1. Production of RTAC: Transferring V5-tagged RTAC to HEK293 cells.

2. Stability testing:

a) Method: Western Blotting

b) Purpose: To demonstrate Fc tag can improve the stability, we let RTAC, LGR4 ECD (extracellular domain), ZNRF3 ECD or LGR4-ZNRF3 ECD persist at 37 Celsius in FBS-containing medium and compare their degrading speed.

c) Result:

The data showed that RTAC can stably exist in this condition with stronger stability compared to other subjects.

1. Binding affinity to 4 RSPOs

a) Method: We transiently transfected HA-tagged RSPOs and V5-tagged RTAC into HEK293 cells and collected RSPOs and RTAC from the conditioned medium after 48 hours. Then, we incubated RTAC with 4 RSPOs in vitro and used α-HA antibody-conjugated agarose to pull down the RSPOs, respectively. As control, V5-tagged LGR4 and ZNRF3 were also collected and tested in RSPO1 and RSPO3 (RSPO4 is similar to RSPO1 and RSPO2 is similar to RSPO3 molecularly).

b) Purpose: To investigate the binding capacity of RTAC to RSPO and compare the affinity of different proteins.

c) Result:

RTAC presented pan-RSPO binding capability:

RTAC showed stronger binding affinity comparing to ZNRF3 or LGR4 ECD alone:

2. Capability to inhibit RSPO:

2.1 RSPO-mediated Wnt signaling

2.1.1 Transcriptional level of gene downstream TCF/LEF:

a) Method: TOPFlash (TCF/LEF operated promoter luciferase assay)

b) Purpose: To evaluate RTAC’s capability to neutralize Wnt/β-catenin activating effect by RSPO3.

c) Results: RTAC can effectively inhibit the function of RSPO3 in luciferase assay

2.1.2 Level of cytosolic β-catenin:

a) Method: Western Blotting

b) Purpose: To confirm RTAC’s capability to decrease the accumulation of β-catenin.

c) Results: RTAC can effectively inhibit the function of RSPO3 in Western Blotting assay.

2.1.3 Transcription level of Wnt target gene:

a) Method: RT-qPCR

b) Purpose: To demonstrate RTAC’s capability to nullify the Wnt target gene expressions caused by RSPO3.

c) Results: RTAC can effectively inhibit the function of RSPO3 in RT-qPCR assay

2.2 RSPO/Wnt-dependent cell growth

a) Method: MTT cell growth

b) Purpose: To investigate the broad effectiveness of RTAC in inhibiting multiple Wnt-dependent tumor cell proliferation.

c) Results: RTAC can effectively inhibit RSPO/Wnt dependent cell growth in several cancer cell lines.

a) Method: Western Blotting

b) Purpose: To show our success of mating pathway reprogramming

c) Results: RTAC can be induced and secreted by eATP treatment

1. Level of cytosolic β-catenin:

a) Method: Coculture of R-yeast with RKO cell and test by Western Blotting

b) Purpose: To confirm R-yeast’s capability to decrease the accumulation of β-catenin.

c) Results: R-yeast inhibits the β-catenin accumulation of cocultured-RKO cell upon eATP treatment.

2. Transcription level of Wnt target gene:

a) Method: Coculture of R-yeast with RKO cell and test by RT-qPCR

b) Purpose: To demonstrate R-yeast’s capability to nullify the Wnt target gene expressions caused by RSPO3.

c) Results: R-yeast can inhibit the elevated Wnt-target gene expression by induced RSPO3 in the cocultured RKO cells.

a) Method: MTT cell growth

b) Purpose: To investigate the anti-colon tumor effect of R-yeast.

c) Results: R-yeast can inhibit the accelerated cell growth caused by RSPO3 of the cocultured RKO cells.