Cd133

Mitochondria were isolated from PK1 cells using a mitochondria isolation kit (Thermos Fisher, USA), following vendor’s instructions, as we have shown³⁷ . Protein concentration was measured with Bradford Protein Assay. Specific antibodies against Drp1 (1:1000; Abcam), Mfn2 (1:1000; Abcam) and OPA1 (1:500; Abcam) were used with blotting protocols. COXIV (1:5000; Abcam) was used as loading controls. Cell surface, EV or kidney protein expression was studied in homogenate. Specific antibodies against CD9 (1:5,000; Abcam), CD81 (1:1,000; Abcam), CD29(1:1000, Abcam), CD24(1:500, Abcam), CD133(1:500, Aviva Systems Biology), COX I (1:1000, Abcam), COX II (1:1000,Abcam), Caspase3 (1:1000, Abcam), Bax(1:1000,Abcam), Bcl-XL (1:1000, Abcam), COX IV, Drp1, p-Drp1(1:1000, Abcam) antibodies were used with blotting protocols, and GAPDH (1:1000, Abcam) as loading controls.The density of each band was analyzed by Image-Pro Plus 6.0 software.

EV were obtained from supernatants of STC-like cells as previously described^{17 (link),35 (link)}. Briefly, STC-like cells were cultured in M199 medium without serum for 48 hours. The CM was centrifuged at 2000 g for 20 minutes to remove debris, and then ultracentrifuged at 100,000 g in a SW41 swing rotor (Beckman Coulter, CA) for 1 hour at 4 °C. The supernatant was collected and used as CM-EV. The EV were washed once with M199, and submitted to a second ultracentrifugation. Protein content and size of EV were assessed by the Bradford assay and Nanosight technology (Nanosight, London, UK), respectively. Western Blot was used to characterize the CD24 (Abcam), CD9 (Bio-Rad), CD81, CD133, (both AVIVA Systems Biology), and CD29 (AbD Serotec) markers. To evaluate proteins carried by EV, Western Blotting of VEGF, IL-6, IL-10, TGF-β1 were performed and compared to EV isolated from adipose tissue-derived MSC isolated from comparable healthy pigs^{15 (link)}.