Mortalin

In brief, polyacrylamide gel electrophoresis was used to separate proteins which extracted from tissues or cells, and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with primary antibodies including: Mortalin(1:1000; Santa Cruz Biotechnology), β-Actin(1:1000; Proteintech), E-cadherin(1:1000; Proteintech), Vimentin(1:1,000; Santa Cruz Biotechnology), Slug(1:1000; Santa Cruz Biotechnology), Twist(1:1,000; Santa Cruz Biotechnology), VEGF (1:1000; Santa Cruz Biotechnology), MMP2 (1:1000; Santa Cruz Biotechnology), MMP9 (1:1000; Santa Cruz Biotechnology), β-catenin(1:1000; Santa Cruz Biotechnology), C-myc(1:1000; Santa Cruz Biotechnology), Cyclin1-D1(1:1000; Santa Cruz Biotechnology), and with secondary antibody, then quantified by gel imaging system (Bio-RAD, Hercules, CA, USA) and photographed.

For immunostaining, cells were cultured on (24 × 24 mm) slides, washed with PBS, fixed with 2% paraformaldehyde for 30 min, permeabilized with 0.5% TritonX-100 (CWBIO, Beijing, China), sealed with BSA (Solarbio, Beijing, China). Primary antibodies specific for the following proteins were used: Mortalin(1:100; Santa Cruz Biotechnology), β-Actin(1:100; Proteintech), E-cadherin (1:100; Proteintech.), Vimentin (1:100; Santa Cruz Biotechnology), C-myc(1:100; Santa Cruz Biotechnology), Cyclin1-D1(1:100; Santa Cruz Biotechnology). Following incubation with the primary antibodies, the cells were then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:400; cat. no. A11008; Invitrogen; Thermo Fisher Scientific, Inc.) or Alexa Fluor 568 goat anti-mouse IgG (1:400; cat. no. A11004; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Sections were sealed with an anti-fading fluorescent fixator containing 4ʹ,6diamino-2-phenylindole (DAPI, Solarbio). The intensity of fluorescence image was observed and photographed under a microscope.

The tissue microarray was manufactured by Shanghai Outdo Biotechnology Corporation (Shanghai, China). Clinicopathological data of the samples were shown that 42 cases were under 60 years of age and 48 cases were over 60 years of age. All cases were pathologically confirmed to be in accordance with clinical pathologic diagnostic criteria of prostate cancer.
According to the Gleason score, there were 36 cases with Gleason ≤ 6 and 54 cases with Gleason ≥ 7. According to the TNM stage, there were 57 cases in stage I-II and 33 cases in stage III-IV. IHC staining scores were assessed by two pathologists who had no knowledge of the patient's clinicopathological data. The IHC staining scores used were '0' (negative, -), '1-3' (weak, +), '4-6' (moderate, ++) and "8-12" (strong, +++).
Immunohistochemistry staining was performed on all tissues using the DAKO LSAB kit (DAKO A/S, Glostrup, Denmark). The tissue microarrays were baked at 60 °C, dewaxed with xylene, hydrated through a gradient concentration and repaired by sodium citrate buffer, then incubated at 3% H 2 O 2 for 20 min. The tissue sections were incubated with Mortalin, Ki-67, C-myc, Cyclin-D1 antibody (1:100, Santa Cruz Biotechnology) overnight at 4 °C, followed by exposure to secondary antibody. Finally, the sections were visualized by 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin.