Knockdown of TauT Expression Impairs Human Embryonic Kidney 293 Cell Development - Advances in Experimental Medicine and Biology - page 291

in tissue culture medium and cultured at 37°C with 5% CO 2. Two weeks later, the
top layer of cells was stained with 0.2% p-iodonitrotetrazolium violet (Sigma).
Cultures were analyzed in triplicate, and colonies larger than 100 mm in diameter
were counted.

28.2.10 Apoptosis Assays

Internucleosomal DNA fragmentation was detected primarily by a DNA laddering
assay. An equal number of cells were suspended in 500 ml of lysis buffer (1% sodium
dodecyl sulfate, 25 mM ethylenediaminetetraacetic acid, and 1 mg/ml proteinase K)
and incubated overnight at 50°C. Ribonuclease A (10 mg/ml) was then added for an
additional 2-h incubation at 37°C. The chromosomal DNA was extracted with phe-
nol/chloroform, precipitated with ethanol, and analyzed by agarose gel electropho-
resis. This was followed by staining the DNA with ethidium bromide to reveal the
fragmentation pattern.

28.2.11 Statistics

All experiments using tissue cultures were performed in triplicate. Statistical com-
parisons were made using one-way ANOVA and Student's t test to determine
significant differences in the means between experimental groups.

28.3 Results

28.3.1 Inhibition of TauT Expression in 293 Cells by RNAi

The SureSilencing TM Pre-Designed shRNA plasmids (SuperArray, Frederick, MD)
were stably transfected into 293 cells, which were selected for neomycin resistance
to G418 (3 mg/ml). To access the success of the TauT knockdown, taurine uptake
was measured. The TauT-deficient 293 cell line showed a 30-35% reduction in tau-
rine transport activity, the intensity of which reflected the relative quantity of func-
tional TauT in the cellular membrane. As shown in Fig. 28.1a , taurine uptake by the
TauT-deficient cells was significantly less than that of the mock group (control),
which expressed a clone NC (negative control) plasmid.
The inhibition of TauT expression was further confirmed by the immunostaining
of TauT by a TauT-specific antibody. As shown in Fig. 28.1b , TauT expression was
detected in control cells, which were characterized by a darker brown in comparison
to less intense staining in TauT-deficient cells. Western blot analysis of TauT protein