Fig. 5

VHL-KO cells induce the EMT and motility of VHL-WT cells by POSTN. a EMT markers (E-cadherin, N-cadherin, MMP-9 and alpha-SMA) assessed by qRT-PCR in RC-VHL-WT cells alone or co-cultured with VHL-KO cells, separated by a transwell plate (VHL-WT + VHL-KO). b–g The motility of fluorescently marked RC cells was measured in a 2D scratch assay by time lapse live-cell microscopy, monitored over 20 h. b The motility of mStrawberry marked RC-VHL-WT cells and EGFP marked RC-VHL-KO cells were measured. c The motility of a 1:1 mixed culture of RC-VHL-WT and RC-VHL-KO cells (top panels). TRITC channel revealed the motility of RC-VHL-WT cells in the mixed culture (lower panel). Respective videos are in Supplementary Movie S1. d The quantified migration speed of the three cell groups is shown. e The migration speed of VHL-WT cells with (right) or without (left) the addition of conditioned medium of VHL-KO cells, cultured for 2 days at 90% confluence. Respective videos are shown in Supplementary Movie S3. f The migration speed of VHL-WT cells (mStrawberry⁺) co-cultured with VHL/POSTN-KO cells (EGFP⁺) or VHL-KO cells (EGFP⁺) was shown (also see Supplementary Movie S5). g Anti-POSTN mAb MPC5B4 (1 µg/mL) was added to VHL-WT cells co-cultured with VHL-KO cells. The migration speed of VHL-WT cells without and with MPC5B4 was shown (see Supplementary Movie S6). h The migration speed of VHL-WT cells alone or with the addition of recombinant POSTN protein or with the addition of POSTN and the integrin inhibitor cilengitide was assessed. i Scratch assay of a 1:1 mixture of VHL-WT and VHL-KO cells was assessed at 18 h. The migration speed of VHL-WT cells in the mixture was measured with 0, 2, 5 or 10 μM cilengitide added (see Supplementary Movie S7). Scale bar: 100 μm. Student t-test was used in (a), (e), (f) and (g) and one-way ANOVA was used in (d), (h) and (i) with triplicate repeats and presented with mean ± SD. (*p < 0.05, **p < 0.01)