Plasmids and transfection
The human PAFAH IB2 full length expression plasmid (pEGFP-C1-PAF-AHIB2-WT) was got as gift from Prof. Xueliang Zhu  and used to co-express GFP as a marker. The mouse PAFAH IB2 WT, functional mutants (E39D, S48C, E39D/S48C) and Lis1(pcDNA3.1-3xFlag vector) were bought from NovoPro and confirmed by sequencing. The HOSE (1 x 103) cells growing in complete medium were transfected with human PAFAH IB2 and mouse PAFAH IB2 wild type and mutant constructs for relative functional assay.
Explore PAF-AH IB2 gene with ovarian carcinoma cases through TCGA
The PAF-AH IB2 genes were explored in the Cancer Genomics dataset (TCGA) through Gepia and investigated the genetic alterations associated with serous ovarian carcinoma’s patient’s cases, which provides large-scale cancer patients genomics data sets from TCGA to research community for visualization, analysis and downloads . Kaplan–Meier plots were generated from an online dataset (http://www.kmplot.com) [GSE15459 and GSE62254]. The disease-free survival (PFS) analysis was performed by using patient’s information. The patient’s population was split by median value.
Ovarian Cancer Cell lines
The human ovarian cancer cell lines, represent the Endometrioid (Tov112D), Clear cells (RMG1), Serious (SKOV3, OVCA3, OVCA420, OVCA432, OVCA633 and OVCA 810) and Mucinous (MCAS, RMUG-L), and the normal human ovarian surface epithelial (Hose 11 and Hose 17-1) cells have been described previously . These cells were bought from National collection of authenticated cell cultures (Shanghai, China) for research purpose only. HOSE cells were immortalized by an HPV E6/E7 gene introduction for research use purpose. Ovarian cancer cell lines were cultured in a medium mixture of MCDB105 medium and 199 (1:1) (Sigma, St. Louis, MO), and supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) and maintained in a 37 °C humidified atmosphere [95% O2+ 5% CO2].
Whole transcriptome expression profiling
RNA was extracted from control and PAFAH1B2 KD cells by TRIzol reagent kit (Invitrogen, Carlsbad, CA). The RNA quality and quantity of samples were tested using spectrophotometric analysis and Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA was extracted from cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA). 1 ug of RNA per each sample were used for target labelling by a two-round amplification protocol. Expression profiles were determined using 4.5μg of fragmented, labelled and hybridized with per Chip (Human Gene whole transcript 1.1 ST Arrays, Affymetrix) The expression data were normalized by RMA pre-processing protocol, background-corrected, and log2-transformed for parametric analysis. All internal control genes were removed and the remaining probe clusters were imported into the Affymetrix Power Tools software (APT package) for next step analsysi. Differentially expressed genes were identified using significance analysis of microarrays (SAM) with the R package ‘samr’ (false discovery rate (FDR) <0.05; fold change >2) and determining the gene list based on the number of significant genes that were identified by fold change. Two-dimensional hierarchical clusters are generated.
Metascape pathway Analysis
Gene ontology (GO) and pathway enrichment analysis of PAF-AH IB2 KD - associated significantly changed genes were performed using Metascape (http://metascape.org/). In this study, an ordered list of genes was first generated by GSEA based on correlation with PAF-AH IB2 KD. The significant survival difference observed between control and PAF-AH IB2 KD was elucidated. Gene set permutations were performed 1,000 times each analysis. The nominal p-value and normalized enrichment score (NES) were used to classify the pathways enriched in each phenotype.
Tumour tissue array Immunohistochemistry analysis
Ovarian cancer tumour tissue microarray was bought from bioaitech (product ID: F100Ov01, xi’an, China), which contained formalin-fixed, paraffin-embedded normal, benign, and cancerous ovarian tissues with identified pathological diagnosis. The array included specimens of 100 ovarian malignancies of surface epithelial origin that representing 5 different histologic types. Sections (5 mm) were applied to detect expression of PAFAH1B2 in ovarian tumour tissues. Briefly, slides were deparaffinized in xylene and rehydrated by a series of graded alcohols buffers, and then 3 min boiled process in a pressure pot to retrieve the antigens. The 3% hydrogen peroxidase 10 min-treatment was used to block endogenous peroxidases. The sections were incubated with PAFAH1B2 antibody (20365-1-AP, Proteintech, China; overnight, 4°C). The peroxidase conjugated secondary antibody (37 °C, 30 min) was incubated with sections and performed the chromogenic with a DAB Substrate Kit, and then counterstained with hematoxylin. The slides were then dehydrated in graded alcohol buffers and covered with coverslips. Staining intensity and percentage of PAFAH1B2-positive tumour cells were observed by microscope and assessed. The staining tumour tissue images were observed and evaluated by ImageJ software and IHC Profiler plugin. The intensity of slide immunohistochemistry was scored automatically after the slides counting. The IHC scored values are represented as means±SEM. The ANOVA analysis was used to compare the mean values of IHC scores between benign and different tumour histological types.
Lentiviral knockdown and plasmid transfection
The lentiviral PAF-AH Ib2-targeting and non-target control shRNA transduction particles (Mission™) were purchased from Sigma-Aldrich (St. Louis, MO). To generate stable knockdown of PAF-AH IB2, ovarian cancer cells (MCAS, SKOV3, OV432, RMGUL; 1 x 105 cells) were growing in complete medium and infected with lentivirus containing pLKO short-hairpin RNA (ShRNA) constructs for PAF-AH IB2 (Sigma). After 48 h infection, cells were screened with medium containing puromycin (2 mg/ml) as the lentivirus vector contained this selection resistance marker for 2 weeks. Stable PAF-AH1b2 knockdown cell lines were validated by Western blot.
Proliferation and scratch wound healing assay
The cell proliferation and cytotoxicity of the drugs to ovarian cancer cells were tested by tetrazolium-based MTT method  in time point manners. Briefly, at the beginning, the single cells solution was (5,000 cells /well) allocated into each well of 96 wells. For proliferation assay, the cells were cultured as normal, and MTT dye solution was added to each well (10μl/well) after per 24h cultured to incubate at 37°C for 4 hours in a humidified chamber. For drug toxicity assay, the drugs were added into plate wells after cells were completely attached. After 48 hours of treatment, MTT dye solution was added into and incubated (37°C, 4 hours) in a humidified chamber. After incubation, solubilization/stop solution (100μl/well) was added and incubated for one hour, the content of wells was mixed and read by 96-well plate scanning spectrophotometer (μQuant) and quantitative software (KC-junior, Bio-Tek Instruments, Inc.) (Absorbance value in 630 nm) for quantitative analysis. The scratch wound healing was performed using a 6 well plate. The cells were cultured as for 24h to form a confluent monolayer, then scratches were performed using a 10-l tip and the culture medium was replaced with fresh complete medium. At the start of experiment, after 12h, 24 h and 48h of incubation, the plates were checked under microscope and took images to track the scratches width. All the images were converted as 8-bit images and analysed using Image J programmer to quantitative calculate the scratches width.
Cell proliferation, invasion/migration in real time by xCELLigence system
The dynamic of cell proliferation, adhesion and migration were assessed by measuring cell amount in real time manner through a xCELLigence system and E plates (Roche). It could monitor cellular events in real time through measuring electrical impedance across interdigitated gold micro-electrodes integrated on the bottom of tissue culture plates. This dynamic measurement provides quantitative data about the biological status of the cells, including cell number, viability and morphology . Briefly, for determination of cell survival and proliferation, E-plate 96 (Roche Applied Science) assemblies were seeded with MCAS/SKOV3 cells (2.0 x 104 cells/well). Plate was assembled on the RTCA DP analyzer, and collecting data with 5-min intervals for 20 h (37 °C, 5% CO2). To examine cell adhesion and migration, serum free medium was added to E-plate 16 to obtain background readings, and cells were added to wells of a CIM plate 16 (Roche Applied Science; 8-m pore size), and dried the membranes at 25 °C for 1 h. The lower chambers were added with fresh medium (10% FBS or with serum-free medium), whereas the upper chambers were filled with serum-free medium (30l/well) [37 °C, 5% CO2, 1 h]. The cells were added to each well and balance for a while [25 °C, 30 min], then assembled the CIM plate onto the RTCA DP analyzer. The cell migration was assessed for 24 h (37 °C, 5% CO2) with 5-min intervals. The data were analysed using the provided RTCA software. The extent of change is proportional to the cell number, morphological and adhesive features. The more cells that are growing on the electrodes, the higher value of electrode impedance increases . Cell index (CI) slope is defined to represent cell status according to the measured relative change in electrical impedance that occurs in the presence or absence of cells in the wells, which is calculated by the following formula: CI=(Zi–Z0)/15, where Zi represents the impedance at an individual time point during the experiment, and Z0 is the impedance at the start of the experiment .
Colony-Forming Assays in agar gel
The scramble control and stable PAF-AH IB2 KD of MCAS Cells were cultured in soft agar gel for additional 30-day cultured followed the protocol. The cancer cells formed colonies were stained (0.5% crystal violet/20% ethanol) and taken image by light microscope. The colonies numbers were calculated by using Image J software.
Cells were washed with cold PBS (phosphate buffered saline) for twice, and the cellular lysates were prepared in ice-precold lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.5 mM DL-dithiothreitol and proteinase inhibitor cocktails, Pierce), and homogenized using a Sonic Dismembrator 100 (Fisher Scientific Inc., MA). The cell lysates protein concentration was determined by MicroBCA kit (Pierce) and equal amount of total proteins from different cell lysates were resolved by SDS-PAGE (4 ~12%) for Western blotting with antibodies against PAF-AH 1B1/B2/B3, beta-actin, phosphorylated p44/42 (Thr202/Tyr204), phosphorylated Akt (Ser473), phosphorylated p53 (Ser15), phosphorylated p21 Waf1, phosphor-Chk2 (Thr68), phosphorylated-CDC2 (Tyr15) and CDC (Cell Signaling, CA). Primary antibodies were visualized by secondary antibodies of goat-anti mouse (IRDye 680CW) or goat-anti rabbit (IRDye 800CW) through an Odyssey scanner (Li-cor biosciences).
The total tyrosine kinases profiles of target cells, including 62 of the 90 tyrosine kinases in the human genome, were performed by Luminex xMAP microspheres (Luminex Corporation, Austin, TX) system, which was coupled individual bead-type of antibody to capture target. According to manufacturer’s recommended procedure, each bead-type of Luminex xMAP microspheres (100 ul, Luminex Corporation, Austin, TX) were coupled separately to antibodies and performed the assays as previously described . Briefly, test data were acquired through a Luminex FlexMAP 3D instrument (Luminex Corporation). The background readings value for each capture antibody were normalized by microspheres with 1x cell lysis buffer (Cell Signaling Technology). Reading values were defined as positive only that higher threefold over the background. The results were normalized against unstimulated EGFR and presented as a fold change in relative phosphorylation. Final average results were generated from three independent experiments.
Flow cytometry for cell apoptosis analysis
Samples were measured by BrdU-488/PI through flow cytometry (Accuri C6 Biosciences) for cell apoptosis analysis. The cells were stained exactly as recommended by the manufacturer of the Annexin kit (Promega, MA). Briefly, cells (5-10x104) were cultured and labelled in the anoxic treatment groups and the normal oxygen groups in their medium. The cells were washed with PBS, and incubated with serum free medium for the desired times. Then, the cells were harvested with trypsin solution and washed twice with PBS. BrdU-488/PI were added into the tube and gently mixed with cells in dark condition [room temperature, 10 min]. Stained cells were washed 3 times with cold PBS and fixed with then permeabilized with 0.5% Triton X-100 in PBS [5 minutes, room temperature]. Finally, cells were analysed by using the flow cytometer and collected data for result analysis.
The cells were seeded in a Chamber Slides (Nalge Nunc International) and normally cultured overnight. For the Hose cells were transfected with GFP-plasmid, cells were observed by microscopy after 24hour. Cells were treated with drug (PAF, C-PAF, ET-18) for 24 hours and then washed twice with PBS. FITC-VAD-fmk (CaspACE™ FITC-VAD-FMK in Situ Marker, Promega) was used to test caspases activation in cells, which is a cell-permeant fluorochrome derivative of caspase inhibitor Val-Ala-DL-Asp-fluoromethylketone. Cells were washed twice by PBS and FITC-VAD-fmk (5 mM) was incubated with cells (20 min, room temperature) in the dark. Immediately after FITC-VAD-fmk staining procedure (see above), cell was co-stained with Hoechst 33342 (1 mg/ml, 10 min) for counterstaining of nuclei in the dark. Then, washing twice in PBS, cells were then fixed with 0.5% paraformaldehyde (20 min, RT) in the dark. PBS washed twice and cells were resuspended in Vectashield H-100 mounting medium (Vector Laboratories, Burlingame, CA). Cells were blocked overnight at 4°C with blocking buffer (0.1% Triton X-100, 2% BSA in PBS). The Annexin V staining to detect the cell apoptosis was followed the related protocol. Images were visualized using Zeiss Axiovert 200 inverted fluorescence microscope (40 x oil objectives) equipped with 14-bit ECCD camera and argon and krypton gas excitation asters at 488 and 568 nm. Z-stack acquisition using optimal slice distancing was performed on each microscope image.
Significance of differences for the associations between cytotoxicity and enzyme activity, pathway activation status and metabolite profile will be determined using ANOVA with Prism software (GraphPad Software, Inc. San Diego, CA). Significance of the test was defined (i.e. p-value ≤ 0.05).