Five-day-old C57BL/6 mice were purchased from SLAC Laboratory Animal Co. (Shanghai, China). All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Shanghai and conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.
C28 was synthesized by Professor Huchen Zhou from the Schools of Pharmacy and Medicine, Shanghai Jiao Tong University. The chemical structure of C28 is shown in Figure 1A.
FGSC culture in vitro
The mouse FGSC line used in this study was characterized and established previously. FGSCs were cultured on mitotically inactivated SIM mouse embryo-derived, thioguanine and ouabain-resistant (STO) cells. The culture medium for FGSCs was alpha-minimum essential medium (Invitrogen Life Sciences) supplemented with 10% fetal bovine serum (Life Technologies), 30 mg/ml pyruvate (Amresco), 2mM L-glutamine (Amresco), 0.1mM β-mercaptoethanol (Biotech), 1mM non-essential amino acids (Invitrogen Life Sciences), 20ng/ml mouse epidermal growth factor (PeproTech), 10ng/ml human basic fibroblast growth factor (PeproTech), 10ng/ml mouse glial cell line-derived neurotrophic factor (PeproTech), 10ng/ml mouse leukemia inhibitory factor (Santa Cruz Biotechnology), and 15mg/ml penicillin (Amresco). The medium was changed every 2–3 days. Cells were passaged at ratios of 1:2 or 1:3 by enzymatic digestion every 4–5 days. The cells cultured at 37°C with 5% CO2.
Cell viability was assessed by cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). FGSCs were seeded at approximately 2000 cells/well in 96-well plates and grown at 37°C with 5% CO2 for 24h. The cells were treated with 0, 0.25, 0.5, 1, 2 and 4μM C28 for 24 and 48h, and then reacted with 10μL CCK8 solution for 1h. Then, the absorbance was measured at 450nm using a microplate reader (Bio-Tek Instruments, Thermo Fisher Scientific, Winooski, VT, USA). All assays were conducted in triplicate.
A Cell-Light EdU Apollo® 567 in vitro imaging kit (RiboBio, Guangzhou, China) was used to assay cell proliferation, according to the manufacturer’s protocol. The cells were incubated with 50μM EdU for 2h at 37°C after C28 treatment for 24h. Then, the cells were fixed in 4% paraformaldehyde for 20min at room temperature and washed with 2mg/ml glycine for 5min on a shaker. Permeabilization was conducted by incubation in 0.5% Triton X-100 for 1h. Then, 1×Apollo was added, followed by incubation for 30min on a shaker. Subsequently, cells were washed three times with PBS. Then, 1×Hoechst 33342 was used to stain nuclei. Images were obtained under a DM2500 microscope (Leica, Germany) and analyzed by Image J.
Apoptotic cells were quantified using an annexin V-FITC/PI detection kit (Invitrogen, Carlsbad, CA) and flow cytometry. Briefly, FGSCs were treated with 0.5μM C28 for 3 and 24h, washed with PBS, collected, and resuspended in 1×Binding Buffer. The cells were then stained for 15min at room temperature with annexin V and propidium iodide, and analyzed by flow cytometry (BD FACSCalibur, BD Biosciences) to quantify apoptosis.
Reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction
FGSCs were cultured in vitro and treated with 0.5µM C28 for 24 and 48h. Total RNA was extracted from ovarian tissues of neonatal mice and FGSCs treated with C28 using TRIzol reagent (Life Technologies, CA), according to the manufacturer’s instructions. RNA quantity and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). HiScript II Q RT SuperMix for qPCR kit (Vazyme Biotech) was used to obtain cDNA. Approximately 1µg RNA was used to synthesize cDNA in a 20µl reaction volume. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed with Taq DNA polymerase. The RT-PCR products were separated on 1% agarose gels and imaged with a bioimaging system (Alpha Innotech). RT-PCR products were confirmed by sequencing.
Quantitative real-time polymerase chain reaction (qPCR) was conducted with SYBR Premix Ex Taq (Takara, Shanghai, China) and an ABI 7500 Real-Time PCR System (Applied Biosystems) to measure expression levels of Trib3, DDIT3, and ATF4. The qPCR reaction volume of 20μl included 10μl SYBR Green PCR Master Mix (Roche, Basel, Switzerland), 1μl cDNA template, 2μl primer mixture, and 7μl water. qPCR conditions were as follows: 95°C for 30s 40 cycles of 95°C for 15s and 60°C for 60s, and then then 95°C for 15s, 60°C for 60s, and 95°C for 15s. All assays were repeated three times. Fold changes in expression were calculated using the 2−ΔΔCt method. ΔΔCt = ΔCt experimental group – ΔCt control group, and ΔCt = Ct target gene – Ct Gapdh. Primers are listed in Additional file 1: Table S1.
FGSCs were cultured in vitro and treated with 0.5µM C28 for 3 and 6h. Cells were lysed with RIPA buffer (Shanghai Yeasen Biotechnology Co., Ltd) containing a protease inhibitor cocktail. Protein concentrations were measured using the bicinchoninic acid (BCA) assay. The western blotting procedure was as follows. A total of 20µg protein was separated by 15% w/v sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% dry nonfat milk in Tris-buffered saline with Tween 20 (TBST) at a room temperature for 1h, followed by overnight incubation with anti-LC3 (1:1000; Abcam), anti-SQSTM1 (1:1000; Abcam), and anti-β-tubulin (1:6000; Abcam) primary antibodies in 5% nonfat milk in TBST for 1h. Membranes were washed three times with TBST for 10min each wash. Subsequently, the membranes were incubated with the secondary antibody (1:2000; Proteintech) in 5% dry nonfat milk in TBST at room temperature for 2h, and then washed three times with TBST for 10min each wash. Finally, the protein bands were scanned with a chemiluminescence imaging system (ProteinSimple, Santa Clara, CA, USA). Densitometry of the bands was performed by Quantity One software.
FGSCs were treated with 0.5µM C28 for 3h, washed with 1× phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 30min at room temperature, and then washed three times with PBS. Subsequently, the cells were blocked in goat serum at 37°C for 10min, incubated overnight with the primary rabbit anti-LC3B antibody (1:150; Abcam) at 4°C, and then washed three times with PBS. Then, the cells were incubated with the tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1:150, goat anti-rabbit IgG; ProteinTech). Finally, the cells were washed with PBS three times and stained with 4′, 6-diamidino-2-phenylindole at room temperature for 5min. Images were acquired using a Leica digital camera under a fluorescence microscope (DMI3000B; Leica). Various fields of view (>three regions) were analyzed for each labeling condition, and representative results are shown.
Total RNA was extracted as described above. RNA integrity was assessed using the Agilent Bioanalyzer 2100. RNA quantity and concentration were assessed using the NanoDrop 2000 spectrophotometer. The total RNAwas treated with DNase to remove genomic DNA contamination. Isolation of mRNA was performed using the Oligotex mRNA Mini Kit (Qiagen GmbH, Hilden, Germany). The mRNA was used for RNA sequencing (RNA-Seq) library preparation with the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The library was then subjected to Illumina sequencing with paired-end 2 × 150 as the sequencing mode. Raw sequence data were submitted to the NCBI Gene Expression Omnibus under accession number GSE136386.
Chromatin immunoprecipitation sequencing
The preparation of chromatin immunoprecipitation(ChIP) and input DNA libraries were performed as described previously. First, cells were crosslinked with 1% formaldehyde for 10min at room temperature and quenched with 125mM glycine for 5min. Then, the cells were resuspended in cold SDS lysis buffer (140mM NaCl, 1mM EDTA, pH 8.0, 1% TritonX-100, and 0.1% SDS protease inhibitors) and sheared with a Bioruptor water bath sonicator (Diagenode). The chromatin fragments were precleared and then immunoprecipitated with Protein A+G Magnetic beads coupled with anti-H3K27ac (ab4729, Abcam). After reverse crosslinking, immunoprecipitated DNA and input DNA were end-repaired and ligated with adapters using the NEBNext Ultra End-Repair/dA-Tailing Module (E7442, NEB) and NEBNext Ultra Ligation Module (E7445, NEB). Highthroughput sequencing of the ChIP fragments was performed using Illumina NextSeq 500, following the manufacturer’s protocols. We used bowtie2 to align the reads to the mm9 reference genome. PCR duplicates were removed using Samtools (version 2.0.1). Normalized genome coverage tracks were generated from uniquely mapped reads using deepTools2 (version 3.1).
Gene Ontology analysis
Gene Ontology (GO) enrichment was performed using GREAT analysis (version 1.8). After clustering the active enhancer, the biological processes were determined using the whole genome as the background and positives as the nearest gene within 10kb for Figure 4 and a false discovery rate (FDR) (significant by both) cutoff of 0.05.
Each experiment was performed three times. Data are expressed as the mean ± SEM. Statistical analysis was performed by the Student’s t-test using SPSS statistics (version 17.0; IBM Corp., Armonk, NY, USA). P< 0.05 was considered as statistically significant.