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. 2019 Aug 21;20(4):3728–3734. doi: 10.3892/mmr.2019.10601

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Copyright: © Chen et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 2. — Increased Sam68 expression in cultured podocytes with HG is inhibited by Sam68 knockdown. (A) The effects of siRNA sequences targeting Sam68 on the mRNA expression of Sam68 in in vitro cultured podocytes. Three pairs of siRNA sequences that targeted Sam68 and one pair of control-siRNA were designed and synthesized. The third pair of siRNA sequence was selected for further experiments based on the interference effect. NG (5.3 mM) group. Values are expressed as the mean ± SEM. *P<0.05 NG+Sam68-siRNA1 or NG+Sam68-siRNA2 vs. NG; ^#P<0.01, NG+Sam68-siRNA1-3 vs. NG+Con-siRNA. (B) The effects of Sam68 siRNA on the mRNA expression of Sam68 in HG-cultured podocytes. The mRNA level of Sam68 was examined with real-time-PCR, and was calculated with the 2^−ΔΔCq method. (C) Double immunofluorescence staining of Sam68 (red), synaptopodin-identified podocytes (green), DAPI-stained nuclei (blue) and merged images (purple) in cultured podocytes treated with HG for 48 h. (D) The protein expression of Sam68 was detected by immunoblotting in the nuclear extract of podocytes incubated with HG (30 mM) for 48 h. Histone H3 was used as the nuclear protein loading control. (E) Densitometric analysis of three independent experiments displayed in D. NG (5.3 mM) group; HG (30 mM) group; MA, NG (5.3 mM)+MA (24.7 mM) group, as an osmolality control; HG+Sam68-siRNA, HG (30 mM)+Sam68-siRNA (50 nM) group; HG+Con-siRNA, HG (30 mM)+non target-siRNA (50 nM) group. Values are expressed as the mean ± SEM. *P<0.05, HG or HG+Con-siRNA vs. NG; ^#P<0.05, HG+Sam68-siRNA vs. HG. Sam68, Src-associated substrate during mitosis of 68 kDa; HG, high glucose; PCR, polymerase chain reaction; NG, normal glucose; HG, high glucose; MA, mannitol.