Additional file 2 of The down-regulation of XBP1, an unfolded protein response effector, promotes acute kidney injury to chronic kidney disease transition
Creators: name: Chen, Jia-Huang
name: Wu, Chia-Hsien
name: Jheng, Jia-Rong
name: Chao, Chia-Ter
name: Huang, Jenq-Wen
name: Hung, Kuan-Yu
name: Liu, Shing-Hwa
name: Chiang, Chih-Kang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Marine Biology
subject: Hematology
subject: Computational Biology

description: Additional file 2: Figure S1. UIRI causes prominent renal damage and development of fibrosis. (a) Diagram illustrates the timeline of the experiment. The left kidney of male C57BL/6 mice was subjected to renal ischemia/reperfusion injury (UIRI) and then sacrificed at different days as indicated. UDx: x days after UIRI. (b) PAS staining represents the accumulation of debris in the tubular lumen after UIRI. The arrowhead in the lower panel indicates debris. Scale bar indicates 200 μm in 40x, 50 μm in 200x. (c) qPCR assessment of the relative expression level of Kim-1 mRNA. (d) Masson’s trichrome staining shows the increased fibrosis fraction in kidney section after UIRI. Scale bar indicates 50 μm in 200x. (e) Quantitative scores of interstitial fibrosis were assessed. (f and g) The expression of α-SMA was examined with western blot analysis and quantified. Data are expressed as means ± SEM, n = 3 ~ 6 in each group. * P &lt; 0.05 and *** P &lt; 0.001, as compared with sham group. Figure S2. Loss of XBP1 expression is a universal characteristic in renal fibrosis models. (a) Western blot analysis showed the protein expression of α-SMA, XBP1u and XBP1s in UUO mice model. GAPDH was used as an internal control. (b-d) Quantification of relative protein expression levels of α-SMA, XBP1u and XBP1s. (e) Western blot analysis showed the protein expression of α-SMA, XBP1u and XBP1s in adenine diet mice model. GAPDH was used as an internal control. (f–h) Quantification of relative protein expression levels of α-SMA, XBP1u and XBP1s. N = 3–4 for each group, * P &lt; 0.05, ** P &lt; 0.01, and *** P &lt; 0.001, as compared with sham or chow diet group. Figure S3. Proximal tubular conditional knockout mice blocked XBP1s activation. (a) Diagram illustrates SLC5aCreERT2; XBP1fl/fl mice. (b) After tamoxifen administration, mice were subjected to IP injection of 500 ng/g of Tunicamycin for 12 h. Western blot analysis showed protein expression of XBP1s after Tunicamycin induction in XBP1fl/fl or XBP1cKO mice. GAPDH was used as an internal control. (c) Immunofluorescence staining demonstrated XBP1 expression in Tunicamycin treated mice kidneys. Scale bar: 250 μm. (d) XBP1s mRNA expression level was determined by semi-quantitative PCR. (e) qPCR assessment of the relative expression level of XBP1s mRNA. Figure S4. Proximal tubular XBP1 specific knockout mice were vulnerable to UIRI-induced kidney injury. (a) Diagram illustrates the experimental timeline of tamoxifen administration and UIRI with contralateral nephrectomy (Nx) surgery in XBP1fl/fl and XBP1cKO mice. (b and c) Blood urea nitrogen (BUN) and serum creatinine (Scr) levels were measured after 1 day of contralateral Nx. N = 3 for each group. ** P &lt; 0.01, and *** P &lt; 0.001, as compared with XBP1fl/fl Sham group. Figure S5. UIRI induces cell cycle arrest in G2/M phase. (a-d) The expression of chk1 and p21 in mice kidneys was evaluated with western blotting and quantified. GAPDH was used as an internal control. Data are expressed as means ± SEM, n = 3 ~ 6 in each group. * P &lt; 0.05, ** P &lt; 0.01, and *** P &lt; 0.001, as compared with sham group. (e and f) Representative images of Ki67+ pHH3+ renal sections in (e) WT mice or (f) XBP1cKO and XBP1fl/fl mice subjected to UIRI or Sham operation. Selected areas indicated highly expressed double-positive tubules. Scale bar: 50 μm. (g) Number of Ki67+pHH3+ tubular cells. Data are expressed as means ± SEM. * P &lt; 0.05, ** P &lt; 0.01, and *** P &lt; 0.001, as compared with XBP1fl/fl sham group. ### P &lt; 0.001 compared between indicated groups.