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. 2017 Apr 26;21(11):2732–2747. doi: 10.1111/jcmm.13189

Figure 7.

Figure 7

Effects of β‐catenin overexpression on MALAT1 promotor activity. (A) β‐catenin overexpression was examined by Western blot and real‐time PCR analysis. Podocytes under normal conditions or transfected with empty vectors were the controls. Values denote the mean ± S.D. *P<0.01 versus NC or control vector. (B) Luciferase reporter system was used to determine the MALAT1 promotor activity. Relative luciferase activity was enhanced after β‐catenin overexpression. Podocytes under normal conditions or transfected with empty vectors were the controls. Values denote the mean ± S.D. *P<0.01 versus NC or control vector. (C) Podocytes overexpressing β‐catenin or control cells were cotransfected with pGL3‐MALAT1 promotor‐luciferase reporter and pGL3‐Renilla luciferase reporter, and were further incubated with LG or HG for 48 hrs. Relative luciferase activity was measured. Overexpression of β‐catenin resulted in enhanced luciferase activity under either LG or HG conditions; HG led to more active luciferase activity than LG in cells where β‐catenin was overexpressed; and overall luciferase activities were significantly up‐regulated in HG‐treated podocytes than LG counterparts. Values denote the mean ± S.D.; aP < 0.05 versus control vector under LG,bP < 0.01 versus control vector under HG,cP < 0.01 versus β‐catenin vector under LG using one‐way anova+LSD‐t test, *P < 0.01 versus LG using two‐way anova. (D) MALAT1 levels were determined by real‐time PCR after cotranfection with luciferase reporter and incubation with either LG or HG for 48 hrs in mouse podocytes overexpressing β‐catenin or control podocytes. β‐catenin overexpression caused a significant upmodulation of MALAT1 levels under LG conditions, whereas showed no further promoting effects after HG treatment; MALAT1 levels were reduced in control podocytes under HG conditions; and overall mature MALAT1 levels were significantly lower in HG‐treated podocytes than LG counterparts. Values denote the mean ± S.D.; aP < 0.05 versus control vector under LG,bP < 0.01 versus control vector under HG using one‐way anova+LSD‐t test, *P < 0.05 versus LG using two‐way anova. (E) Effects of β‐catenin shRNA on SRSF1 protein expression. SRSF1 protein levels were promoted after HG stimulation; β‐catenin knock‐down had limited impacts on SRSF1 protein expression, suggesting β‐catenin might serve as a downstream effector of SRSF1. Podocytes under culture of LG or transfected with cont siRNA were the controls. Values denote the mean ± S.D.; aP < 0.05 versus LG.