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. 2011 Dec 5;6(12):e28509. doi: 10.1371/journal.pone.0028509

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Dubey et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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The total cholesterol levels after STAT6 knockdown was checked at different time points in NCI-H460 and A549 cells. Scrambled siRNA was used as negative control. The data is expressed as the mean ± S.D. of 3 independent experiments performed in triplicates. * indicates p value < 0.05 in comparison to untransfected cells. a) Western blot analysis was done to analyze the change in expression of pSTAT6 protein upon IL-4 treatment in NCI-H460 cells. GAPDH was used as a loading control for densitometric analysis. Graph represents the fold change in protein expression compared to untransfected NCI-H460 cells. The data is expressed as the mean ± S.D. of 3 independent experiments. * indicates p value < 0.05 in comparison to untransfected cells. b) Real Time PCR was performed to determine the changes in expression level of genes related to cholesterol biosynthesis and homeostasis in NCI-H460 cells and A549 cells 48h post transfection of STAT6 specific siRNA. The samples were normalized to 18s rRNA expression. The real time data is expressed as the mean ± S.D. of 3 independent experiments performed in triplicates. * indicates p value < 0.05 in comparison to untransfected cells. c) Cholesterol assay was carried out to determine the effect of IL-4 treatment on total cholesterol levels in untransfected and siRNA transfected NCI-H460 cells and A549 cells. The data is expressed as the mean ± S.D. of 3 independent experiments performed in triplicates. * indicates p value < 0.05 in comparison to untransfected cells. d) The western blot of HMGCS1, HMGCR and IDI1 was done on cell extracts from untransfected and siRNA transfected NCI-H460 cells at different time points. GAPDH was used as a loading control for densitometric analysis. Graph represents the fold change in protein expression compared to untransfected NCI-H460 cells. The data is expressed as the mean ± S.D. of 3 independent experiments. * indicates p value < 0.05 in comparison to untransfected cells.