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3 In-Tether Chiral Center Induced Helical Peptide Modulators …
3.4.6 Cell Viability Assay
Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenylt-etrazolium bromide, Sigma) assay. Cells were seeded in a 96-well plate
at a density of 5 × 103 cells/well and incubated with p53 peptides and nutlin-3a in
serum-free media for 4 h, followed by serum replacement and additional incubation
for 44 h. MTT (5 mg/mL, 20 μL) in PBS was added and the cells were incubated
for 4 h at 37°C with 5% CO2. DMSO (Dimethylsulfoxide, 150 μL, Sigma) was
then added to solubilize the precipitate with 5 min of gentle shaking. Absorbance
was measured with a microplate reader (Bio-Rad) at a wavelength of 490 nm. Cell
viability was obtained by normalizing the absorbance of the sample well against
that of the control well and expressed as a percentage, assigning the viability of
non-treated cells as 100%.
3.4.7 Western Blot Analysis
For western blot analysis, cells were seeded in 6-well plates and treated for 48 h with
p53 peptides and nutlin-3a. To isolate the protein, cells were washed with PBS and
harvested using lysis buffer (50 mM Tris·Cl PH = 6.8, 2% SDS, 6% Glycerol, 1% βmercapitalethanol, 0.004% bromophenol blue). Total cellular protein concentrations
were determined by a spectrophotometer (Nano-Drop ND-2000). 20 μg of denatured
cellular extracts were resolved using 10% SDS-PAGE gels. Protein bands in the
gel were then transferred to nitrocellulose blotting membranes and incubated with
the appropriate primary antibody. The antibody dilutions were as follows: 1:500 for
MDM2, MDMX, p53 and 1:1000 for actin, H3. Membranes were incubated overnight
at 4°C and washed the next day with buffer (1 × PBS, 0.05% Tween 20). Goat antirabbit or anti-mouse secondary antibodies were used for secondary incubation for
1 hours at room temperature. Proteins were then visualized with chemiluminescent
substrates.
3.4.8 RNA Extraction and RT-PCR and Microarray Analysis
For western blot analysis, cells were seeded in 6-well plates and treated for 48 h with
P53 peptides and nutlin-3a as described for the western blot assay. Then total RNA
was extracted from cells using TRIzol reagent (Invitrogen) and the amount of RNA
was quantified by a spectrophotometer (Nano-Drop ND-2000). Total RNA (2 μg)
was reverse transcribed to cDNA using the reverse transcriptase kit from Promega
according to the manufacturer’s instructions. The mRNA levels of the target genes
were quantified by real-time PCR using SYBR green (Promega) in an ABI Prism
7500 real-time PCR system (Applied Biosystems). The primers used are listed in
3 In-Tether Chiral Center Induced Helical Peptide Modulators …
3.4.6 Cell Viability Assay
Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenylt-etrazolium bromide, Sigma) assay. Cells were seeded in a 96-well plate
at a density of 5 × 103 cells/well and incubated with p53 peptides and nutlin-3a in
serum-free media for 4 h, followed by serum replacement and additional incubation
for 44 h. MTT (5 mg/mL, 20 μL) in PBS was added and the cells were incubated
for 4 h at 37°C with 5% CO2. DMSO (Dimethylsulfoxide, 150 μL, Sigma) was
then added to solubilize the precipitate with 5 min of gentle shaking. Absorbance
was measured with a microplate reader (Bio-Rad) at a wavelength of 490 nm. Cell
viability was obtained by normalizing the absorbance of the sample well against
that of the control well and expressed as a percentage, assigning the viability of
non-treated cells as 100%.
3.4.7 Western Blot Analysis
For western blot analysis, cells were seeded in 6-well plates and treated for 48 h with
p53 peptides and nutlin-3a. To isolate the protein, cells were washed with PBS and
harvested using lysis buffer (50 mM Tris·Cl PH = 6.8, 2% SDS, 6% Glycerol, 1% βmercapitalethanol, 0.004% bromophenol blue). Total cellular protein concentrations
were determined by a spectrophotometer (Nano-Drop ND-2000). 20 μg of denatured
cellular extracts were resolved using 10% SDS-PAGE gels. Protein bands in the
gel were then transferred to nitrocellulose blotting membranes and incubated with
the appropriate primary antibody. The antibody dilutions were as follows: 1:500 for
MDM2, MDMX, p53 and 1:1000 for actin, H3. Membranes were incubated overnight
at 4°C and washed the next day with buffer (1 × PBS, 0.05% Tween 20). Goat antirabbit or anti-mouse secondary antibodies were used for secondary incubation for
1 hours at room temperature. Proteins were then visualized with chemiluminescent
substrates.
3.4.8 RNA Extraction and RT-PCR and Microarray Analysis
For western blot analysis, cells were seeded in 6-well plates and treated for 48 h with
P53 peptides and nutlin-3a as described for the western blot assay. Then total RNA
was extracted from cells using TRIzol reagent (Invitrogen) and the amount of RNA
was quantified by a spectrophotometer (Nano-Drop ND-2000). Total RNA (2 μg)
was reverse transcribed to cDNA using the reverse transcriptase kit from Promega
according to the manufacturer’s instructions. The mRNA levels of the target genes
were quantified by real-time PCR using SYBR green (Promega) in an ABI Prism
7500 real-time PCR system (Applied Biosystems). The primers used are listed in