Keratin 18

Cells were grown on glass cover lips in the presence or absence of flubendazole for 24 hr. After fixed in 4% (v/v) formaldehyde/PBS, Cells were permeabilized (0.5% TritonX-100/PBS) and blocked for 1 hr in 5% BSA/PBS. IF was performed at room temperature using Vimentin (Merck Millipore, 1:200), Keratin 18 (Cell Signaling Technology, 1:100), α-tubulin (Santa Cruz, 1:400) and γ-tubulin (Sigma Aldrich, 1:800) antibodies. Alexa-conjugated anti-IgG antibodies (Invitrogen Corp, 1:200) were used for secondary detection. DAPI (Sigma Aldrich, 1 μg/ml) was used for nuclear staining. Images were acquired using an Olympus microscope.

The cells were lysed in ice-cold whole-cell extract buffer (50 mM Tris-HCl pH 8.0, 4 M urea, and 1% Triton X-100). The cell extracts were resolved by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% non-fat milk in Tris-buffered saline containing 0.2% Tween-20, the membranes were probed with the following antibodies: keratin 18 (Cat. 4548, Cell Signaling, MA, USA), p-AKT (Cat. 9271, Cell Signaling), AKT (Cat. 9272, Cell Signaling), p-ERK (Cat. 4695, Cell Signaling), ERK (Cat. 4370, Cell Signaling), p-MEK (Cat. 9154, Cell Signaling), MEK (Cat.9122, Cell Signaling), p-mTOR (Cat. 5536, Cell Signaling), β-tubulin (Cat. 5346, Cell Signaling), α-tubulin (Cat.sc-5286, Santa Cruz, CA, USA), keratin 8 (Cat. 53280, Abcam, Cambridge, UK), keratin 19 (Cat. 56625, Abcam) and GAPDH (Cat. 2251, Abcam). Following incubation with horseradish peroxidase-coupled secondary anti-mouse (Cat. 074-1806, KPL, Gaithersburg, MD, USA) or anti-rabbit antibodies (Cat. 474-1506, KPL), the protein bands were visualized using ECL Blotting Detection Reagents (Cat.54-61-00, KPL). Densities of the immunoreactive bands were evaluated using ATTO Densitograph Software Library CS analyzer (ATTO instruments, Tokyo, Japan).

Primary human prostate specimens were collected from 118 PCa patients from Renji Hospital. Tumor differentiation was defined according to the Gleason grading system. Tumor staging was defined according to the 7th edition of the American Joint Committee on Cancer TNM staging manual [20 ].
Tissue slides were stained with primary antibody against AMACR (ab219309), CXCL1 (ab86436), LCN2 (ab125075) from Abcam (Cambridge, UK), Keratin18 (#4548, Cell Signaling Technology, Massachusetts, USA) and CD177 (LS-B1953, LifeSpan BioSciences, Washington, USA). IHC results were evaluated independently by two pathologists who were blinded to the patients’ details. The staining intensity was scored on the following scale: negative (0), weak (1), moderate (2), or strong (3). The staining extent score was on a scale of 0–3, corresponding to the percentage of immunoreactive cells (< 5, 5–25%, 26–50%, 50–100%, respectively). An aggregate score ranging from 0 to 6 was calculated by adding the intensity score and staining extent score together, resulting in a negative (0–1), low (2), moderate (3–4), and high (5–6) expression for each specimen. PerkinElmer Opal™ Tyramide Signal Amplification system was employed for multiplex protein co-staining and images were captured by VectraPolaris™ Quantitative Slide Scanner (PerkinElmer, Massachusetts, USA).