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. 2019 Nov 26;9:17610. doi: 10.1038/s41598-019-53855-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Establishment and characterization of primary renal cell lines from mouse. (a) Morphology of mouse tubular epithelial cells during in vitro culture. (b) Quantitative RT-PCR analysis of proliferation markers related to or downstream targets of hTERT and SV40 gene. n = 4 for primary cells at the 2^nd passage, immortalized cells at the 2^nd passage, and immortalized cells at the 12^th passage, respectively. (c) Crystal violet staining of primary cells and immortalized cells after the 15^th passage. (d) Relative mRNA expression of Prominin1, Sox9, and Lgr5 in the immortalized cells at the 5^th and 15^th passages compared to that in mouse kidney lysate (mKidney). n = 4 for immortalized cells at the 5^th and 15^th passages, respectively, and n = 3 for mouse kidney lysates. (e) Quantitative RT-PCR analysis of kidney-specific segment markers in mouse cells at the 1^st, 8^th, and 17^th passages. n = 5 for 1^st, 8^th, 17^th passage immortalized cells, respectively. (f) FACS analysis of tubule segment markers in cells at the 4^th and 20^th passages. Immortalized cells at the 4^th and 20^th passages were stained with anti-AQP1, PNA, nephrin, or AQP2 (open histograms) or isotype control mAb (grey histograms). (g) GGT activity of mouse immortalized primary cells compared to a mouse tubular cell line (TCMK-1). n = 6 for TCMK-1 and mouse renal primary cells, respectively. (h) Dextran uptake by mouse immortalized primary cells at the 8^th passage after 48 h of incubation with Alexa Fluor 488-conjugated dextran. (i) Representative images of immunostaining for E-cadherin and Zo-1 in CTL and AQP1-, AQP2-, podocin-, or THP-depleted cells. Cells were at the 5–7^th passages and the depleted population was obtained from negative fractions via magnetic activated cell sorting. (j,k) Western blotting (j) and quantitative RT-PCR (k) analyses for epithelial-mesenchymal transition of cells of the CTL and AQP1-, AQP2-, podocin-, or THP-depleted population. The grouping of gels/blots cropped from different parts of the same gel. (l,m) The amounts of BMP7 (l) and TGFb1 (m) secreted by cells of the CTL and AQP1-, AQP2-, podocin-, or THP-depleted population. n = 4 for CTL and AQP1-, AQP2-, podocin-, or THP-depleted cells, respectively. All data are shown as the means ± s.e.m. *p < 0.05 compared with primary cells, TCMK-1 or CTL by unpaired Student’s t-test. Scale bar, 20 μm.