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. 2024 Jan 8;8:7. doi: 10.1038/s41698-023-00495-5

Fig. 4. CAFs signal via Wnt5a.

Fig. 4

A, B ALDEFLUOR assay of OC-CAF coculture with Wnt5a inhibition. OVCAR3 cells were seeded with CAFs and cocultured for a week with 200 μM Wnt5a inhibitor Box5. ALDEFLUOR assay was performed to label CSCs (green). A Fluorescent imaging of OC-CAF coculture labeled by ALDEFLUOR. Scale bar: 100 μm. B Flow cytometry analysis was done to quantify CSCs in control/CAF cocultured groups with/without treatment. Mean ± SD from three independent experiments. *p < 0.01 (t-test). C Spheroid formation assay of OC-CAF coculture with Wnt5a inhibition. OVCAR3 cells were seeded with/without CAFs in ultra-low adhesion plates and cocultured for 14 days with 200 μM Wnt5a inhibitor Box5. Representative mages (left) and quantification of number of spheroids (right) are shown. Scale bar: 400 μm. Mean ± SD from three independent experiments. *p < 0.01 (t-test). D WNT5A was knocked out in CAFs (WNT5A-KO) using CRISPR Cas9, and the WNT5A-KO CAFs were cocultured with OVCAR3 cells followed by ALDEFLUOR assay to determine the effect on CSC. Fluorescent imaging and quantification of CSCs are shown. CSCs were quantified by ImageJ counting. Scale bar: 100 μm. Mean ± SD from three independent experiments. *p < 0.01 (t-test). E qPCR for CSC markers in OVCAR3 cells treated with Box5 for 3 days. Mean ± SD from three independent experiments. *p < 0.01 (t-test). F Immunofluorescent staining of heterotypic spheroids of OVCAR3 + CAF or OVCAR3 monoculture. OVCAR3 + CAF (1:1) were seeded in ultra-low adhesion plate for 7 days. The spheroids were isolated, fixed, and stained for respective markers. ALDH1 was labeled green, Wnt5a was labeled cyan, and vimentin (CAF marker) was labeled red. Scale bar: 200 μm.