. (L, M) Relative mRNA levels of proinflammatory cytokines Il1b, Il6 (L) and Epo (M) in sorted PDGFRß+ REPs in WT or Fundc1-/- mice after 48 hr of PBS or PHZ treatment, respectively. For each experiment, n = 3–5 mice for each group. Individual mice are represented by symbols. Data shown are representative of at least three independent experiments. Similar results were found in each experiment. All data are mean ± SEM; *p<0.05, **p<0.01. Statistical significance was analyzed by using the two-tailed unpaired Student’s t-test. Figure 4—source data 1.Inflammation and Epo expression in REPs.

Fig 5: Impaired mitophagy and reduced EPO production in Fundc1-/- mice during cisplatin-induced renal anemia.(A) Schematic diagram showing the generation of renal anemia disease model by cisplatin. (B) Hemogram parameters of red blood cell (RBC) counts and hemoglobin (HGB) in the peripheral blood of WT controls and Fundc1-/- mice after PBS or cisplatin treatment. (C) The serum EPO concentration in Fundc1-/- and WT mice after PBS or cisplatin treatment. (D) Western blotting (left) and the corresponding quantification (right) showing the protein levels of mitochondrial membrane proteins (TOMM20 and COXIV) and autophagic proteins (P62 and ratio of LC3II/I) in kidneys of WT and Fundc1-/- mice with PBS or cisplatin treatment. (E) Relative mRNA levels of Il6, Il1b, and Tgfb1 in kidneys of WT and Fundc1-/- mice with or without cisplatin treatment. (F, G) Flow cytometry analysis of mitophagy in PDGFRß+ REPs of mt-Keima/WT and mt-Keima/Fundc1-/- mice after PBS or cisplatin treatment. The representative FACS plots (F) and the quantification (G) are shown. (H) Mitochondrial mass detected by using MitoTracker Green via FACS in PDGFRß+ REPs of WT or Fundc1-/- mice after PBS or cisplatin treatment. (I) Mitochondrial membrane potential detected by using TMRM by FACS in PDGFRß+ REPs of WT or Fundc1-/- mice after PBS or cisplatin treatment. (J) Mitochondrial ROS level detected by using MitoSOX via FACS in PDGFRß+ REPs of WT or Fundc1-/- mice after PBS or cisplatin treatment. (K) Relative mRNA levels of proinflammatory cytokines Il6 (left) and Tnfa (right) in sorted PDGFRß+ REPs in WT or Fundc1-/- mice after PBS or cisplatin treatment. (L) Relative mRNA levels of Epo in sorted PDGFRß+ REPs in WT or Fundc1-/- mice after PBS or cisplatin treatment, respectively. (M) A hypothetic model depicting the role of FUNDC1 in REPs. FUNDC1-mediated mitophagy is required for mitochondrial steady-state homeostasis. However, under stresses, the damaged mitochondria are accumulated in Fundc1-/- REPs due to impaired mitophagy. Consequently, elevated ROS levels from these damaged mitochondria of REPs incur inflammatory responses by enhancing the expression of proinflammatory cytokines TNFa, IL6 and IL1b, which in turn promote myofibroblastic transformation of REPs, resulting in the loss of EPO generation and subsequently anemia. For each experiment, n = 3–5 mice for each group. Individual mice are represented by symbols. Data shown are representative of at least three independent experiments. Similar results were found in each experiment. All data are mean ± SEM; *p<0.05, **p<0.01, ***p<0.001. Statistical significance was analyzed by using the two-tailed unpaired Student’s t-test. Figure 6—source data 1.Mitophagy and EPO production during cisplatin-induced renal anemia.