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. 2021 Dec 16;22(24):13511. doi: 10.3390/ijms222413511

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Less starvation-induced cell death in PC1KO mIMCDs. (A) Knockout of polycystin-1 (PC1KO) in mouse inner medullary collecting duct cells (mIMCDs) was confirmed by Western blot. (B) Reduced Pkd1 mRNA expression was confirmed by quantitative PCR (qPCR) (N = 7). (C) Representative microscopic brightfield images of wild-type (WT) and polycystin-1 knockout (PC1KO) mouse inner medullar collecting duct cells (mIMCDs) after 80 h of starvation by incubation in Hank’s balanced salt solution (HBSS). WT mIMCDs are less confluent and show more dark rounded detached cells. Scale bar = 300 µm. (D) Analysis of confluence of WT (black) and PC1KO (grey) mIMCDs during incubation in HBSS using the IncuCyte Live Cell Analyzer. Left: quantification of % confluence over time in HBSS with linear fit and 95% confidence interval (dashed line) superimposed; right: quantification of the slope of the linear fit of the confluence over time. Paired observations of each independent experiment are represented by the same symbol (N = 3). (E) Analysis of Cytotox Green signal increase during incubation in HBSS in WT (black) and PC1KO (grey) mIMCDs. Left: quantification of % of Cytotox Green signals over the total area of the image over time in HBSS; right: quantification of the slope of the linear fit over time (N = 3). (F) Percentage live cells over time in HBSS as analyzed by Trypan Blue exclusion in WT (black) and PC1KO (grey) mIMCDs (N = 4). (G) Apoptosis was analyzed by Western blotting of cleaved Caspase 3 in WT and PC1KO mIMCDs. Left: Representative Western blot of protein lysates following 0 h or 72 h of nutrient starvation; right: quantification of cleaved Caspase 3 levels over Actin (N = 3). (H) Percentage live cells following 72 h of incubation in HBSS as analyzed by Trypan Blue exclusion in WT and PC1KO mIMCDs, transfected either with Vehicle (+Veh) or human PC1 (+hPC1). Paired observations of each independent experiment are represented by the same symbol (N = 3). (I) Analysis of confluence of WT (black) and PC1KO (grey) mIMCDs during incubation in HBSS, followed by recovery in normal medium (DMEM) using the IncuCyte Live Cell Analyzer. Left: quantification of confluence (normalized to the initial time of recovery), with the linear fit in DMEM superimposed. The dashed line is the 95% interval of the linear fit; right: quantification of the slope of the linear fit in DMEM during recovery. Paired observations of each independent experiment are represented by the same symbol (N = 3). (J) Analysis of phosphorylated S6 (pS6) levels in WT and PC1KO mIMCDs in basal conditions. Left: representative Western blot; Right: quantification of pS6 levels over total S6 (N = 9); (K) Analysis of pS6 levels in WT and PC1KO mIMCDs following 48 h of HBSS and 24 h of recovery in DMEM. Left: representative Western blot; Right: quantification of pS6 levels over total S6 (N = 4). NS: not significant, ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001.