Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis
Fig 7
Detection of CTR-ZO-1 interaction in PC cell lines and tumor specimens.
A: Representative micrographs of CTR-ZO-1 interactions in PC-3CTRwt cells in the absence/presence of CT stimulation for 30 seconds. Each red dot represents an interaction between activated CTR and ZO-1, detected by in situ PLA™ using the Duolink® kit. Nuclei are stained with DAPI (blue). The graph presents quantitative data of CTR-ZO-1 interactions in three separate, identical experiments (mean ±SEM for n = 6, p<0.001, paired t-test). B: Representative micrographs of CTR-ZO-1 interactions in PC-3CTR∆ESS cells as described in A. The graph presents quantitative data of three separate, identical experiments described in Fig 7B (mean ±SEM for n = 6). C: Representative micrographs of CTR-ZO-1 interactions in LNCaP cells as described in A. The graph presents quantitative data of three separate, identical experiments described in Fig 7C (mean ± s.e.m. for n = 6, *p<0.001, paired t-test). D: Representative micrographs of CTR-ZO-1 interactions in PC specimens of metastatic (T3bN0M1; Gleason Score 10; Stage III, Prostate Adenocarcinoma, Malignant) and non-metastatic tumors (T2aN0M0; Gleason Score 6; Stage II, Prostate Adenocarcinoma). The graph presents quantitative data (mean ±SEM for n = 3 of each condition; *p<0.001, paired t-test). E. Snap-frozen human prostate tumor specimens were homogenized (10 mg by weight), and same amount of clarified supernatant was used for immunoprecipitation of CTR from all tumors. CTR IPs were fractionated on SDS-PAGE and ZO-1 was detected by immunoblotting (Rabbit Anti-ZO-1 serum, Invitrogen, Life Technologies, Grand Island, NY). The blots were normalized by the immunoprecipitation antibody (Rabbit Anti-CTR serum, Abcam, Cambridge, MA). The bands on the left side of molecular weight markers were from frozen prostate tissues (1- BPH; 2–4 Prostate Adenocarcinoma localized, Gleason Score 6–7). The bands on the right side of molecular weight markers were also from frozen prostate tissues- 1–3 were localized tumors (T2cN0M0; Gleason Scores 6–7) and 4–6 were metastatic (T3aN0Mx or greater; Gleason Scores-7-9). The tissues on the right side were limited in availability, and we could not do IP controls. The amount of tissues was limited for just one experiment. Therefore, we obtained multiple tissues to perform this experiment.