Recombinant human rspo1

To measure WNT reporter activity in HAP1-7TGP cells or derivatives thereof, ~24 hr before treatment cells were seeded in 24-well plates at a density of 8 × 10⁴ per well and grown in 0.5 ml of CGM 2. Cells were treated for 16–24 hr with the indicated concentrations of WNT3A CM, L cell CM, recombinant mouse WNT3A (R and D Systems Cat. # 1324-WN) recombinant human RSPO1 (R and D Systems Cat. # 4645-RS), LiCl, CHIR-99021 (CT99021) (Selleckchem Cat. # S2924) or XAV-939 (Selleckchem Cat. # S1180) diluted in CGM 2. Cells were washed with 0.5 ml PBS, harvested in 150 μl of 0.05% Trypsin-EDTA (0.05%) (Thermo Fisher Scientific Cat. # 25300054), resuspended in 450 μl of CGM 2, and EGFP fluorescence was measured immediately by FACS on a BD LSRFortessa cell analyzer (BD Biosciences) using a 488 laser and 505 LP, 530/30 BP filters, or on a BD Accuri RUO Special Order System (BD Biosciences). Typically, fluorescence data for 5000–20,000 singlet-gated cells was collected and, unless indicated otherwise, the median EGFP fluorescence ± standard error of the median (SEM = 1.253 σ/√n, where σ = standard deviation and n = sample size) was used to represent the data.

HEK293 or A549 cells seeded in a 24-well culture plate were transfected with 200 ng/well of STF luciferase reporter and 20 ng/well of pRL-SV40 mixed with 1.5 μl/well TransIT-2020 (Mirus Bio), with or without plasmids encoding the Wnt1 or Wnt3a gene. Varying amounts of ICAM-1 or EphA2 plasmid DNA were transfected into HEK293 to create a graded expression of guide antigens. Six (HEK293) or 24 h (A549) post transfection, cells were treated with testing agents (purified phages or antibodies) diluted in growth media and further incubated for an additional 16 h. Firefly luciferase (FL) and Renila luciferase (RL) activities were separately measured using Dual-Luciferase Reporter Assay System (Promega) and each FL value was normalized with the corresponding RL value. Normalized data were expressed as a percentage relative to the control experiment without antibody treatment. To evaluate EphA2-targeted bispecific antibodies, HEK293-EphA2 cell model that stably expresses high levels of EphA2 was generated by lentiviral transduction and STF reporter assays were performed as described above. For RSPO1-mediated reporter activity stimulation, 100 ng/ml of recombinant human RSPO1 (R&D Systems) was to the culture medium in addition to ligands, and STF report assays were performed as described above.