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Figure 3


Fig. 3. P311 gene silencing does not affect lipid accumulation in NIH3T3-L1 cells. (A) P311 mRNA expression levels during adipogenic differentiation of NIH3T3-L1 cells as determined by quantitative real-time RT-PCR. There is almost a complete lack in P311 transcripts at the beginning of induction (Day 2) followed by a gradual increase to base-line levels at the completion of differentiation (Day 8). Mean ±s.d. from triplicate samples are shown. Asterisks indicate statistically significant (P<0.01) pair wise comparison between Day 0 and Day 2 samples (*) and between Day 0 and Day 4 samples (**). There is no significant difference between Day 0 and Day 8 samples. (B) NIH3T3-L1 preadipocytes infected with control lentivirus or lentivirus expressing a short hairpin RNA (shRNA) targeting P311 were examined for relative P311 RNA expression levels by quantitative real-time RT-PCR. There is an ~90% decrease in P311 transcripts in P31-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<0.05). (C-F) Phase contrast (C and E) and epifluorescence (D and F) images of control (C,D) and P311-shRNA-lentivirus-infected NIH3T3-L1 (E,F) cells following induction of adipogenic differentiation demonstrate that both control and P311-shRNA-lentivirus-infected cells (marked by GFP) accumulate lipid droplets (arrows) to similar extent. Scale bar, 10 µm. (G) The relative amount of lipids in control versus P311-shRNA-lentivirus-infected NIH3T3-L1 cells following induction of adipogenic differentiation, assessed by Nile-Red-stained cells followed by flow cytometry, indicates no significant change in lipid accumulation in the P311-shRNA-lentivirus-infected population. Control and P311-shRNA-lentivirus-infected NIH3T3-L1 cells were sorted for GFP and Nile Red fluorescence. The mean fluorescence intensities of the Nile Red channel in the GFP+ populations were determined. Values are normalized to the average of the mean fluorescence intensities of the control GFP+ samples. Mean ± s.d. from triplicate samples are shown.





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