Cell culture and treatments
293T cell line was donated by Professor Sheng Wang of Beijing University of Technology. CEC2 and EC109 cell lines were provided by Dr. Jintao Li at Beijing University of Technology. These cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Beyotime Institute of Biotechnology), 100 mg/mL streptomycin and 100 U/mL penicillin (Beyotime Institute of Biotechnology) in a 37 ℃ and 5% CO2 atmosphere.
Tissue microarray and immunohistochemistry (IHC)
The tissue microarray was prepared by Wuhan Iwill Biological Technology Co.Ltd. It contains 34 ESCC tissues (tumor) and corresponding adjacent normal esophageal tissues (AT). The scores are based on the sum of the percent staining area plus staining intensity. Clinical information was provided by the commercial source. The expression of p62 in tissue microarray was summarized in Table 1. The paraffin-embedded esophageal tissues were obtained from the Department of Oncology, Affiliated Taihe Hospital of the Hubei University of Medicine. These include poorly differentiated (n=5), moderately differentiated (n=5), and highly differentiated (n=5) esophageal squamous cell carcinomas (It's classified by pathology). Briefly, 4-μm-thick sections mounted on glass slides were processed for immunohistochemistry (IHC). After dewaxing and hydration, endogenous peroxidase was inactivated by 3% methanol hydrogen peroxide for 10 min. Then 10% goat serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) was used to block nonspecific binding by incubating sections for 2 h at room temperature while gently tilting the sections without washing them, followed by incubation with p62 (1:200; cat. no. 55274; ProteinTech Group, Inc.), Nrf2 (1:200; cat. no. 55274; ProteinTech Group, Inc.) and Vimentin (1:200; cat. no. AF1975; Beyotime Institute of Biotechnology) anti-bodies at 4 °C overnight in a moist chamber. After being washed three times with phosphate-buffered saline (PBS), the sections were incubated with a secondary antibody (1:200; cat. no. A0208; Beyotime Institute of Biotechnology at room temperature) for 1 h and rinsed in PBS. Diaminobenzidine (DAB) was used as a chromogen, and sections were counterstained with hematoxylin. Negative controls were obtained by incubating specimens with PBS instead of the primary antibody. All patients signed informed consent forms prior to the study. Brown particles in cells are thought to be positive signals; the staining intensity was determined and scored by two pathologists. Please refer to Table 2 for scoring criteria.
Establishment of p62 knockdown, Nrf2 knockdown, p62 and Nrf2 double knockdown stable cell lines
To produce p62 knockdown, Nrf2 knockdown, p62 and Nrf2 double knockdown stable cell lines, p62 and Nrf2 deletion mutants were first constructed. Oligo DNA sequences were designed in the target DNA region using the CRISPR Design online tool, the sequences were shown in table 2. The primer dimers of oligo DNAs were annealed with the pGL3-U6-sgRNA-PGK plasmids. The plasmids were transfected into cells using an electro cell manipulator electroporator (Boston Industries, Inc.), and after 48h, 2 μg/mL puromycin (cat. no. ST551; Beyotime Institute of Biotechnology) at room temperature was added to the medium for 3 days. When the cells reached the logarithmic stage, they were digested and a single cell was cultured in a 96-well plate for 15 days, and then transferred to a 6-well plate for culture. Cells were collected and genomic DNA was extracted. T7E1 endonuclease kit (cat. no. E001S; VIEWSOLID BIOTECH) was used to detect the target effeciency. Positive cell lines were selected for subsequent experiments.
RNA extraction and reverse transcription PCR
Total RNA was extracted from cells by TRIzol (Beyotime Institute of Biotechnology). Reverse transcription was carried out using reverse transcriptase (Takara Bio, Inc.) according to the manufacturer's instructions. The levels of E-cadherin, N-cadherin, Vimentin and Snail were detected by PCR (95 ℃ for 3 min;95 ℃ 30 SEC; 58 ℃ 30 SEC;72 ℃ 30 sec; 72 ℃ for 5 min; for 30 cycles),GAPDH serves as an internal reference. Gene-specific primers are shown in table 2 in the supplemental file.
Western blot analysis
Cells of different treatment groups were incubated in T25 culture bottles. Total protein was isolated from the cells using RIPA buffer with 1% phenylmethyl sulfonylfluoride (PMSF). The protein concentration was determined by BCA protein detection kit (Beyotime Institute of Biotechnology). 30μg total protein in each sample was isolated in 12% tris-glycine SDS gel, and transfer to 0.22 μm polyvinylidene fluoride (PVDF) membrane (Beijing BeiFang Tongzheng Biotechnology Development Co. Ltd). Then the membrane was sealed in TBST with 5% skim milk powder for 2 h, and incubated overnight with the corresponding primary antibody (diluted according to instructions), TBST was used for washing three times, each wash took about 10 min, The membrane was incubated at room temperature for 2 h with HRP-conjugated secondary antibody (1:5000; Beyotime Institute of Biotechnology). The binding antibody was observed with an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) and imaging system (Bio-Rad, USA).
F-actin staining
Cells from different treatment groups were cultured in 96-well plates until 50% confluent, and fixed with 4% paraformaldehyde. Cells were permeated with 0.5% Triton X-100 and incubated in the dark for 30 minutes with 100 nM of rhodamine-phalloidin (Cytoskeleton, Inc.) at room temperature. Then the nuclei were stained using DAPI (Thermo Fisher Scientific, Inc.). Lastly, the cells were observed and photographed using High-Content Analysis (HCA; PerkinElmer, Inc.), and analyzed using the harmony software.
Wound healing assay
Cell migration was detected by wound healing assay. Cells were cultured in 6-well plates until arrived at 80 to 90 percent of confluence. A 200 μL pipette tip was used to produce scratches. Then the cells were cultured in serum-free medium and photographed with an inverted microscope (Leica Corporation) at 0, 24, 48, 72 and 96h after scratching. The wound healing rate was measured as follows: [(empty area 0 h -empty area x h)/ empty area 0 h] ×100.
Invasion assay
The matrigel (BD Biosciences, CA, USA) was added to each upper chamberaccording to the manufacturer’s instructions. 1×105 ESCC cells were inoculated into the upper chamber. Medium containing 10% FBS was added to the lower chamber. After incubation at 37 ℃ for 72 h, the non-invasive cells were gently wiped with a cotton swab from the top of the matrigel. The invasive cells at the bottom of the chamber were fixed in methanol and stained with 0.1% crystal violet. The cells in five random fields were counted with inverted microscope (Leica Corporation) and quantified.
Colony formation assay
A total of 500 cells were cultured in 6-well plates for 2 weeks. After the cells form visible clones, the cells were fixed with methanol for 20 min and stained with crystal violet for 30 min. The numbers of clonesin each group were counted with an inverted phase contrast microscope (Leica Corporation). Three independent replicates were performed.
Xenograft assay
All animal experiments were approved by Animal Ethics Committee at Animal ethics committee of the Hubei University of Medicine, all animal procedures and animal care were conducted in accordance with the guidelines for institutional animal research. Female BALB/c-nu mice aged 4 weeks were selected (purchased from Shanghai experimental animal center of Chinese academy of sciences). They were fed under specific, non-pathogenic conditions and given sterilized food and water. 1×107 wild , p62KD, Nrf2KD, and p62KD:Nrf2KD EC109 cells were subcutaneously injected into the left armpit, right armpits, left groin and right groin of 4 nude mice, respectively. After 4 weeks, the mice were killed, the subcutaneous tumor was removed and fixed with 10% paraformaldehyde for further analysis. Similarly, The same number of wild, p62KD, Nrf2KD, and p62KD:Nrf2KD EC109 cells were inoculated into the tail vein of 4-week-old female nude mice. After 8 weeks, the visceras of all the mice were dissected and fixed in formalin for further analysis.
Statistical analysis
The values are shown as the means±S.D. for triplicate experiments. To statistically analyze the significance between two groups or greater than two groups, the Student’s t-test or the ANOVA test was appropriately used, respectively. The Fishers exact test was used to analyze the relationships among PHLDA2 expression and clinical characteristics of ESCC patients. P<0.05 was considered statistically significant. SPSS 25.0 software was used for all statistical analyses.