Skim milk

Immunoblotting was performed as described previously (Li et al. 2015). After MCF-7 and Hela cells were treated with different concentrations of ICBCP (0, 40, 80 and 160 µg/mL) for 48 h. They were then resuspended in cold PBS after removal of the nutrient solution, and then centrifuged at 1500 rpm for 5 min and lysed in RIPA lysis buffer with freshly added 100 mM PMSF by ice bath for about 30 min; afterwards, the protein supernatant was centrifuged at 12,000 rpm for 10 min at 4^°C. The protein supernatant was quantified using a BSA standard curve. The proteins were denatured with loading buffer at 100^°C. A known value of 40 μg protein was separated by using 10–12% SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, USA). The membranes were blocked with 5% skim milk (BD Biosciences, Franklin), which dissolved in pH-adjusted TBST at room temperature for 2 h. The membranes were incubated with diluted primary antibodies in skim milk at 4^°C with gentle shaking, overnight, included Caspase-3 (1:1000), Caspase-8 (1:1000), Caspase-9 (1:1000), PARP (1:1000), Bcl-2 and Bax, β-actin was considered as the internal control. The membranes were incubated with HRP-linked anti-rabbit and anti-mouse IgG secondary antibodies diluted by TBST at room temperature for 2 h. The protein bands were visualised with Ultra ECL by supersensitive chemiluminescence imaging (Tanon Co., China).

The brain samples were heated at 95 °C in 1% SDS sample buffer with 100 mM DTT for 5 min, separated by SDS-PAGE on 4–20% precast polyacrylamide gels (Biorad, Feldkirchen, Germany), and transferred to PVDF membranes (Biorad, Feldkirchen, Germany). The membranes were blocked in 5% skim milk (Becton Dickinson, Heidelberg, Germany) in PBST (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and incubated with anti-Sez6 antibody [24 (link)] or anti-Actin antibody (Merck Millipore, Darmstadt, Germany) at 4 °C overnight. The membranes were washed with PBST three times and incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) at room temperature for 1 h. After final washes with PBST, the membranes were incubated with ECL prime for a few minutes, and signal was detected with LAS-3000 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Densitometry quantification of Western blot signal was conducted using Multi Gauge V3.1 software (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Pyocyanin production was assessed qualitatively by visual inspection of cultures grown on Cetrimide agar (Becton, Dickinson, MD, USA). Production was classified as low (+), or high (++), and isolates that exhibited growth but lacked pyocyanin production were scored as (+/−).
Extracellular proteases and siderophores were assessed in a similar manner by examining clearing zones produced on either skim milk (Becton Dickinson, MD, USA), or chrome azurol S agar38 , respectively. Isolates lacking either trait were classified as negative (−), while those with a moderate diameter (+) or a large diameter (++) were scored accordingly.
Amino acid auxotrophy was screened by growth on M9 minimal salts agar (Sigma-Aldrich, ON, Canada) supplemented with 20% (wt/vol) glucose. Auxotrophic isolates were identified by a lack of growth following 48–72 h of incubation at 37 °C. The identification of auxotrophies for specific amino acids was characterized as described earlier, with the addition of one of 18 amino acids to the agar at physiologically relevant concentrations39 (link).
Mucoidy was assessed visually following 48 h incubation on LB and yeast extract mannitol (YEM) agar. Growth was scored as as non-mucoid (−), lightly mucoid (+), moderately mucoid (++), or heavily mucoid (+++), which was noted by the transfer of the exopolysaccharide alginate onto the opposing lid of the Petri dish40 (link).

PUE (production batch number: 150303) was purchased from Guangzhou Baiyun Mountain Tianxin Pharmaceutical Limited Company (Guangzhou, China). The coating antigen PUE-bovine serum albumin (PUE-BSA) and anti-PUE-MAb from ascitic fluid were produced in our lab as described previously [15 (link)]. Goat anti-mouse immunoglobulin conjugated to horseradish peroxidase (GaMIgG-HRP, whole molecule) was purchased from GE Healthcare (Little Chalfont, UK). Ninety-six-well immunoplates were purchased from Corning Inc. (Corning, NY, USA). Skim milk was purchased from Becton Dickinson (Franklin Lakes, NJ, USA). 3,3′,5,5′-Tetramethylbenzidine (TMB) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other commercial chemicals were of analytical grade (AR) and were obtained from Sinopharm Chemical Reagents Beijing Co., Ltd. (Beijing, China).

Four-micrometer-thick paraffin sections were treated with 0.01 M citrate buffer at 90°C and heated for 5 min in a microwave oven at 750 W. IHC staining for CXCR4 and TGF-β1 was performed using the avidin–biotin immunoperoxidase technique (Histofine SAB-PO kit, Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Anti-CXCR4 and anti-TGF-β1 polyclonal Ab (Chemicon, Temecula, CA, USA) were used at a dilution of 1:100.
Vimentin (dilution 1:200; Chemicon) was used to identify isolated cells originating from the mesenchyme. The cells were fixed in 4% paraformaldehyde and permeabilized with PBS containing 5% skim milk (Becton Dickinson) and 0.1% Triton X-100 for 30 min. The cells were subsequently incubated with mouse antihuman monoclonal Abs overnight. After washing with PBS containing 0.5% Tween 20, the cells were incubated with fluorescein isothiocyanate-conjugated secondary Abs for 30 min. Negative and positive control slides were prepared by incubating the sections with isotype controls instead of primary Abs. Nuclei were counterstained with Hoechst 33342. The cells were then washed three times with PBS and observed through fluorescence microscopy (Olympus, Tokyo, Japan).