2.1 | Cell culture
The human pancreatic cancer cell lines (PANC-1 and BxPC-3) were obtained from the National Collection of Authenticated Cell Cultures and cultured according to standard ATCC protocols. In brief, PANC-1 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). BxPC-3 cells were cultured in Roswell Park Memorial Institute (RPMI) containing 10% FBS.
2.2 | RNA isolation and quantitative real-time PCR
Briefly, total RNA was extracted using TRIzol reagent (EZBioscience B004D) according to the manufacturer's protocol. NanoDrop 2000 (Thermo Fisher Scientific) was used to test the concentration of RNA. Complementary DNA was synthesized using reverse transcription kits (Takara, RR036A). Quantitative real-time RT-PCR (qRT-PCR) was performed with QuantStudio 6 Flex (Thermo Fisher Scientific). The 2−ΔCt method was used to analyze qRT-PCR data. More information on primers used are listed below.
PITX2: 5'- GTG TGG ACC AAC CTT ACG GAA G -3' (forward), 5'- CGA AGC CAT TCT TGC ATA GCT CG -3' (reverse);
LRP6: 5'- CAG TTG GAG TGG TGC TGA AAG G-3' (forward), 5'- CCA TCC AAA GCA GCC CGT TCA A-3' (reverse);
CTNNB1: 5'- CAC AAG CAG AGT GCT GAA GGT G-3' (forward), 5'- GAT TCC TGA GAG TCC AAA GAC AG-3' (reverse).
AXIN1: 5'- GTA TGT GCA GGA GGT TAT GCG G-3' (forward), 5'- CAC CTT CCT CTG CGA TCT TGT C-3' (reverse);
LEF1: 5'- CTA CCC ATC CTC ACT GTC AGT C-3' (forward), 5'- GGA TGT TCC TGT TTG ACC TGA GG-3' (reverse);
WNT2: 5'- AGG ATG CCA GAG CCC TGA TGA A-3' (forward), 5'- AGC CAG CAT GTC CTG AGA GTA C-3' (reverse);
GPX4: 5'-AAG TTC AGT CAG AGA CCT GCG-3' (forward), 5'-ATA TCC GAG CCC TCC TCC TTC − 3' (reverse);
β-actin: 5'- CAC CAT TGG CAA TGA GCG GTT C-3' (forward), 5'- AGG TCT TTG CGG ATG TCC ACG T-3' (reverse).
2.3 | Western blotting
Cells were lysed with RIPA buffer (Beyotime P0013D), followed by lysis of nuclei by an ultrasonic breaker (Ningbo Xinzhi Co., Ltd.) at a power of 50w for 15s. Samples were heated at 100°C for 5 min in SDS-PAGE protein uploading buffer, separated with 4%-12% SDS-polyacrylamide gels, and transferred to PVDF membranes by a wet transfer device (Bio-Rad). The membranes were closed with 5% skimmed milk powder for 4h at room temperature and then incubated at 4°C. The secondary antibodies were implemented to incubate membranes after washing for three times using TBST solution. We obtained antibodies targeting PITX2 (Abcam, ab98297), GPX4 (ABclonal, A1933), LRP6 (ABclonal. A22661), CyclinD1 (ABclonal A19038), AXIN1 (Affinity, DF9264), β-Catenin (Affinity, AF6266), LEF1 (Affinity, DF7570), β-actin (Santa Cruz, sc-47778). Next, the membranes were probed with secondary antibodies conjugated to HRP Goat Anti-Mouse IgG (H + L) (ABclonal, AS003) and HRP Goat Anti-Rat IgG (H + L) (ABclonal, AS028). Antigen-antibody complexes were visualized using the Omni-ECL™ Enhanced Pico Light Chemiluminescence Kit (Epizyme, SQ101L).
2.4 | Stable knockdown and overexpression of PITX2 by lentiviral vectors
Stable knockdown of PITX2 was induced by lentiviral infection. PITX2 interference lentivirus suspension and negative control (NC) were constructed by HANBIO (Shanghai, China). The sequences were as follows: shPITX2-1: 5′-GCTGTGTGGGACCAACCTTA-3′; shPITX2-2: 5′-CCAACTCTATCTCGTCCAT-3′; and NC: 5′-TTCTCCGAACGTGTCACGT-3′. Puromycin was used to screen infected stable cells after infection. The coding sequences of human PITX2 were cloned into the vector pCMV-c-flag (Beyotime) to generate PITX2 expression plasmids.
2.5 | Chemicals
RSL3 (S815502), Ferrostatin-1 (S724302), and gemcitabine (S114910) were purchased from Selleck. BODIPY™ 581/591 C11 was purchased from Invitrogen (Invitrogen™, D3861).
2.6 | CCK-8 cell viability assay and colony formation assay
PANC-1 and BxPC-3 cell lines were grown at a concentration of 5000/well into 96-well plates and incubated in 100 µl of medium with 10 µl of CCK-8 (Beyotime, C0040) for 1 h at 37°C. Cell viability was evaluated by measuring the absorbance values using an enzyme labeling instrument (MOLECULAR, SpectraMax ABS) at a wavelength of 450 nm. For the colony formation experiments, PANC-1 and BxPC-3 cells were inoculated at 500 cells per well in 6-well plates and cultured for 14 days. The colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet for 30 min and then counted.
2.7 | 5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
An EdU assay was conducted to detect the proliferation of PANC-1 and BxPC-3 cell lines. The BeyoClick™ EdU Cell Proliferation Kit (Beyotime, C0078S) containing Alexa Fluor 594 was used. PANC-1 and BxPC-3 cell lines were seeded in 24-well plates. After 48 h culture, the cells were fixed with 4% paraformaldehyde, incubated with 10 µM EdU solution for 2 h, and then stained with Click Reaction Solution and with DAPI. EdU-labeled cells were photographed and counted under an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
2.8 | Migration and invasion assay
Transwell chambers were used to detect cell migration ability. PANC-1 and BxPC-3 cell lines were resuspended in serum-free medium at a concentration of 5 × 105 cells/ml. 200 µl of cell suspension was added to the upper chamber, and 500 µl of serum-containing medium was added to the lower chamber. Then, the non-migrated cells were removed using a cotton swab after 24h of incubation at 37°C. The cells in the lower chamber were fixed with 4% paraformaldehyde for 20 min and then stained using 1% crystal violet. The cells were fixed with 4% paraformaldehyde for 20 min, then stained with 1% crystal violet for 30 min and observed under the microscope. Transwell chambers were also applied to detect cell invasion ability. PANC-1 and BxPC-3 cell lines were resuspended in serum-free medium at a concentration of 5 × 105 cells/ml, and 200 µl of cell suspension was added to the upper chamber pre-coated with Matrigel, and 500 µl of serum-containing medium was added to the lower chamber. After 48h of incubation, the cells were removed from the upper layer with a cotton swab and fixed in 4% paraformaldehyde for 20min and then stained with 1% crystal violet for 30min and observed under a microscope.
2.9 | BODIPY™ 581/591 C11 staining and Reactive oxygen species assay
Cells were inoculated in 6-well plates at a density of 600,000/well. After 24h of culture, cells were stained with 2 µmol/L C11 BODIPY (Thermo Fisher Scientific, D3861) for 30 min and observed by flow cytometry or confocal microscopy. For ROS assay, cells were also inoculated at 600,000/well in 6-well plates and cultured for 24h. The concentration of DCFH-DA (Beyotime, S0033S) was diluted to 10 µM using serum-free medium, incubated at 37°C for 20 min, then washed with serum-free medium and detected by flow cytometry.
2.10 | MDA and GSH/GSSG Assay
The PANC-1 and BxPC-3 cell lines were inoculated in 6-well plates. Cells were lysed to obtain cell homogenates, and the protein concentration was determined using the BCA protein analysis kit (Epizyme, ZJ101), followed by the Lipid Peroxidation MDA Assay Kit (Beyotime, S0131M) to measure the MDA concentration. The ratio of MDA to protein concentration was subsequently calculated. The ratio of GSH/GSSG was determined according to the GSH and GSSG Assay Kit (Beyotime, S0053) and normalized using the protein concentration of cell lysates.
2.11 | Enhanced mitochondrial membrane potential assay
400,000 cells were seeded in confocal dishes and cultured for 24h. With an enhanced mitochondrial membrane potential assay kit with JC-1 (Beyotime, C2003S), the cells were incubated at 37°C for 20min and then washed 3 times, followed by observation under a confocal microscope (Leica, STELLARIS 8 CRS).
2.12 | Transmission electron microscope (TEM) imaging
PANC-1 and BxPC-3 cell lines were placed in 10-cm cell dishes and pretreated with the appropriate conditions for 24 h. Then, cells were collected and fixed with 2.5% glutaraldehyde. TEM imaging was performed by Servicebio, Wuhan, China.
2.13 | RNA-seq analysis
RNA-seq was conducted at BioProfile Biotechnology (Shanghai, China).
2.14 | Cell death
Apoptosis was assessed by FACS (Beckman Coulter) using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™, 556547).
2.15 | Chromatin immunoprecipitation (ChIP) assay and promoter activity assessment by a dual-luciferase assay
Chromatin immunoprecipitation assays were conducted using the Chromatin immunoprecipitation Kit (BersinBio, Bes5001). The primers used to detect GPX4 promoter occupancy are listed as follows: Primer1: 5'- TTG GCC GGA TGC GGT GGC TC-3' (F), 5'- AAG CGA TTC TCC TGA CTC AGC − 3' (R); Primer2: 5'- TAG CGC CAC TGC ACT CCA GC-3 ' (F), 5'- CAT TCC TGG CTA ATT ATT G-3' (R). The GPX4 promoter region, spanning from − 2000 to + 200 of the transcription start, was cloned into the pGL3-Basic vector (Promega). A dual-luciferase system (Promega, E1910, USA) was used to measure firefly and renilla luciferase activities according to the manufacturer's protocol.
2.16 | Glutathione peroxidase (GPX) activity assay
5 × 106 cells were taken from each of the PANC-1 and BxPC-3 cell lines and lysed by sonication to obtain the lysis products according to the instructions of the Glutathione Peroxidase (GPX) Activity Assay Kit (Sangon, D799618). The absorbance was measured at 412 nm using a spectrophotometer after the reaction.
2.17 | Xenograft tumors in nude mice and immunohistochemical staining (IHC)
We conducted all animal experiments in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. The animal experiments were approved by our institutional animal care. To establish a tumor xenograft model, 3 × 106 PANC-1 cells were injected into the pancreatic peritoneum of nude mice. After 4 weeks, tumors were removed from executed nude mice, and tumor size was measured. In paraffin-embedded tissue sections, anti-PITX2 and GPX4 antibodies were used and stained according to standard IHC procedures. The dilution ratio of the anti-PITX2 antibody (Affinity, DF13574) and anti-GPX4 antibody (ABclonal, A1933) was 1:1000.
2.18 | Statistical analysis
All statistical analyses were performed using GraphPad Prism version 8.0 software (GraphPad Software, USA). All data were reported as the means ± SD of triplicate experiments, and the differences between the two groups were compared using the two-tailed Student’s t-test. Comparisons between multiple groups were performed using one-way ANOVA, and a P-value < 0.05 was statistically significant.