Fig 1: Characterization of EPO knockdown mice. (A) Renal Epo mRNA expression and (B) serum EPO levels at month 3 after DOX initiation and (C) hematocrit changes at months 0 to 3 after DOX initiation in shEPOrtTAPOS (n=9) and shEPOrtTANEG controls (n=7). (D) Total splenocytes, (E) CD8+ T cells, (F) CD4+ T cells, and (G) Treg in the spleen at 3 months after DOX initiation. Data were analyzed using (A, B) t test, *P < 0.05; (C) Multiple Mann–Whitney test, ****P <0.00001; (D–G) Mann–Whitney U test, *P <0.01, ns: not significant.
Fig 2: Downregulating endogenous EPO production increases IL-6 and MCP-1 production in macrophages. Intracellular cytokine production in CD11b+ splenic macrophages isolated from B6.MRL/lpr shEPOrtTAPOS (n=12) and shEPOrtTANEG controls (n=10) at day 145 after DOX food initiation (flow cytometry). Mann–Whitney U test; ns, not significant, **P < 0.01; ***P < 0.001 between B6.MRL/lpr shEPOrtTAPOS and shEPOrtTANEG groups.
Fig 3: Effects of endogenous EPO downregulation on GC B-cell and T-cell subsets. (A, B) Representative plots and data quantification for splenic B220+Fas+GL7+ GC B cells; (C, D) CD4+PD1+CXCR5+Foxp3- TFH and CD4+PD1+CXCR5+Foxp3+ TFR; (E, F) total CD4+ T cells and (G, H) CD8+ T cells; (I, J) CD4+CD25+Foxp3+ Treg in B6.MRL/lpr shEPOrtTAPOS (n=14) and shEPOrtTANEG (n=14) at day 145 after DOX food initiation. Mann–Whitney U test; ns, not significant, *P < 0.05; **P < 0.01; ***P < 0.001 between B6.MRL/lpr shEPOrtTAPOS and shEPOrtTANEG groups.
Fig 4: Validation of inducible Epo downregulation in B6.MRL/lpr mice. (A) Experimental design. (B) Epo mRNA expression in kidney from B6.MRL/lpr shEPOrtTAPOS (n=23) and shEPOrtTANEG controls (n=15). (C) Hematocrit levels in B6.MRL/lpr shEPOrtTAPOS and shEPOrtTANEG mice at 2 months after DOX initiation. t test; *P < 0.05.
Fig 5: Downregulating endogenous EPO production increases disease severity and shortens survival in a murine lupus model. (A) Anti-dsDNA autoantibody levels in B6.MRL/lpr shEPOrtTAPOS (n=12) and shEPOrtTANEG (n=9). (B) Clinical score of skin lesions developed in B6.MRL/lpr shEPOrtTAPOS (n=23) compared with shEPOrtTANEG controls (n=14). (C) Albumin-to-creatinine ratio (ACR) in B6.MRL/lpr shEPOrtTAPOS (n=19) and shEPOrtTANEG (n=15) mice. (D) Representative images of PAS staining of kidney tissue at sacrifice; scale bars: 20–100 μm; magnification 10×–40×. Multiple Mann–Whitney test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between B6.MRL/lpr shEPOrtTAPOS and shEPOrtTANEG. (E) Survival (%) of B6.MRL/lpr shEPOrtTAPOS (n=20) and shEPOrtTANEG (n=14) mice; log-rank (Mantel–Cox) test; *P < 0.05.
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