Page 9 - 《南京医科大学学报（自然科学版）》2025年第9期

第45卷第9期 苏 宁，曹 颖，张淑平，等. PARP1在K192位点的乳酸化抑制卵巢癌细胞的迁移和增殖［J］.
2025年9月 南京医科大学学报（自然科学版），2025，45（9）：1219-1228，1241 ·1221 ·

from the China Cell Bank（Shanghai，China）. The were transfected with empty（no PARP1 insert），
A2780 cells were cultivated in high⁃glucose Dulbecco’s PARP1⁃WT，or PARP1⁃K192A plasmids using Lipo⁃
modified Eagle medium（Gibco，USA）supplemented fectamine 2000 reagent（Fisher Scientific，USA）. The
with 10% fetal bovine serum（Gibco，USA）and 1% pen⁃ cells were then cultured for 24 h for PARP1 expression.
1.2.4 Colony formation assays
icillin/streptomycin（Sigma，USA）at 37 ℃ in a 5% CO2
incubator with 90% humidity. Twenty⁃four hours after the transfection，200 cells
1.1.2 Tumor samples and normal tissues were seeded into each sub⁃well of a six⁃well plate and
Clinical OC samples，including primary OC tis⁃ cultivated for additional six days. The wells were then
sues（n=37）and normal ovarian tissues（n=21），were fixed with 1% paraformaldehyde，stained with 0.1%
collected from the Second Affiliated Hospital（SAH）of crystal violet dye，and analyzed under a light micro⁃
Nanjing Medical University（NJMU）. The tissue sam⁃ scope（Ti2⁃A，Nikon，Japan）.
ples were examined by qualified clinicians to ensure 1.2.5 Wound healing assay
5
the correct classification. This study was conducted in Twenty⁃four hours after transfection，5×10 cells
strict accordance with the 1975 Declaration of Helsin⁃ were seeded into each sub⁃well of the six⁃well plates
ki and was approved by the Ethics Committees of the and cultured untill 80%-90% confluence. Next，a ster⁃
SAH of NJMU（authorization no.：［2023］⁃KY⁃007⁃01）. ile 10 μL pipette tip was used to create a liner scratch
1.2 Methods across the cell monolayer. Then，three randomly selected
1.2.1 Dot blotting areas were imaged at assigned time points（0，24，48 h）
Proteins were isolated from clinical tissue sam⁃ using a light microscope and measured with ImageJ
ples by lysing with PIPA buffer，separated by sodium software.
dodecyl sulfate ⁃ polyacrylamide gel electrophoresis 1.2.6 ROS detection
（SDS⁃PAGE），and then transferred to polyvinylidene Reactive oxygen species（ROS）were quantified
difluoride（PVDF）membranes. Next，the membranes using a ROS Detection Kit（Beyotime，China）. First，
were washed and blocked with blocking buffer（Tris ⁃ cells were spread on glass slides and immobilized，
buffered saline with Tween 20 containing 5% non⁃fat then incubated with a ROS detection probe tagged with
milk）. Subsequently，they were incubated with primary dichlorofluorescein diacetate for 20 min at 37 ℃ in the
and secondary antibodies against target proteins. Finally， dark. Then，ROS signals were imaged under a spinning
the target proteins were visualized using an enhanced disk confocal microscope（Oxford Instruments，UK）.
chemiluminescence（ECL）kit（Yeasen，Shanghai，Chi⁃ 1.2.7 Detection of early apoptosis
na）and an ECL detection system（Tanon，Shanghai， A2780 cells were cultured in dishes with a glass
China）. bottom and stained with 5 μL of an Annexin V⁃FITC/
1.2.2 PTM identification by mass spectrometry PI Kit（Yeasen，China）for 15 min in the dark at room
Human OC and normal ovarian tissue samples temperature. Finally，apoptosis signals were imaged un⁃
（three per group）were preserved in dry ice and trans⁃ der a spinning disk confocal microscope.
ported to a specialized mass spectrometry company（Bio⁃ 1.2.8 Immunofluorescence
techPack，Beijing，China）for PTM site identification. Immunofluorescence was performed as previously
［9］
1.2.3 Plasmid construction and transfection described . Briefly，A2780 cells were grown on glass
Enhanced green fluorescent protein（EGFP）with slips and then permeated，fixed，and blocked with 1%
a Strep Ⅱ peptide tag was attached to the C⁃terminal bovine serum albumin in PHEM. Then，they were incu⁃
end of human PARP1 and inserted into the mammalian bated with primary antibodies against marker of prolif⁃
expression plasmid pcDNA3.1（+）. The PARP1⁃K192A eration Ki ⁃ 67（MKI67；Cell Signaling Technology，
and ⁃K192Q mutant constructs were generated by site⁃ USA）and proliferating cell nuclear antigen（PCNA；
directed mutagenesis（Vazyme，Nanjing，China）using Sangon Biotech，Shanghai，China）and then relevant
the wild⁃type（WT）plasmid as a template. A2780 cells fluorophore ⁃ tagged secondary antibodies. DNA was