Cells, transfection, plasmids, antibodies, reagents

AGS, AGS/DDP and HGC27, HGC27/DDP (human gastric cancer) cell lines were routinely maintained in RPMI 1640 media and DMEM medium high glucose (BDBIO, China) respectively which was fortified with 10% fetal bovine serum under standard culture conditions (Lonsera, China). For transfection, cells were transfected with PolyJet™ DNA transfection reagent when the cell density reached 80–90% confluence (SignaGen Laboratories, Gaithersburg, MD). The HA-Ubiquitin, pLV3-CMV-PARL (human)-CopGFP-Puro, pCMV-GFP-NEDD4L(human)-3 × FLAG-Neo, pCMV-MARCHF8(human)-3 × FLAG-Neo were obtained from MiaoLingBio (Wuhan, China), pcDNA3.1-NPR1(human)-3 × FLAG-C was obtained from Genepharma (Shanghai, China). MG-132 (HY-13259) and Cycloheximide (CHX, HY-12320), Ferrostatin-1 (Fer-1, HY-100579), 3-Methyladenine (3-MA, HY-19312), and Mitochondrial division inhibitor 1 (Mdivi-1, HY-15886) were purchased from MedChemExpress. Chloroquine (CQ, CSN25650) was purchased from CSNpharm. Cisplatin (D8810) was purchased from Solarbio (Beijing, China). PARL Antibody (F-3) and NPR1 Antibody (G-2) were obtained from Santa Cruz Biotechnology. Anti-PINK1, anti-Parkin, anti-LC3B and anti-p62 Antibodies were obtained from abcam. Anti-GPX4, anti-SLC7A11, anti-FTH, anti-HO-1, anti-COX2, anti-TFRC, anti-VDAC1, anti-TOMM20, anti-MARCH1, anti-DRP1, anti-MFN1, anti-β-actin, iFluor™ 647 & 488 Conjugated Goat anti-rabbit & mouse IgG, HRP Conjugated Goat anti-Rabbit & Mouse IgG Antibodies were obtained from Huabio (Hangzhou, China). Anti-NEDD4L and anti-MARCH8 Antibodies were obtained from Proteintech (Wuhan, China).

The quantitative real-time RT-PCR method (qRT-PCR)

Collect total RNA from the cells of each group according to the instructions of Eastep® Super Total RNA Extraction Kit (LS1040, Promega, China). Then, use RevertAid™ First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, USA) to reverse transcribe the obtained cellular RNA into cDNA. Add the cDNA and its corresponding primers and Universal SYBR Green Fast qPCR Mix (4993626, QIAGEN, China)into an 8-tube strip, and use a PCR instrument to detect the expression of each gene.

Small interfering RNA (siRNA)

The target cells, which were approximately 30% to 50% confluent, were transfected with 100 nM of siRNA molecules, mixed with 4.5 μl GenMute (SignaGen, SL100568) in a cell culture plate. Cells were cultured for 48 h before plating for protein or RNA isolation and various assays.

si-NPR1-1: 5′- GUGGAACCGAAGCUUUCAATT-3′;

si-NPR1-2: 5′- CCUGUACACUUGCUUUGAUTT-3′;

si-NPR1-3: 5′-GCCUCAAGAAUGGAGUCUATT-3′;

si-NPR1-4: 5′- GGUACUCACUCACCAAUGATT-3′;

si-PARL: 5′-AUUAACAAGUGGUGGAAUA-3′;

si-MARCH1: 5′- GGUAGUGCCUGUACCACAATT-3′;

si-MARCH8: 5′- GGAAGAGACTCAAGGCCTA-3′;

si-NEDD4L: 5′- AACCACAACAAAGUCACAG-3′.

Cell counting kit-8 (CCK-8) assay

An Enhanced Cell Counting Kit-8 (CCK-8) (BL1055A, Biosharp, China) was used in the cell proliferation assay, wherein 3000 cells/well was seeded in 96 well plates. According to manufacturer’s instruction, we examined the cell proliferation.

Colony formation assay

The GC cells underwent enumeration and were subsequently seeded in triplicate fashion into a 6-well plate, with each well containing precisely 1000 cells. Following a 10-day incubation period, the cells were fixed utilizing 4% paraformaldehyde for a duration of 15 min. Each sample of fixed cells was stained three times with crystal violet solution, ensuring that the staining process was replicated in triplicate for consistency and accuracy. After washing out the dye, we counted colony numbers and compared the results.

Transwell assay

We placed 1 × 10^4 transfected GC cells into the upper chamber of a 24-well transwell insert (8-micron pore size, Corning, USA), utilizing 200 μL of medium devoid of serum. In the lower chamber, 600 µl of complete medium containing 15% FBS was placed. After 24 h of coculture in an incubator, after fixation with 4% paraformaldehyde, the cells were subsequently stained using 0.1% crystal violet. Following drying, the stained cells were photographed using a Nikon optical microscope manufactured in Japan. ImageJ software was employed for counting the stained cells.

EdU assay

The transfected cells were subjected to an incubation process with 50 mM EdU reagent in a 37 °C incubator that was supplemented with 5% CO2 for a period of 2 h. Post-incubation, the cells underwent rigorous PBS washes and were fixed with 4% paraformaldehyde for 30 min. After additional PBS washes, the GC cells were permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, the samples were exposed to an anti-EdU working solution for staining. Following this, the nuclei were counterstained with Hoechst33342 for 10 min and visualized using a Nikon optical microscope from Japan. Subsequently, the enumeration of the stained cells was conducted employing the ImageJ software for analysis.

Analysis of malondialdehyde (MDA), glutathione (GSH), lactate Dehydrogenase (LDH) and Fe 2+levels

For assessing MDA levels, the MDA Assay Kit (A003-4–1, Nanjing Jiancheng Bioengineering Institute, China) was employed. Centrifuged cell lysates (12,000 g, 4 °C, 10 min) were utilized, and the MDA detection working solution was subsequently added, thoroughly mixed, and heated to 100 °C for 15 min. After cooling to room temperature, the supernatant was centrifuged. Utilizing the standard curve method, the MDA content was quantified by measuring the optical density (OD) at 532 nm. For determining GSH levels, the GSH Assay Kit (A006-2–1, Nanjing Jiancheng Bioengineering Institute, China) was applied. Cell lysates, centrifuged at 12,000 g, 4 °C for 10 min, served as the starting material. The GSH detection working solution was added and thoroughly mixed. Following incubation to room temperature, the solution3 was added. Subsequently, the GSH content was quantified through the standard curve method by measuring the optical density (OD) at 405 nm. To analyze Fe2+ levels, the Fe2+ Assay Kit (E-BC-K772-M, Elabscience, China) was employed. Cell lysates, subjected to centrifugation at 12,000 g, 4 °C for 10 min, were utilized. The supernatant was mixed with an iron reductase reagent and incubated for 40 min. Subsequently, the optical density was measured at 593 nm utilizing a microplate reader for Fe2+ quantification. The LDH-Glo™ Cytotoxicity Assay Kit (J2380, Promega,China) was used to conduct LDH + level assay. Optical density readings at 490 nm were obtained using a microplate reader to analyze the plates.

Analysis of mitochondrial membrane potential

To assess the mitochondrial membrane potential, we employed the Mitochondrial Membrane Potential Assay Kit with JC-1 (M8650, Solarbio, China). The transfected cells were first rinsed with PBS and then incubated with JC-1 dye at 37 °C for 20 min. Following the incubation period, the cells were promptly washed with PBS. DAPI (BL105A, Biosharp, China) was used to counterstain nuclei. Fluorescence images were captured by an inverted Routine Microscope (ECLIPSE Ts2, Japan).

Detection of reactive oxygen species (ROS) levels

We counted GC cells, planted them in wells, digested them with DCFH-DA ROS probe (Sigma-Aldrich, St. Louis, Missouri, USA), stained them with the dye, and analyzed the results by flow cytometry. (CytExpert, Beckman, Bremen, Germany).

Western blot assay

Protein content of cells was extracted using Laemmli 2 × Concentrate (S3401; Sigam). By electrophoresis on polyacrylamide gels, proteins were separated from samples and transferred to nitrocellulose membranes (NC) (66,485, PALL, USA) and detected with the antibodies. Using Bio-Rad's ChemiDoc Touch Imaging System (Hercules, USA) to capture the protein bands after incubation with an enhanced chemiluminescence kit (ECL, Millipore, Burlington, USA).

Co-localization assays

The transfected cells were washed with PBS. Incubate the cells with 100 nM LysoTracker Green (C1047S, Beyotime, China) and 100 nM MitoTracker Red(C1035-50 μg, Beyotime, China) at 37 °C for 1 h to label mitochondria and lysosomes, respectively. After staining, replace the staining solution with fresh medium. Fluorescence detection is performed by Confocal Laser Scanning Microcopy (LSM 800, Zeiss, Germany).

Immunofluorescence (IF) assay

The GC cells were seeded onto a confocal dish. Underwent fixation with 4% paraformaldehyde for a period of 15 min, followed by three thorough washes in PBS. To permeabilize the cells, they were exposed to 0.1% Triton X-100 from Solarbio, China, for 10 min. Following this, the cells were blocked in PBS supplemented with 1% Bovine Serum Albumin for 30 min. The cells were incubated with the designated primary antibodies at 4 °C for an overnight period. Subsequently, they were incubated with the secondary antibodies at room temperature for a duration of 1 h. Nuclei counterstaining was achieved using DAPI (acquired from Biosharp, China). Finally, the fluorescence detection was conducted utilizing a Confocal Laser Scanning Microscope, model LSM 800, manufactured by Zeiss in Germany.

Immunoprecipitation (IP)

The cellular material was gathered using an IP lysis buffer which included the protease inhibitor PMSF from Beyotime (product code ST505) and a protease inhibitor cocktail sourced from Biosharp, China (product code P1005). Subsequently, 500 µg of the whole cell lysate protein underwent pre-clearing by being incubated overnight at 4℃ with 1.0 µg of the PARL antibody, utilizing IgG as the control. This mixture was then incubated with Protein A + G Agarose beads (Beyotime, China, product code P2012) for 5 h. Following three washes with chilled PBS buffer, the beads were subjected to boiling in a 2 × SDS loading buffer. Finally, protein expression analysis was carried out using the Western blot assay methodology.

Ubiquitination assay

To the collected cell samples, an adequate volume of cell lysis buffer was introduced. The cell lysate was incubated on ice for a period of time, and then the lysed cells were centrifuged. The supernatant was carefully transferred to a new centrifuge tube, whereupon PARL antibody was introduced to the protein samples. These were then incubated overnight at a temperature of 4 °C. The following day, A/G agarose beads were added to the mixture to sequester the formed antigen–antibody complex. The agarose beads underwent a washing procedure using RIPA buffer. Lastly, the agarose bead-antigen–antibody complex was resuspended in 2 × loading buffer, and the sample was boiled for 5 min to release the antigen, antibody, and beads. Subsequently, the ubiquitination level was detected by Western blot assay using ubiquitin antibody.

LC–MS/MS

Hydrolyze all protein samples from co-IP by protein endonucleases (Trypsin). Subsequently, the hydrolyzed samples were subjected to detection and analysis utilizing Liquid Chromatography-Tandem Mass Spectrometry (nanoLC-QE). Upon completion, the LC–MS/MS data were processed by means of mass spectrometry matching software, specifically MASCOT, to attain qualitative identification information pertaining to the peptide molecules of the target protein.

Nude mouse xenograft animal assay

Male nude mice, aged approximately 4–5 weeks and weighing between 18 and 20 g, were procured from Qinglongshan Animal Breeding Farm located in Jiangning District, Nanjing, China. To explore the in vivo function of NPR1, the mice were randomly assigned to three distinct groups: an NPR1 knockdown group, an overexpression group, and a control group, with an equal number of 5 mice in each. From each group, a quantity of 5 million cells (5 × 10^6) were harvested and subsequently subcutaneously injected into distinct locations within the inguinal area of each mouse. One week following the injection, cisplatin (administered at a dose of 5 mg/kg) dissolved in PBS, or solely PBS, was injected intraperitoneally three times per week. After a period of 5 weeks, the xenograft tumors were extracted. Throughout the study, the tumor volume was regularly monitored on a weekly basis, and after 5 weeks, the tumors were excised and weighed for analysis.

Statistical analysis

All experimental findings are presented in the form of the mean, accompanied by the standard deviation (SD), derived from three independent and replicate experiments. The statistical evaluation of these data was conducted utilizing the GraphPad Prism 6 software package (La Jolla, CA, USA). For comparisons between two groups, Student's t-test was employed to assess the significance of observed differences. For evaluating the statistical significance of biological effects in more complex scenarios, either one-way or two-way Analysis of Variance (ANOVA) with multiple comparison tests are utilized. These methods allow for the comprehensive assessment of differences between multiple groups, providing insights into the underlying biological mechanisms and effects. A p value < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01 and ***P < 0.001.