Journal: Science advances
Article Title: PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases.
doi: 10.1126/sciadv.adr8648
Figure Lengend Snippet: Fig. 3. Loss of Ppdpf impairs mitochondrial function. (A) The stress score of control, UUO 1d, and UUO 5d kidney samples from scRNA-seq. (B) UMAP plot of the stress score. (C) KEGG enrichment analysis in UUO 1d-specific PT cells by scRNA-seq. (D) Representative transmission electron microscopy images of WT and Ppdpf KO kidney tubules (8 weeks old). (E) Quantifications of damaged mitochondria in (D). (F) Relative mtDNA levels in WT and Ppdpf KO kidneys analyzed by amplifying ND1 and 16S genes and normalized against the hexokinase 2 (HK2) gene. (G) Complex I enzyme activity of kidneys from WT and Ppdpf KO mice. (H) Left: Western blots of complex I (NDUFB9 and NDUFS3) and complex II (SDHA and SDHC) in primary cells of WT and Ppdpf KO kidneys. Right: Quantifications of NDUFB9 and NDUFS3. (I) Quantifications of fluorescent JC1 signaling (green/red ratio) and fluorescent mitoSOX signaling in primary cells from WT and Ppdpf KO mice. (J) Representative images and quantifica- tions of MitoTraker of primary cells from WT and Ppdpf KO mice. Scale bars, 10 μm. (K) Analysis of oxygen consumption rate (OCR) in primary cells from WT and Ppdpf KO mice. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. **P < 0.01; ****P < 0.0001. (A) n = 3, control; n = 3, UUO 1d mice; n = 2, UUO 5d mice. (D and E) n = 6, WT; n = 6, KO. (F to H) n = 3, WT; n = 3, KO. (I) n = 4, WT; n = 4 KO. (J) n = 10, WT; n = 10, KO. (K) n = 3, WT; n = 3, KO. TNF, tumor necrosis factor; a.u., arbitrary units; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; mOD, measure optical density.
Article Snippet: Protein extracts were quantified by the Micro BCA Protein Assay Kit (Thermo Fisher Scientific), resolved by SDS–polyacrylamide gel electrophoresis, and probed with the following antibodies: PPDPF (Proteintech, #19912- 1- AP), NDUFB9 (Proteintech, #15572- 1- AP), NDUFS3 (Proteintech, #15066- 1- AP), SDHA (Proteintech, #14865- 1- AP), SDHB (Proteintech, #10620- 1- AP), LAMIN B1 (Proteintech, #66095- 1- Ig), TOMM20 (Proteintech, #66777- 1- Ig), Flag (Proteintech, #66008- 3- Ig), vimentin (Proteintech, #10366- 1- AP), ACTA2 (Sigma- Aldrich, #SAB5700835), KIM1 (Proteintech, #83221- 2- RR), fibronectin (Abcam, #ab2413), DRP1 (Abcam, #ab184247), OXPHOS (Abcam, #ab110413), HA (Proteintech, #51064- 2- AP), NMNAT1 (Sigma- Aldrich, #HPA059447), NMNAT2 (Sigma- Aldrich, #HPA015708), NMNAT3 (Sigma- Aldrich, #HPA039077), ACTIN (Proteintech, #HRP- 60008), GAPDH (Proteintech, #HRP- 60004), and TUBULIN (Proteintech, #66031- 1- Ig).
Techniques: Control, Transmission Assay, Electron Microscopy, Activity Assay, Western Blot, Two Tailed Test
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