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Published in final edited form as: Hepatology. 2011 Aug 9;54(4):1371–1378. doi: 10.1002/hep.24496

3A-B. rASBT3’-luciferase Assay. 3A. The rASBT3’-luciferase constructs were transfected into IEC-6 cells and luciferase activity was assessed in control cells, those with siRNA mediated reduction in HuR expression (siHuR) or those with overexpression of wild type mouse HuR (wtHuR). Overexpression of HuR enhances luciferase activity, while silencing induces the opposite effect in all constructs except the basal SV-40 driven construct that does not contain rASBT 3’UTR. Bars represent the mean of nine measurement in three separate sets of experiments. Error bars represent standard of deviation. The y-axis is logarithmic. 3B. The rASBT3’-0.3kb luciferase construct was transfected into IEC-6 cells that were treated with siTTP, siHuR, scrambled control (siScr) or vehicle (control). Luciferase activity was markedly increased when TTP was silenced, while it was reduced when HuR was silenced. 3C. rASBT3’-ßglobin Assay. The ß-globin control reporter construct and one containing 0.3kb of rASBT 3’UTR were transfected into Caco-2 cells treated with either scrambled anti-sense RNA or HuR siRNA. At the specified time points after RNA transcription was terminated RNA was extracted and analyzed by Northern blotting for either ß-globin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Natural log transformation of the data was used to derive half-lives. Silencing HuR had no effect on the control reporter but reduced the rASBT 3’UTR mRNA’s half-life from 2.4 to 0.3 hours. 3D. Effect of HuR and TTP on endogenous Caco-2 ASBT mRNA stability. Caco-2 cells were treated with either siHuR (panel A) or siTTP (panel B). Scrambled antisense (siScr) was used as a control. 48 hours after this transfection, RNA synthesis was terminated using Actinomycin D and cycloheximide. RNA was prepared from cells at timed intervals after RNA synthesis was terminated. In control cells, ASBT mRNA was detectable 2 to 4 hours after RNA synthesis was terminated, while in siHuR treated cells ASBT mRNA could barely be detected at 1 hour and in siTTP cells ASBT mRNA was detectable at 8 hours.

3A-B. rASBT3’-luciferase Assay. 3A. The rASBT3’-luciferase constructs were transfected into IEC-6 cells and luciferase activity was assessed in control cells, those with siRNA mediated reduction in HuR expression (siHuR) or those with overexpression of wild type mouse HuR (wtHuR). Overexpression of HuR enhances luciferase activity, while silencing induces the opposite effect in all constructs except the basal SV-40 driven construct that does not contain rASBT 3’UTR. Bars represent the mean of nine measurement in three separate sets of experiments. Error bars represent standard of deviation. The y-axis is logarithmic. 3B. The rASBT3’-0.3kb luciferase construct was transfected into IEC-6 cells that were treated with siTTP, siHuR, scrambled control (siScr) or vehicle (control). Luciferase activity was markedly increased when TTP was silenced, while it was reduced when HuR was silenced. 3C. rASBT3’-ßglobin Assay. The ß-globin control reporter construct and one containing 0.3kb of rASBT 3’UTR were transfected into Caco-2 cells treated with either scrambled anti-sense RNA or HuR siRNA. At the specified time points after RNA transcription was terminated RNA was extracted and analyzed by Northern blotting for either ß-globin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Natural log transformation of the data was used to derive half-lives. Silencing HuR had no effect on the control reporter but reduced the rASBT 3’UTR mRNA’s half-life from 2.4 to 0.3 hours. 3D. Effect of HuR and TTP on endogenous Caco-2 ASBT mRNA stability. Caco-2 cells were treated with either siHuR (panel A) or siTTP (panel B). Scrambled antisense (siScr) was used as a control. 48 hours after this transfection, RNA synthesis was terminated using Actinomycin D and cycloheximide. RNA was prepared from cells at timed intervals after RNA synthesis was terminated. In control cells, ASBT mRNA was detectable 2 to 4 hours after RNA synthesis was terminated, while in siHuR treated cells ASBT mRNA could barely be detected at 1 hour and in siTTP cells ASBT mRNA was detectable at 8 hours.