Fig 2: Hypoxia-induced CHK1 upregulation in RTECs is depend on FAT10. (A) Upon hypoxia treatment for 24 h, mass spectroscopic analysis was performed to detect protein expression in RTECs from FAT10+/+ mice (n = 3) and FAT10-/- mice (n = 3). (B) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in FAT10+/+ RTECs or FAT10-/- RTECs after hypoxia injury. Tubulin was used as a loading control. *P < 0.05, **P < 0.01. (C) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in HK-2 cells following treatment with hypoxia or without hypoxia. *P < 0.05, **P < 0.01. (D) The protein and mRNA levels of FAT10 and CHK1 in HK-2 cells following treatment with hypoxia or without hypoxia. **P < 0.01. (E) Determination (left) and quantification (right) of the FAT10 and CHK1 protein levels in HK-2 cells transfected with shFAT10 following hypoxic injury. **P < 0.01. (F) Upon hypoxia treatment for 24 h, the mRNA levels of FAT10 and CHK1 in HK-2 cells transfected with shFAT10. **P < 0.01.

Fig 3: FAT10 is required for CHK-1-mediated G2/M arrest in RTECs under hypoxia treatment. (A) Western blotting showing the protein expression of CHK1, CDKN1A, TGF-ß and CTGF in CHK1-silencing HK-2 cells following hypoxia injury. Tubulin was used as a loading control. (B) TGF-ß and CTGF in the culture supernatants was measured in culture supernatants by ELISA assay. **P < 0.01. (C) Detection for cell cycle of CHK1-silencing HK-2 cells following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). *P < 0.05. (D) Upon hypoxia treatment, western blotting of FAT10, CHK1, CDKN1A, TGF-ß and CTGF in HK-2 cells stably transfected with shFAT10 in the presence or absence of Flag-CHK1. (E) Detection for cell cycle of FAT10-silencing HK-2 cells in the presence or absence of Flag-CHK1 following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). *P < 0.05. (F) Western blotting showing the protein expression of FAT10, CHK1, CDKN1A, TGF-ß and CTGF in FAT10+/+ RTECs and FAT10-/- RTECs following treatment with hypoxia or without hypoxia. (G) Detection for cell cycle of FAT10+/+ RTECs and FAT10-/- RTECs following treatment with hypoxia or without hypoxia. Results are expressed as peak diagram (left) and calculated distribution for cells in G0/G1, S and G2/M phases (right). *P < 0.05.

Fig 4: FAT10 was up-regulated and positively correlated with renal fibrosis in patients with calculi related chronic kidney disease. (A) Representative photos of renal sections from normal kidney and biopsy samples from patients with calculi related chronic kidney disease. IHC, immunohistochemistry; scale bar = 50 µm. (B) The expression of renal FAT10 at initial biopsy positively correlated with the tubular interstitial fibrosis index in a partial correlation analysis. (C–F) Scatter plots show a positive correlation between FAT10 and USP7, CHK1, CDKN1A, TGF-ß, respectively. (G–I) The statistical analysis data show that the expression level of USP7 was positively correlated with the expression level of CHK1, CDKN1A and TGF-ß. *P < 0.05, **P < 0.01.

Fig 5: UUO-induced kidney fibrosis was suppressed in FAT10-deficient mice. (A and B) Representative HP and FAT10 staining in the kidneys from FAT10+/+ (A) and FAT10-/- (B) mice subjected to either UUO or sham operation; scale bar = 50 µm. (C and D) Representative Masson’s trichrome (C) and picrosirius red staining (D) of kidney sections from FAT10+/+ and FAT10-/- mice with or without UUO for 7 days. (E and F) Immunohistochemistry of protein expression of a-SMA (E) and Collagen IV (F) in obstructed kidneys from FAT10+/+ and FAT10-/- mice subjected to either UUO or sham operation. (G and H) Bar graph (right) shows quantification of fibrotic areas in histological sections; **P < 0.01 versus FAT10+/+ mice at the same time point; n = 6. (I and J) Bar graph shows quantification of areas of positive cells; **P < 0.01 versus FAT10+/+ mice at the same time point; n = 6.