ROR2 induces JNK signaling in receiving AGS cells. (A) Schematic depicting the JNK signaling assay (adapted from ref. 25). Upon activation of the JNK signaling pathway, the mCherry reporter protein translocates from the nucleus to the cytoplasm of the cell. (B) Representative confocal images of AGS-B18 cells cocultured with wild-type pCAF2 transfected with either membrane GFP (Left), ROR2 BFP (Center), or dCD ROR2 BFP (Right). The scale bar represents 10 μm. (C) Quantification of the cytoplasmic to nuclear ratio of the JNK reporter signal in receiving AGS-B18 reporter cells cocultured with pCAF2 cells as described in (B). Reporter cells were cocultured with wild-type (WT) AGS cells as a control. Boxes represent 95% quartile, the centerline indicates the mean, and the whiskers indicate the range. n = number of receiving cells quantified. Results are from three independent experiments. Significance was calculated using an unpaired t test between two conditions. (D) Quantification of the cytoplasmic to the nuclear ratio of JNK reporter signal in AGS-B18 reporter cells cocultured with AGS cells transfected with either membrane GFP (WT AGS), ROR2, ROR2, and WNT5A, ROR2 lacking the cytoplasmic domain (dCD ROR2) or dCD ROR2plus WNT5A as indicated. n = number of receiving cells quantified. Results are from three independent experiments. Significance was calculated using an unpaired t test between two conditions. (E) Quantification of the cytoplasmic to nuclear ratio of JNK reporter signal in AGS-B18 reporter cells cocultured with AGS cells transfected with either membrane GFP (WT AGS), ROR2 and WNT5A, or ROR2, WNT5A, and dnIRSp534K to block cytoneme formation as indicated. AGS-B18 reporter cells were also cultured in media only taken from AGS cells transfected with ROR2 and WNT5A for 24 h as indicated by MEDIA. n = number of receiving cells quantified. Significance was calculated using an unpaired t test between each two conditions.
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