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. 2014 Apr 9;15(4):5952–5969. doi: 10.3390/ijms15045952

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© 2014 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 license (http://creativecommons.org/licenses/by/3.0/).

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Figure 4. — The effect of LeuRS knockdown on leucine-induced proliferation and expression of lactation-associated proteins in DCMECs. DCMECs were divided into three groups: nontransfected group without siRNA and Lipofectamine™ 2000 reagent, control group with a negative siRNA and Lipofectamine™ 2000 reagent as well as transfected group with a LeuRS-specific siRNA and Lipofectamine™ 2000 reagent, all groups were treated with 1.2 mmol/L leucine. (A) Cell viability and cell proliferation after LeuRS knockdown for 0, 24 and 48 h were measured by using CASY-TT; (B) Measurement of the cell cycle progression by flow cytometric analysis of PI-stained DCMECs after LeuRS knockdown for 24 h; (C) Changes in the level of lactation- and mTOR signaling-related gene expression in response to LeuRS knockdown for 24 h as measured by using qRT-PCR; (D) Representative western blotting of mentioned proteins. DCMECs were treated as mentioned above for 24 h. β-Actin was assessed as a loading control; (E) Quantitation of (D); (F,G) Lactose and triglyceride secreted into the culture medium were detected after LeuRS knockdown for 48 h; (H) β-Casein expression in DCMECs was detected by using qRT-PCR after LeuRS knockdown for 24 h; (I) Level of β-Casein protein in DCMECs after LeuRS knockdown for 24 h as assessed by western blotting analysis. β-Actin was assessed as a loading control. A representative blot and quantitation of three independent experiments are shown. In all panels, data represent the mean ± SD of three independent experiments, and three or six wells per treatment within each independent experiment. ^* and ^** indicate p < 0.05 and p < 0.01, respectively.

Figure 4. — The effect of LeuRS knockdown on leucine-induced proliferation and expression of lactation-associated proteins in DCMECs. DCMECs were divided into three groups: nontransfected group without siRNA and Lipofectamine™ 2000 reagent, control group with a negative siRNA and Lipofectamine™ 2000 reagent as well as transfected group with a LeuRS-specific siRNA and Lipofectamine™ 2000 reagent, all groups were treated with 1.2 mmol/L leucine. (A) Cell viability and cell proliferation after LeuRS knockdown for 0, 24 and 48 h were measured by using CASY-TT; (B) Measurement of the cell cycle progression by flow cytometric analysis of PI-stained DCMECs after LeuRS knockdown for 24 h; (C) Changes in the level of lactation- and mTOR signaling-related gene expression in response to LeuRS knockdown for 24 h as measured by using qRT-PCR; (D) Representative western blotting of mentioned proteins. DCMECs were treated as mentioned above for 24 h. β-Actin was assessed as a loading control; (E) Quantitation of (D); (F,G) Lactose and triglyceride secreted into the culture medium were detected after LeuRS knockdown for 48 h; (H) β-Casein expression in DCMECs was detected by using qRT-PCR after LeuRS knockdown for 24 h; (I) Level of β-Casein protein in DCMECs after LeuRS knockdown for 24 h as assessed by western blotting analysis. β-Actin was assessed as a loading control. A representative blot and quantitation of three independent experiments are shown. In all panels, data represent the mean ± SD of three independent experiments, and three or six wells per treatment within each independent experiment. ^* and ^** indicate p < 0.05 and p < 0.01, respectively.