96
3 In-Tether Chiral Center Induced Helical Peptide Modulators …
and 1 μg of DAPI (Sigma)/ml at room temperature for 30 min in a dark environment.
Cells were washed three times with PBS, and the stained cells were mounted with
mounting medium and the coverslips were sealed with nail polish. Fluorescence was
recorded using a confocal microscope.
3.4.10 Preparation of Paraffin Section Histological Analysis
(IHC)
For histological experiments, organ tissues were collected on the final day treatment
and fixed in 4% buffered formalin-saline at room temperature for 24 hours. Following
this, tissues were embedded in paraffin blocks and 4 mm thick paraffin sections were
mounted on a glass slide for hematoxylin and eosin (H&E) staining. The H&E
staining slices were examined under a light microscopy (Olympus BX51).
3.4.11 Antitumor Efficacy in Human Xenograft Model Using
PhR Peptide
Athymic nude mice (BALB/c ASlac-nu) were obtained from Vital River Laboratory
Animal Technology Co. Ltd. of Beijing, People’s Republic of China and allowed an
acclimation period of 1 week. Mice were maintained in an isolated biosafety facility
for specific-pathogen-free (SPF) animals with bedding, food, and water. All operations were carried out in accordance with the National Standard of Animal Care and
Use Procedures at the Laboratory Animal Center of Shenzhen University, Guangdong
Province, People’s Republic of China (the permit number is SZU-HC-2014-02). For
tumor suppression assay, athymic nude mice (female; 6 weeks old) were inoculated
with 1 × 10
7 PA-1 cells (PA-1 cells were trypsinized, harvested, and resuspended
in DMEM, with 100 mL volume of each) propagated in vitro subcutaneously in the
lower flank of mice. After 10–15 days, mice with tumors exceeding 100–150 mm
3 in
volume were randomly divided into 3 groups of 5–6 mice per treatment group. Mice
bearing PA-1 tumors were injected with PhR (10 mg/Kg) or nutlin-3a (10 mg/Kg)
using PBS as negative control. Mice were injected once every 2 days starting on day
0. Tumor volumes were measured by calipers (accuracy of 0.02 mm) every other
day and calculated using the following formula: V = L × W
2 /2 (W, the shortest
dimension; L, the longest dimension). Each tumor was independently measured and
calculated by changes in volume (folds) relative to day 0. Statistical significances
between groups were tested by one-way analysis of variance.
3 In-Tether Chiral Center Induced Helical Peptide Modulators …
and 1 μg of DAPI (Sigma)/ml at room temperature for 30 min in a dark environment.
Cells were washed three times with PBS, and the stained cells were mounted with
mounting medium and the coverslips were sealed with nail polish. Fluorescence was
recorded using a confocal microscope.
3.4.10 Preparation of Paraffin Section Histological Analysis
(IHC)
For histological experiments, organ tissues were collected on the final day treatment
and fixed in 4% buffered formalin-saline at room temperature for 24 hours. Following
this, tissues were embedded in paraffin blocks and 4 mm thick paraffin sections were
mounted on a glass slide for hematoxylin and eosin (H&E) staining. The H&E
staining slices were examined under a light microscopy (Olympus BX51).
3.4.11 Antitumor Efficacy in Human Xenograft Model Using
PhR Peptide
Athymic nude mice (BALB/c ASlac-nu) were obtained from Vital River Laboratory
Animal Technology Co. Ltd. of Beijing, People’s Republic of China and allowed an
acclimation period of 1 week. Mice were maintained in an isolated biosafety facility
for specific-pathogen-free (SPF) animals with bedding, food, and water. All operations were carried out in accordance with the National Standard of Animal Care and
Use Procedures at the Laboratory Animal Center of Shenzhen University, Guangdong
Province, People’s Republic of China (the permit number is SZU-HC-2014-02). For
tumor suppression assay, athymic nude mice (female; 6 weeks old) were inoculated
with 1 × 10
7 PA-1 cells (PA-1 cells were trypsinized, harvested, and resuspended
in DMEM, with 100 mL volume of each) propagated in vitro subcutaneously in the
lower flank of mice. After 10–15 days, mice with tumors exceeding 100–150 mm
3 in
volume were randomly divided into 3 groups of 5–6 mice per treatment group. Mice
bearing PA-1 tumors were injected with PhR (10 mg/Kg) or nutlin-3a (10 mg/Kg)
using PBS as negative control. Mice were injected once every 2 days starting on day
0. Tumor volumes were measured by calipers (accuracy of 0.02 mm) every other
day and calculated using the following formula: V = L × W
2 /2 (W, the shortest
dimension; L, the longest dimension). Each tumor was independently measured and
calculated by changes in volume (folds) relative to day 0. Statistical significances
between groups were tested by one-way analysis of variance.