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Fig. 6. P311 gene silencing affects retinoic-acid-mediated lipid accumulation in lung fibroblasts. (A) Lung fibroblasts cultured for 1 week and infected with control lentivirus or P311-shRNA lentivirus were examined for relative P311 mRNA expression levels by quantitative real-time RT-PCR. There is an 
80% decrease in P311 transcripts in P311-shRNA-lentivirus-infected cells relative to control-lentivirus-infected cells. Mean ± s.d. from triplicate samples are shown. The differences between P311-shRNA and control samples are statistically significant (P<0.01). (B) Epifluorescence images of control and P311-shRNA-lentivirus-infected cells exposed to fatty-acid complexes (1 µM) and RA (1 µM) for 48 hours, showing that P311-shRNA-lentivirus-infected cells (identifiable by co-expression of GFP) accumulated less lipid droplets (stained with Nile Red) relative to control-lentivirus-infected cells (arrows). (C) The relative amount of lipids in control- versus P311-shRNA-lentivirus-infected cells, assessed by Nile-Red-stained cells followed by flow cytometry, indicates an 85% decrease in lipid accumulation in the P311-shRNA-lentivirus-infected population. Fatty-acid- (1 µM) and retinoic-acid- (1 µM) treated control- and P311-shRNA-lentivirus-infected cells were sorted for GFP and Nile-Red fluorescence after 48 hours. The mean fluorescence intensities of the Nile-Red channel in the GFP-positive populations were determined. Values were normalized to the average of the mean fluorescence intensities of the control GFP+ samples. Mean ± s.d. from triplicate samples are shown. The differences between P311 shRNA and control samples are statistically significant (P<0.01).
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