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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Biochim Biophys Acta. 2013 Apr 28;1829(9):905–915. doi: 10.1016/j.bbagrm.2013.04.009

Figure 2.

Figure 2

Depletion of hnRNP K by siRNA causes an increase in the splicing of G6PD mRNA. HepG2 cells stably expressing the G6PD reporter shown in (A) were transfected with 100nM siRNA against hnRNP K, hnRNP L, or a non-targeting (NT) control. After 48 hours, RNA and protein were isolated. The Exon 12(+) reporter contains mouse genomic DNA from exon 12 (nt 37–93), intron 12, and exon 13 including the entire 3’-untranslated region (3’-UTR) of the G6PD gene ligated to β-galactosidase (β-gal) and transcription is driven by the CMV promoter. The open arrows indicate the location of the primers used to detect overall reporter expression. The closed arrows represent primers used to detect the splicing of the reporter. The Exon 12(−) vector also contains mouse genomic DNA ligated to β-galactosidase but contains only the last 8 nt of exon 12 to preserve the 5’splice site (5’SS) of intron 12. (B) A representative immunoblot of hnRNP K depletion in HepG2 cells is shown. (C) Expression of mRNA produced by the Exon 12(+) reporter in HepG2 cells that were transfected with the indicated siRNA was measured with the primers to the β-galactosidase sequence (open arrows). The data are normalized to the amount of GAPDH mRNA. The data are the mean ± SE of n=6 independent siRNA knockdowns. (D) Following knockdowns with the indicated siRNA, splicing of the reporter RNA was with the primers indicated by black arrows. The products of a representative RT-PCR are shown. Samples were also amplified without prior treatment with reverse transcriptase (RT) to test for DNA contamination. The percentage of mRNA that was spliced is calculated as the intensity of the spliced band over the combined intensities of the bands for spliced and unspliced RNA. The data represent the mean ± SE of n=6 independent siRNA transfections. (E) Expression of the Exon 12(−) reporter in HepG2 cells that were transfected with the indicated siRNAs was measured by RT-qPCR and primers to the β-galactosidase portion of the reporter RNA. The amount of reporter mRNA is expressed relative to the amount of GAPDH mRNA. Each bar represents the mean ± SE n=3 independent siRNA transfections. (F–H) Knockdown of hnRNP L and measurement of Exon 12(+) reporter expression and splicing were carried out as described for hnRNP L. Each bar represents the mean ± SE n=4 independent siRNA transfections. The “*” symbol indicates a significant difference (p<0.05).