Fig 2: Commonalities between ciliating and non-ciliating TNBC cancer cells. (A) Spheroids formed by mouse TNBC MET1 depleted of PRK1 + PRK2 using doxycycline (dox) inducible shRNA (images, top and quantification, below). (B) Western blot analysis (top) and quantification (below) of lysate from cells ± serum. (C) Quantification of total PRK1 levels (± serum) by western blot analysis. Unless stated otherwise n = 3 (±s.e.m.).

Fig 3: Depletion of PKN1 synergizes with WNT3A to elevate Wnt/ß-catenin. A, expression levels of PKN1 in the three Mannheim cohorts. These data were adapted from Hoek et al. (33). The p values (*, p < 0.05; **, p < 0.01) were calculated using a one-way ANOVA with Tukey's post-test. B–D, A375 (B and D) and A2058 (C) malignant melanoma cells stably expressing a ß-catenin (B and C) or NF-?B (D) reporters were transiently transfected with the indicated siRNA oligonucleotides. Forty-eight hours after transfection, the cells were stimulated overnight with WNT3A-conditioned medium (B and C) or medium containing recombinant TNF (1 ng/ml; D) and then assayed for luciferase abundance. Error bars represent the S.D. of technical replicates, and the p value (****, p < 0.0001) was calculated using Student's t test. To assess the knockdown efficiency of the siRNAs, cells were lysed and processed for Western blot (WB) (B and C, bottom panels). CTRL, control.

Fig 4: Human and mouse TNBC cells are more sensitive to PRK2 depletion. (A) PRK2 expression across a panel of breast cancer cell lines (Depmap.org). (B) Sensitivity to PRK2 depletion in a panel of breast cancer cell lines (Depmap.org). (C) Relative proliferation of 3 TNBC cell lines upon siRNA induced PRK2 depletion. Non-targeting (NT) siRNA oligos used as control. (D) Western blot analysis confirming deletion of PRK2 in MET1 cells and the subsequent exogenous expression of PRK2 fused to GFP (PRK2:GFP). (E) Relative proliferation of MET1 (Parental) and MET1 cells null for PRK2 (Null). (F) Relative proliferation of Null cells expressing GFP or PRK2:GFP. Unless stated otherwise, n = 3 (±s.e.m.).

Fig 5: PRK1 and PRK2 co-depletion impairs planer polarity and anchorage independent growth. (A) Example of cleared and non-cleared mIMCD3 spheroid structures. (B) Quantification of cleared spheroids formed by mIMCD3 cells expressing doxycycline (dox) inducible shRNA constructs ± dox (>100 cysts quantified for each condition). (C) Distribution of the different spheroid diameters formed in B. Unless stated otherwise, n = 3 ± s.e.m. shown.