Fig 2: C1orf54 overexpression promoted renal recovery after kidney IRI. WT mice were transfected with C1orf54-overexpressing adenovirus or control vector and then subject to IRI. A, B, BUN and Cre levels after kidney IRI (n = 6). C, D, Ki-67 immunofluorescent staining in renal sections from control and C1orf54-overexpressing mice on Day 3 post-IRI. Ki-67+ cells were quantified (n = 6). Scale bars, 100 µm. E, F, Histological analysis of renal sections from control and C1orf54-overexpressing mice on Day 14 after IRI. Fibrotic areas were quantified (n = 6). G, H, WT mice were transfected with C1orf54-overexpressing adenovirus, and wortmannin (PI3K inhibitor) was intraperitoneally injected into the mice once every other day, and serum BUN and Cre levels were examined at different time-points after kidney IRI (n = 6). Data are expressed as means ± SD. **P < .01, *P < .05

Fig 3: C1orf54 promoted TEC proliferation through PI3K/AKT signalling. A, B, Western blotting analysis of p-P38/P38, p-JNK/JNK, p-STAT3/STAT3 and ß-catenin levels in the kidney of WT and C1 KO mice at Day 3 after IRI (n = 4). C, D, Western blotting analysis of p-AKT/AKT levels in the kidney of WT and C1 KO mice at different time-points after IRI (n = 6). E, F, HK-2 cells were transfected with control siRNA or C1orf54 siRNA and then subject to 6-h hypoxia treatment and reoxygenation (HR) for different periods. Western blotting was performed to examine phospho-AKT (p-AKT) and AKT levels (n = 6). G, H, Control and C1orf54 adenoviruses were injected through the tail vein 24 h before kidney IRI, and the PI3K/AKT-signalling inhibitor wortmannin was injected daily through the peritoneum. C1orf54 adenoviruses injection greatly increased its protein expression in the kidney in vivo (G), PCNA expression (H) was examined at different time-points after IRI (n = 6). Data are expressed as means ± SD. NS, not significant; ***P < .001, **P < .01, *P < .05

Fig 4: C1orf54 expression was transiently decreased after kidney ischaemia-reperfusion injury (IRI). A, Immunofluorescent staining of C1orf54 at different time-points after kidney IRI. Scale bar, 100 µm. B, Quantification of C1orf54-positive cells (n = 6). C, Western blotting analysis of C1orf54 expression in renal tissues post-IRI and D, quantification of C1orf54 expression relative to GAPDH (n = 6). E, Western blotting examination of C1orf54 levels in the serum of WT mice on Day 1 after IR surgery and F, quantification of band densities (n = 4). Data are expressed as means ± SD. ***P < .001, **P < .01

Fig 5: Absence of C1orf54 impaired tubular epithelial cell (TEC) proliferation. A, Ki-67 immunofluorescent staining at the corticomedullary junction in renal sections from WT and C1 KO mice at indicated time-points after kidney IRI. Scale bars, 100 µm. B, Quantification of Ki-67+ cells (n = 6). C, At different time-points after IRI, BrdU was intraperitoneally injected into WT and C1 KO mice, and 2 h after injection, kidney tissues were harvested for BrdU immunofluorescent staining. Scale bars, 100 µm. D, Quantification of BrdU+ cells (n = 6). Data are expressed as means ± SD. NS, not significant; ***P < .001, **P < .01