Skl2001

Foetal bovine serum (FBS) and Dulbecco's Modified Eagle's medium (DMEM) were obtained from Gibco‐BRL (Thermo Fisher Scientific, Inc). β‐Glycerophosphate (β‐GP; G5422) and adenine (V900471) were purchased from Sigma‐Aldrich (Merck KGaA). Ginsenoside Rb1 (41753‐43‐9) was obtained from Fleton Natural Products Co., Ltd. The Wnt/β‐catenin agonist SKL2001 (S8320) and PPAR‐γ inhibitor GW9662 (S2915) were from Selleck Chemicals. The Nuclear and Cytoplasmic Protein Extraction Kit (P0027) was from Beyotime Biotechnology.
Antibodies against calponin 1 (#17819), runt‐related transcription factor 2 (RUNX2; #12556), β‐catenin (#8480), phospho‐β‐catenin (Ser675) (#9567), glycogen synthase kinase‐3β (GSK‐3β; #9315), phospho‐GSK‐3β (Ser9) (#9322) and histone‐H3 (#4499) were from Cell Signaling Technology, Inc. An antibody against α‐smooth muscle actin (α‐SMA; NBP2‐22120) was purchased from Novus Biologicals. An antibody against peroxisome proliferator‐activated receptor gamma (PPAR‐γ; sc‐7273) was purchased from Santa Cruz Biotechnology. An antibody against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; TA‐08) was purchased from ZSGB‐BIO.

Human OC cell lines (OVCAR3, A2780 and SKOV3) and normal ovarian epithelial cell lines (IOSE80) were obtained from the Chinese Academy of Cell Collection (Shanghai, China), and cultured in a humidified conditions at 37 °C with 5% CO₂. IOSE80, SKOV3 and OVCAR3 cells were cultivated in RPMI-1640 (Gibco, USA) with fetal bovine serum (FBS) (ExCell Bio, New Zealand) and 1% penicillin/streptomycin (PS) (NCM Biotech, China). Specifically, 10%FBS was needed to IOSE80 and SKOV3, and 20% to OVCAR3. Dulbecco’s modified Eagle medium (Gibco, USA) with 10% FBS and 1% PS were used to culture A2780 cells.
When the cell fusion degree reached 60–70%, small interfering RNAs (siRNAs) (GenePharma, Suzhou, China) targeting NRSN2-AS1, PTK2, and MG53 as well as overexpression plasmids (GenePharma) pcDNA3.1-NRSN2-AS1, pEX-1-MG53 and an empty vector (EV) were transfected into OC cells by Lipofectamine 2000 (Invitrogen, USA) and X-treme GENE HP DNA transfection reagent (Mannheim, Germany). The nucleotide sequences of all siRNAs are shown on Table S2.
XAV-939 and SKL2001 (Selleck, Shanghai, China) were solubilized in dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) at 10 mM, and used in experiments at 15 μM. Following treatment, cells were harvested for further analysis 48 h later.

SW620, 293T, HCoEpiC, and RKO cells were cultured in DMEM containing 10% FBS (MilliporeSigma) and 1% penicillin/streptomycin. F-12K Nutrient Mixture (Invitrogen) was used to cultivate LoVo cells with 10% FBS and 1% penicillin/streptomycin. All cells were purchased from American Type Culture Collection and were detected by the mycoplasma detecting kit (Yise Medical Technology) to confirm a nonmycoplasma condition. All cells were cultured in the condition of 37°C and 5% CO₂. The small interfering RNAs used in this study were siDicer-1, siDicer-2, and siDicer-3, which were purchased from RiboBio. The lentivirus-based shSURC-575, shSURC-1083, and shAPC (GenePharma) were used to infect SW620 and LoVo cell lines. The overexpression plasmid of SURC and APC were designed and purchased from GeneCopoeia. The miR–185-5p inhibitor and miR–185-5p mimic were purchased from RiboBio. Actinomycin D was purchased from MedChemExpress, while PNU-74654 and SKL2001 were purchased from Selleck.

MCF-7 was maintained in DMEM High Glucose with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. T-47D was cultured in RPMI 1640 medium with 10% FBS and 1% penicillin and streptomycin. MDA-MB-231 and MDA-MB-468 were grown in L15 medium with 10% FBS and 1% penicillin and streptomycin. SUM159 was cultured in DMEM/F12 medium with 10% FBS and 1% penicillin and streptomycin. BT549 was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1 μg/ml insulin and 1% penicillin and streptomycin. Medium, FBS and Penicillin-Streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All the cells were maintained at 37 °C with 5% CO₂. Old cell culture medium was replaced with fresh medium every other day. All the antibodies used in this study can be found in Supplementary Table 1. XAV939 and SKL2001 were purchased from Selleck.

1 × 10⁵ FB cells were seeded in a six-well microplate containing 2 ml 10% FBS/DMEM medium per well. After 24 h, the medium was replaced by 2% FBS/DMED containing 10 UM of ICG-001 (S2662, Selleck), 10 UM of SKL2001 (S8334, Selleck), or an equal volume of DMSO. The medium was changed every other day during culturing. After 7 days, all cells were harvested with TRIzol (15596026; Thermo Fisher) for mRNA sequencing. Library construction and sequencing were performed by Sinotech Genomics Co., Ltd (Shanghai, China). Total RNA was isolated using RNeasy mini kit (74904; Qiagen). The poly(A)-containing mRNA molecules were purified using poly(T)-oligo-attached magnetic beads. Then the mRNA underwent fragmentation, reverse transcription, purification, and enrichment. Clusters were generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA). Paired-end sequence files (fastq) were mapped to the reference genome (GRh38-1.2.0) using HISAT2. Differential expression analysis for mRNA was performed using the R package edgeR. Differentially expressed RNAs with |log₂(FC)| value >1 and q value <0.05, considered significantly modulated, were retained for volcano plot and GO analysis.

BODIPY 493/503 (#D3922) and DAPI (#D1306) were purchased from Invitrogen (CA, USA). SKL2001 was purchased from Selleckchem (Shanghai, China). Dexamethasone (#D4902), 3-isobutyl-1-methylxanthine (#I5879), insulin (#I2643), Oil red O (#O9755), MS-222 (#E10521), and Triton X-100 (#T8787) were purchased from Sigma-Aldrich (MO, USA). Antibodies against P42/44 (#9102), β-catenin (#9562), Phospho-β-catenin (#9561), PPAR-γ (#2443), Ubiquitin (#3936), GAPDH (#5174), HA-tag (#3724) as well as secondary HRP-conjugated antibodies against mouse (#7076) and rabbit (#7074) were purchased from CST (MA, USA) and used at 1:1,000 dilution for western blotting. Anti-FLAG-tag antibody (#F1804) and anti-FLAG-tag M2 affinity gel (#A2220) were from Sigma-Aldrich (MO, USA). Antibodies against Plin1 (#A16294), OSBPL2 (#A14199), α-Tubulin (#AC012), β-catenin (#A11512) as well as secondary HRP-conjugated Mouse Anti-Rabbit IgG Light Chain (#AS061) were from ABclonal (Wuhan, China). Antibodies against β-catenin (#BF0319) for zebrafish were from Affinity Biosciences (Changzhou, China). Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG were purchased from Life Technologies (CA, USA).

The transfected cells were seeded in a 96-well microplate at a density of 3000 cells/well and then cultured overnight. After 1, 2, 3, or 4 days of incubation, 10 μL Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) reagent was added to every well and cells were further cultured for another 2 hours at 37°C. In the SKL2001 (Selleck, Shanghai, china) activated group, this agonist was added into the medium to activate target cells. The optical density of each well at 450 nm was detected using a microplate reader (Thermo Fisher Scientific, MA, USA).