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  • Cancer Letters br RGD motif mutated to RGE was


     Cancer Letters 442 (2019) 320–332
    (RGD motif mutated to RGE) was purchased from Sangon (Shanghai, China). A p38 MAPK inhibitor, SB203580, was purchased from Selleck (Shanghai, China).
    2.3. RNA preparation and quantitative reverse-transcription PCR (qRT-PCR)
    Total-RNA samples were extracted using the TRIzol reagent (TAKARA, Japan) then subjected to reverse transcription with the PrimeScript RT Reagent Kit (TAKARA, Japan). qRT-PCR was performed by means of the SYBR Premix Ex Taq II kit (TAKARA, JPN). Standard curves were generated, and the relative amounts of target gene mRNA were normalised to β-actin. The utilised primer sequences are listed in Supplementary Table 1. All the experiments were performed at least three times.
    2.4. Immunohistochemistry (IHC) and immunofluorescent staining (IF)
    Tumourous and normal tissues were fixed with 10% formalin. Paraffin-embedded specimens were sectioned at 4 μm thickness. The immunohistochemical protocols were described previously [35]. Tissue sections were incubated with a rabbit polyclonal antibody against in-tegrin β3 (1:200; Abcam, UK). The normal rabbit IgG served as a ne-gative control to eliminate non-specific staining. Integrin β3-positive cells were counted in at least 20 fields of view, each section at 200 × magnification. Image-Pro plus 6.0 software (Media Cybernetics, USA) was employed to quantify the IHC staining. Mean optical density (MOD: IOD/area) was used to evaluate integrin β3 expression levels. p-p38 (1:100; Bioworld, USA), Ki67 (1:200; Abcam, UK) were conducted in the same manner.
    For IF staining, cells were grown on pre-prepared coverslips in a 24-well plate and were co-cultured with CM from CAFs for 3 h at 37 °C, then fixed with 4% paraformaldehyde. The subsequent staining was described in detail as done previously [35]. The specific FLAG Peptide against IL32 (Rockland, USA) and integrin β3 (Santa Cruz, USA) were employed in the IF staining. The normal rabbit or mouse IgG served as a negative control. A fluorescein isothyonate-labelled goat anti-rabbit and Cy3-labelled goat anti-mouse IgG antibodies served as a secondary antibody (Sigma, USA).
    2.5. Western blotting
    Western blotting analysis was performed as described previously [36]. Briefly, total cell proteins were extracted in RIPA lysis buffer (Beyotime, China), quantified with BCA protein assay kit (Beyotime, China), resolved in a 6–12% SDS-PAGE gel, and then were incubated with appropriate primary antibodies. β-Actin (ZSGBBIO, China) was used as a loading control. The specific primary antibodies employed in this study were as follows: anti–integrin β3 (Abcam, UK), anti-IL32 (Rockland, USA), anti-p38, FLAG Peptide anti-AKT, anti-STAT1, and the corre-sponding phosphorylated proteins (Bioworld, USA), anti-fibronectin (Bioworld, USA), anti–N-cadherin (Bioworld, USA), and anti-vimentin (Bioworld, USA). Horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG antibody (ZSGBBIO, China) served as a secondary an-tibody, and the protein expression levels were visualised by the en-hanced chemiluminescence system (Bio-Rad, Hercules, EDA USA). Images were captured using Scion image software.
    2.6. Western blotting of immunoprecipitates (IP-WB)
    IP-western blotting was performed with the lysates from HEK293T and BT549 cells. The cells were incubated with rIL32 (20 ng/ml) or CM derived from CAFs for 3 h. The extracts were cleared by centrifugation and then pre-cleared by gentle rocking at 4 °C with washed protein G-Dynabeads (Abcam, UK). The pre-cleared extracts subjected to im-munoprecipitation with 2 μg of an anti-IL32 or anti–integrin β3
    antibody and 20 μl Protein G Dynabeads overnight. Equivalent amounts of immunoglobulin G (IgG) served as the control. The Protein G Dynabead–IgG conjugates were washed five times with lysis buffer and boiled in SDS sample buffer, and the released proteins were resolved by SDS-PAGE. The immunoprecipitates were analysed by western blotting with a primary antibody against IL32 or integrin β3.
    2.7. Enzyme-linked immunosorbent assay (ELISA)
    IL-32 protein levels in CAFs and NFs supernatants were measured by ELISA (R&D, USA). The absorbance (450 nm) of each sample was de-tected on a standard automatic microplate reader (BioTek, USA).
    CAFs or NFs at approximately 80% confluence were washed with PBS, and then cultured in a fresh serum-free DMEM medium for 48 h. The conditioned medium (CM) was collected and filtered for further analysis. For neutralisation experiments, a neutralizing antibody against human IL32 (Rockland, USA, 500 ng/ml) was pre-incubated at 37 °C with the supernatant for 1 h before invasion assays.