Cell culture and CRISPR
HO8910 and ES-2 ovarian cancer cells were cultured in DMEM/RPMI1640 supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37°C under a humidified 5% CO2 atmosphere.
WISP2 deletion cells were generated via CRISPR genomic editing technology. These plasmids were then transfected into HO8910 and ES-2 cells. Twenty-four hours following transfection, transfected cells were enriched via puromycin selection for 3 d and sorted on 96-well plates with only a single cell per well. The clones were screened via western blot using WISP2 antibody (1:2000, Abcam).
Wisp2 sgRNA sequence was as follows:
Wisp2-1: GCTGTGAGGTGAATGGCCGC
Wisp2-2: TTGCCGGCTGCATCACTGCC
Soft agar colony formation assay
One-milliliter layers of 0.5% agar were prepared in a 35 mm cell culture dish. Cells were suspended in 1 ml of 0.35% agar containing 1 × cell culture medium and 10% FBS and poured over these layers. The final cell concentration in each culture was 0.5 × 103 cells/ml. Triplicate cultures were used for each experiment. Plates were placed in a 5% CO2 humidified incubator at 37 °C. Colonies were counted 2–3 weeks after plating using an Omnicon FAS II Image Analysis System.
Wound-healing assay
ES-2 WT and WISP2 deleted cells were grown in DMEM supplemented with 10% FBS until confluence was reached. The medium was then changed to fresh serum-free medium, and the cell monolayers were scraped in a straight line using a P-10 pipette tip to create a scratch. The plates were photographed at 0 and 24 h using a phase-contrast inverted microscope (Nikon Ti, Nikon Corp.).
Transwell Migration Assay
Twenty-four-well tissue culture plate inserts with 8 mm-pore filters and BioCoat Matrigel (BD Biosciences, Bedford, MA, USA) were used to assess the migration and invasive potential of ES-2 and HO8910 WT and WISP2 deleted cells. The cells were suspended in serum-free medium and then added to a transwell (100 μl cell suspension/well at a concentration of 0.5–1 × 105 cells/ml). After incubation for 24 h at 37 °C, cells at the upper surface of the transwell were removed using cotton swabs. Migrated cells that had attached to the lower surface were stained using hematoxylin and eosin. Transwells were rinsed with water and air-dried. Positive cells were quantified using Image-Pro Plus 6.0 software.
PE Annexin V apoptosis detection:
ES-2 WT and WISP2 deficient cells were cultured in 6-well plates overnight. Cells were washed twice with cold PBS and resuspended in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml. Next, 1 × 105 cells were transferred to a 5 ml culture tube, treated with 5 µl of PE Annexin V and 7-amino-actinomycin D, gently vortexed, and incubated for 15 min at 25 ℃ in the dark. After incubation, 400 μl of 1 × Binding Buffer was added to each tube, and cells were analyzed via flow cytometry within 1 h.
Mice and xenograft models
Mice (n = 8) were housed under standard conditions with a 14 h/10 h light/dark cycle and provided with food and water ad libitum. All animal protocols were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. To assess cancer cell proliferation in vivo, we subcutaneously transplanted ES-2 WT or WISP2 deficient cells (1 × 106) into both back flanks of 8-week-old female nude mice. Three weeks later, primary tumor masses were collected from athymic nude mice, fixed in 4% paraformaldehyde, and embedded in paraffin.
Immunohistochemical (IHC) analysis
Primary tumor masses were excised and fixed in 4% paraformaldehyde in PBS overnight. For immunochemistry related studies, sections were deparaffinized, rehydrated with xylene and a descending alcohol gradient, and incubated in 0.3% H2O2. Following antigen retrieval using 10 mM sodium citrate (pH 6.0), sections were incubated with anti-WISP2, anti-p-ERK1/2, anti-p-YAP, anti-p-Histone H3, anti-cleaved caspase-3 antibodies (Cell Signaling Technology, 1:200) using a Vector ABC kit (Vector Laboratories) at room temperature for 1 h. Afterward, the sections were allowed to react with biotin-labeled secondary antibodies for 30 min. Staining was performed using the Vectastain ABC kit and 3,3′-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA).
Immunofluorescence analysis
Cells were cultured in a 24-well plate overnight, washed with PBS, and fixed for 10 min at room temperature with 4% paraformaldehyde in PBS. Cells were permeabilized with 0.3% Triton X-100 in PBS, incubated with the blocking buffer (PBST containing 5% bovine serum albumin), and sequentially probed with anti-p-H2AX, anti-Ki-67, and anti-cleaved caspases-3 antibodies (Cell Signaling Technology, 1:200) and 488-conjugated secondary antibodies (Molecular Probes). Slides were mounted using a VectaShield with 4′, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Digital images were acquired using a laser scanning confocal microscope with 6–100 × magnification.
Western blot analysis
Total proteins were isolated from the cell extracts, and 30 μg of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After probing with primary antibodies, membranes were washed in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated with horse-radish peroxidase-linked secondary antibodies. Finally, the obtained bands were detected using an Enhanced Chemiluminescence Detection Kit ( Millipore, Bedford, MA, USA ).
The primary antibodies used were as follows:
Antibodies
|
Source
|
Indentifier
|
WISP2
|
Abcam
|
Cat#:31317
|
LATS1
|
Cell Signaling
|
Cat#:3477
|
YAP
|
Cell Signaling
|
Cat#:14074
|
YAPS127
|
Cell Signaling
|
Cat#:4911
|
LATS2
|
Cell Signaling
|
Cat#:5888
|
ERK1/2
|
Cell Signaling
|
Cat#:4695
|
p-ERK1/2
|
Cell Signaling
|
Cat#:4370
|
Actin
|
Abcam
|
Cat#:ab3280
|
AKT
|
Cell Signaling
|
Cat#:9272
|
p-AKT
|
Cell Signaling
|
Cat#:4058
|
PARP
|
Cell Signaling
|
Cat#:9532
|
cleaved caspase-3
|
Cell Signaling
|
Cat#:9664
|
KI-67
|
Cell Signaling
|
Cat#:9129
|
p-Histone H3
|
Cell Signaling
|
Cat#:9701
|
RNA extraction and real-time RT-PCR analysis
Total RNA was extracted using TRIZOL, according to the manufacturer’s instructions. Real-time PCR analysis was performed using a KAPA SYBR FAST qPCR kit (Kapa Biosystems, USA) and an Applied 7300 Real-Time PCR System. Relative mRNA levels were determined by normalizing the obtained expression levels to endogenous GAPDH mRNA levels using Microsoft EXCEL. The relative transcript levels of the control sample were set at 1 and compared with the transcript levels of the other samples. Quantitative RT-PCR reactions were performed in triplicate. The following primers were used to amplify target genes:
Gapdh: 5'-GCCTGGAGAAACCTGCCAAGTATG-3' and 5'-GAGTGGGAGTTGCTGTTGAAGTCG-3';
Ctgf: 5'-AGCTGACCTGGAGGAAAACA-3' and 5'-GACAGGCTTGGCGATTTTAG-3';
Cyr61: 5'-GCTCAGTCAGAAGGCAGACC-3' and 5'-GTTCTTGGGGACACAGAGGA-3';
Amotl2: 5'-AGGAGAAGAGTTGCCCACCTATGAG-3' and 5'-TCGAAGAGCTTCATCCTGTCGC-3';
p90RSK: 5'-CAGAGACCTCAAGCCTGAGAAC-3' and 5'-CCACCAGTCCGCACTATGGG-3';
c-Myc: 5'-ACCAGAGTTTCATCTGCGACCC-3' and5'-TGGAGGTGGAGCAGACGCTG-3'
Elk1: 5'-AGGCAATGGCCACATCATCTC-3' and 5'-CGCTCCCTTGCGGATGATG-3';
Mdm2: 5'-CCTGGCTCTGTGTGTAATAAG-3' and 5'-ATCCAACCAATCACCTGAATG-3';
Perp: 5'-GGCTTCATCATCCTGGTGAT-3' and 5'-ACAGCAGCCAAGGCAAGGAG-3';
Snai1: 5'-AGAGTTTACCTTCCAGCAGCC-3' and 5'-GGACAGAGTCCCAGATGAGC-3';
Vimentin-1: 5'-TACATCGACAAGGTGCGCTT-3' and 5'-TCGTTGGTTAGCTGGTCCAC-3';
Skp2: 5'-TTGCGCATGTGTCAGAGACC-3' and 5'-AGGTGTTGGAGGTAGTTGAGC-3';
p16: 5'- CTGCCCAACGCACCGAATAG-3' and 5'- ACCACCAGCGTGTCCAGGAA-3'
Mmp13: 5'- AAGGAGCATGGCGACTTC-3' and 5'- TGGCCCAGGAGGAAAAGC-3'
Htra1: 5'-GGGACTGGTCGTGTTTGTGC-3' and 5'-CATTGACCTTTGGGTGCTGACT-3'
Statistical analyses
All in vitro assays were performed in triplicate. Groups were compared using two-tailed t-tests or ANOVA via the statistical program GraphPad Prism (GraphPad Prism, San Diego, CA, USA). Statistical significance was set at p < 0.05.