Biomedical Engineering Reference
In-Depth Information
Fig. 2
Analysis of lipid droplets fractionation. (
a
) Diagram demonstrated the fractions
sampled after sucrose density-gradient centrifugation. (
b
) Western blotting
analysis of fraction samples. The results demonstrated that both TIP47 and
Rab40c were enriched in the top fl oating LD-containing fraction (lane 1). The
ER-Golgi recycling protein KDEL receptor was detected in the bottom fractions.
Figure (
b
) is reproduced from the open access journal PLoS One [
19
]
fresh medium 1, and maintained for another 24-48 h, then go
to the next step (
see
Note 9
).
3. Replace medium 1 with medium 2, and maintain the culture in
stimulation medium for 2 days, then go to the next step.
4. Replace medium 2 with medium 3, and maintain the culture
for 2 days.
5. Feed the cells with fresh medium every 2 days (
see
Note 10
).
6. To examine the differentiation process, the cells are stained by
BODIPY 493/503 and analyzed by confocal microscopy or
western blot as mentioned above (Fig.
3
).
1. HepG2 cells are cultured as mentioned above.
2. Seed and grow the cells on coverslips for 24 h to 60 %
confl uence.
3. Transfect the cells with GFP-Rab40c plasmids as mentioned in
Subheading
3.1
, and grow the cells for 18-24 h.
4. Replace the medium with induction medium (refer to
Subheading
2.7
), and grow the cells for another 24 h.
5. To examine the formation of lipid droplets, the cells are stained
by Oil Red O and analyzed by confocal microscopy as men-
tioned in Subheading
3.1
. As demonstrated in Fig.
4
, the num-
ber and the size of LDs increased with the stimulation of oleic
acids in a time course dependent manner, and Rab40c staining
was gradually associated with LDs.
3.4 Induction
of LD Biogenesis
in HepG2 Cells
