Skip to main content
. 2010 Jul 22;107(32):14339–14344. doi: 10.1073/pnas.0912701107

Fig. 4.

Fig. 4.

TGF-β-induced miR-21 expression in TEC. (A) Analysis of TGF-β expression in the kidneys of C57BL/6 mice undergoing IRI or sham surgery. Kidneys from naïve C57BL/6 mice were used as a control. Shown is expression on various days after injury or sham surgery. TGF-β expression was analyzed by real-time PCR. Pooled RNA from at least three kidneys per time point was analyzed. (B) Cultures of primary TEC were established from C57BL/6 mice and stimulated with either 20 or 100 ng/mL TGF-β for 24 h. The cells were harvested and total RNA prepared. The levels of mature miR-21 were then determined by real-time PCR. Similar results were observed at 72 h. Shown are data from at least three separate experiments. Relative quantitation (RQ) was calculated by the ddCT method normalizing miRNA expression to the endogenous control SnoRNA202. (C) TEC do not produce TGF-β following ischemia. Cultures of primary TEC were established as above and ischemia induced. Sham cells were not overlaid with mineral oil. The cells were harvested and TGF-β expression analyzed by RT-PCR using standard methods. Spleen cells were used as a positive control. Shown are data from at least three separate experiments. RQ was calculated by normalizing expression to the endogenous control GAPDH. (D) TGF-β stimulation leads to a modest increase in pri-miR-21. Cultures of primary TEC were established and stimulated with the indicated amounts of TGF-β for 24 h. The cells were harvested and total RNA prepared. Levels of pri-miR-21 were then quantitated by real-time PCR using the ABI TaqMan mmu-pri-miR-21 assay kit. (E) Ischemia leads to a decrease in pri-miR-21. Cultures of primary TEC were established, ischemia was induced, and the cells were harvested. Levels of pri-miR-21 were then quantitated as indicated for C. (F) Analysis of pri-miR-21 expression in the kidneys of C57BL/6 mice undergoing IRI or sham surgery. Kidneys from naïve C57BL/6 mice were used as a control. Shown is expression on various days after injury or sham surgery. Pooled RNA from at least three kidneys per time point was analyzed.