Figure 5.

Inhibition of Dot1l alleviated fibrosis induced by H/R in vitro.

Notes: HK-2 cells were pretreated with or without EPZ004777 at different doses (1, 5, and 25 μM) for 24 hrs, and exposed to H/R. (A) The mRNA levels of α-SMA, vimentin, and fibronectin were detected using real-time RT-PCR. (B) Representative bands of Western blotting analyses for the protein expression of α-SMA, vimentin, and fibronectin. The bar graph shows the fold change in protein levels relative to the control group based on three independent samples. (C) HK-2 cells were transfected with an siRNA against Dot1l or a negative control siRNA (si-NC) for 48 hrs, and pretreated with or without EPZ004777 at a dose of 25 μM for 3 hrs before being exposed to H/R. The mRNA levels of α-SMA, vimentin, and fibronectin were detected using real-time RT-PCR. (D) Representative bands of Western blotting analyses for the protein expression of α-SMA, vimentin, and fibronectin. The bar graph shows the fold change in protein levels relative to the control group based on three independent samples. In addition, (E) SOD activity, (F) MDA content, and (G) H₂O₂ production were detected using three independent samples. Values are expressed as the mean±SEM; *P<0.05, between the DMSO and EPZ groups; ^#P<0.05, between the si-NC and si-Dot1l groups.

Abbreviations: H/R, hypoxia/reoxygenation; HK-2, human kidney-2; RT-PCR, reverse transcription-polymerase chain reaction; Dot1l, disruptor of telomeric silencing-1-like; α-SMA, alpha-smooth muscle actin; siRNA, small interfering RNA; SOD, superoxide dismutase; MDA, malondialdehyde; DMSO, dimethyl sulfoxide; EPZ, EPZ004777; SEM, standard error of mean.