Fig 1: ROR1 is required for the prevention of CAV1 being routed to the lysosome.(a) WB analysis showing a gradual decrease and an easily detectable retention of CAV1 expression 24 h after siROR1 transfection in NCI-H1975 cells. (b) IP–WB analysis showing the impaired association between cavin-1 and CAV1 in siROR1-treated NCI-H1975 cells. Cell lysates harvested 24 h after siROR1 transfection, when the siROR1-induced reduction of CAV1 expression was not yet obviously elicited. (c) Two-colour immunofluorescence analysis showing a significant loss of colocalization between CAV1 and cavin-1 24 h after siROR1 treatment. (d) Two-colour immunofluorescence analysis showing significant colocalization of CAV1 with LAMP-1 24 h after siROR1 treatment. Uncropped images of blots are shown in Supplementary Fig. 11.
Fig 2: ROR1 colocalizes with CAV1 and cavin-1 and retains cavin-1 in DRM.(a) Sucrose density-gradient centrifugation confirmed the presence of ROR1 and cavin-1 in the DRM fractions containing CAV1 and CAV2 in the NCI-H1975 cells. (b) ROR1 and CAV1 colocalization shown by two-colour immunofluorescence staining in NCI-H1975 cells. Colocalization was quantified using ImageJ software. Also see Supplementary Fig. 5. (c) The colocalization of ROR1 with CAV1 and cavin-1 shown by three-colour immunofluorescence staining using super-resolution structured illumination microscopy in NCI-H1975 cells. (d) Sucrose density-gradient centrifugation showing the loss of CAV1 as well as marked changes of cavin-1 subcellular distribution in NCI-H1975 cells knocked down for ROR1. Also see Supplementary Fig. 6a. (e) Two-colour immunofluorescence staining showing markedly impaired colocalization between cavin-1 and CAV2 induced by ROR1 knockdown in NCI-H1975 cells. Colocalization was quantified using ImageJ software. Also see Supplementary Fig. 6b. Uncropped images of blots are shown in Supplementary Fig. 11.
Fig 3: ROR1 and CAV1 knockdown results in decreased phosphorylation of multiple RTKs.(a) Phospho-RTK array results showing the inhibitory effects of siROR1 treatment on the phosphorylation state of multiple RTKs in NCI-H1975 cells (left panel). Averages of the mean pixel densities in two independent experiments are given for each of the representative RTKs (right panel). See Supplementary Fig. 1a for data in PC-9 cells. (b) The impairment of growth factor-induced phosphorylation in multiple RTKs in NCI-H1975 cells knocked down for ROR1. See Supplementary Fig. 1b for data in PC-9 cells. (c) Phospho-RTK array results showing the inhibitory effects of siCAV1 treatment on the phosphorylation state of multiple RTKs in NCI-H1975 cells (left panel), and averages of the mean pixel densities of the representative RTKs in two independent experiments (right panel). The siControl blot of a is re-displayed for ease of comparison. See Supplementary Fig. 1c for data in the PC-9 NSCLC cell line. Uncropped images of blots are shown in Supplementary Fig. 11.
Fig 4: ROR1 kinase activity is not required to sustain CAV1 expression.(a) Sustainment of CAV1 expression in the presence of siROR1 by introduction of both siRNA-resistant, wild-type (wt) and kinase-dead ROR1 in the NCI-H1975 cells. Also see Supplementary Fig. 7. (b) Sucrose density-gradient assay showing that ROR1 kinase activity is dispensable for the retention of cavin-1 in DRM fractions. NCI-H1975 cells stably transfected with either wt or kinase-dead forms of siRNA-resistant ROR1 were subjected to siROR1 treatment in this analysis. Uncropped images of blots are shown in Supplementary Fig. 11.
Fig 5: Schematic diagram of proposed model showing how ROR1 sustains caveolae formation.ROR1 facilitates the interaction of cavin-1 and CAV1 at the plasma membrane in a kinase activity-independent manner, which in turn sustains caveolae formation and prosurvival signalling towards AKT through multiple RTKs via its scaffold function for cavin-1 and CAV1 in human cancer cells. Our results also provide mechanistic insight into how ROR1 inhibition can overcome EGFR–TKI resistance caused by bypass signalling via diverse RTKs.
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