Rwnt5a

First, 50 × 10⁴ primary neurons were seeded per well into 6-well plates, and treated at DIV10 for 1 h at 37 °C with control or Wnt5a-containing conditioned medium or with recombinant Wnt5a (rWnt5a, R&D systems) (300 ng/mL) or were pretreated with 1 µM HRI-i or 100 µg/mL CHX (Sigma Aldrich) for 30 min -and then incubated with conditioned medium or rWnt5a. Cells were lysed with RIPA buffer supplemented with phosphatases and proteases inhibitors. Then, 60 µg of protein from hippocampal neurons or synaptosomes was loaded into 8–10% acrylamide:bisacrylamide gels. Primary antibodies used were: GluN2B from NeuroMab (1:5), Abcam (1:700) and Alomone (1:350), actin (Abcam 1:10,000), PS51-eIF2α (Abcam 1:500) and eIF2α (Abcam 1:500). Secondary antibodies (Abcam) were used at a dilution of 1:5000.

MG-63 cells were plated onto 96-well cell culture clusters (Costar) and grown to confluence, and then serum-starved for 24 h. The monolayer cells were scratched manually with a plastic pipette tip, and after two washes with PBS, the wounded cellular monolayer was allowed to heal for 10 h in DMEM containing 100 ng/ml recombinant Wnt5a (rWnt5a) (R&D Systems). Photographs of central wound edges per condition were taken at time 0 and at the indicated time points using digital camera (Nikon, Tokyo, Japan).

Three siRNAs for WNT5A (Ruibo Biotechnology Company, Guangzhou, China) were transfected into cells (30-50% confluency) using Lipofectamine^TM RNAi MAX reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For recombinant WNT5A (rWNT5A; R&D systems, Minneapolis, MN) treatment, the rWNT5A was reconstituted in sterile PBS containing 0.1% BSA to a stock concentration of 10 μg/ml. Based on the literature [15 (link)], we chose a concentration of 0.2 μg/ml and 12 h as the dose and time points.
For protein kinase C (PKC) inhibition studies, GF-10923X (an inhibitor of PKCα, β, δ, and ε) (Calbiochem, San Diego, CA) was used at a concentration of 1 μM in an attempt to inhibit the conventional PKC pathway. Cells were either pretreated with the inhibitors for 1 h for western blot analysis or for 12 h for wound healing assays and then were treated with rWNT5A in the continuing presence of inhibitor or were treated with inhibitor alone for the indicated times. For the PKC activation studies, phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) was used at 200 nM. For vehicle controls, the cells were treated with equivalent amounts of DMSO.

FOSL2- or Wnt5a-knockdown cells were produced by lentivirus-mediated transduction using synthetic short hairpin RNA (shRNA) oligonucleotides (GenePharama, Shanghai, China) according to the manufacturer's protocols. The small interfering RNAs (siRNA) were used to transiently knock down CYP19A1 or FZD5 (GenePharama, Shanghai, China). The sequences of shRNA and small interfering RNA used are listed in Table S1. FOSL2 overexpressing lentivirus were purchased from GeneCopoeia (Guangzhou, China), and viral supernatant was used to infect NFs in the presence of 8 µg/ml polybrene according to the manufacturer's instructions.
The pcDNA3.1(+)-FOSL2 plasmid was created by amplifying FOSL2 from CAFs and was cloned into pcDNA3.1(+) vector. The synthesized nucleotide wild type (WT), truncated or mutant (Mut) Wnt5a promoter was inserted into pGL3 luciferase reporter vector (GenePharma, China), respectively. The reagents used in this study are as follows: the neutralizing antibody against VEGF (Bevacizumab; Roche/Genentech, Switzerland), 0.2 mg/mL; axitinib (Selleck, USA), 5 nM; CCK8 (Beyotime, China), 5 mg/mL; SQ22536 (Selleck, China), 100 µM; H89 (Selleck, China), 5 µM; rWnt5a (R&D Systems, USA), 5 µg/mL; BOX5 (Merck Millipore, USA), 5 µM. The usage of rWnt5a and BOX5 in xenografts was described in animal experiments in detail.

E. chaffeensis recombinant full-length TRP120 (rTRP120-FL), TRP120 TRD (rTRP120-TR), or thioredoxin (rTrx; ctrl) were expressed in Escherichia coli and purified as described previously (9 (link)). rWnt5a (R&D Systems, Minneapolis, MN) and peptides (GenScript, Piscataway, NJ) were obtained from a commercial source. Synthesized peptides include TRP120-TR-Wnt5a (IKDLQDVASHESGVSDQPA; represents the entire homologous Wnt5a sequence), TRP120-TR (−) (SHQGETEKESGITESHQKEDEI; neg ctrl), TRP120-Wnt-SLiM (QDVASH), TRP120-Wnt-SLiM-mut (IKDLGAGAGAESGVS; Gly/Ala substitutions in the Wnt SLiM motif), and TRP120-Wnt-QDVAS (QDVAS).