Fig. 4.
ILK functionally correlates with Fzd7 in OC spheroids and tumors. A IF staining for p-ILKSer246 (Alexa Fluor 488, green) and Fzd7 (Alexa Fluor 647, red) in UHB-1912–02 primary OC cells grown as spheroids and treated or not with Wnt-3A. Protein co-localization is identified by yellow spectra on merged images (upper panel). Quantification of Alexa Fluor 488 (green) and Alexa Fluor 647 (red) proteins was calculated by using Metamorph software (N = 3; *P < 0.05). Quantification of co-localized proteins was calculated by volume area of green over red spectra by using Metamorph software (N ≥ 3; *P < 0.05). Scale bar, 100 µm. B Fzd7/p-ILKSer246 colocalization (red dots) detected by PLA in primary human cells. Representative images are shown. Scale bar, 100 µm. Quantification of the number of total Fzd7/p-ILKSer246 red dots per sample in a diagram (N = 3; *P < 0.05, **P < 0.01). C Equal amounts of lysates of HEY-A8, OVCAR-8, and OVCAR-3 cells grown as spheroids were immunoprecipitated with ILK and Fzd7 Abs. WB was performed using ILK, Fzd7, and GAPDH Abs. D Co-IP with anti-Fzd7 Ab and WB for ILK and Fzd7 using full-length recombinant ILK and Fzd7. E Fzd7 and p-ILKSer246 expression levels detected by IHC in human ovarian tumors, metastatic, and normal surface ovarian epithelium specimens included on a TMA (left. Representative images are shown. Scale bar, 200 µm. Quantification of Fzd7 and p-ILKSer246 protein levels by H intensity in OC TMA analysis (right). Error bars show mean (± STD) (N = 3; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). F Correlation between Fzd7 and p-ILKSer246 protein levels (Pearson r = 0.31, P = 0.019). G Correlation between ILK and Fzd7 mRNA expression levels (Spearman r = 0.3, P = 1.7e-10) in the TCGA OC database obtained from GEPIA. H Fzd7 and ILK mRNA expression levels show a tendency toward co-occurrence (Log2 odds ratio = 2.197, P = 0.02, Q = 0.02) in the OC TCGA datasets obtained from cBioPortal. I OS curves generated using HGSOC tumor microarray data of 14 datasets from 7 different array platforms using OvMark for tumors expressing higher than median levels of ILK and of Fzd7 versus those expressing lower than median levels of ILK and of Fzd7 (HR = 1.763 and P = 0.00427)