Bcl 2

Cells were suspended in lysis buffer (20 mM Tris-HCl, pH 7.8, 50 mM NaCl, 5 mM EGTA, and 1% v/v Triton X-100) containing freshly added protease and phosphatase inhibitors (Roche). Lysates were clarified by centrifugation at 4°C, and protein concentration was determined by Bio-Rad protein assay.
To verify the protein contents of the BMSC-ECM after alkaline detergent extraction, BMSC-ECM were scratched from culture plate and dissolved in buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol and 4% SDS. Samples were homogenized by pass through 27 g needle for at least 7 times, incubated on ice for 60 minutes, cleared by centrifugation and subjected to protein concentration assessment. Then 0.2% glycerol and 0.2% bromophenol blue (final concentration) were added and samples were heated at 95°C for 5 minutes.
The following antibodies are used: phosphor-AKT Ser 473 and were from Cell Signalling. γ-H2AX, osteopontin, β-actin, MnSOD2, BCL-2, and BCL-xL were from Millipore. p27, p21, collagen 1, fibronectin, MCL-1, Gpx1/2, were from Santa Cruz Biotechnology.

MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF-7, BT-474 and SK-BR-3 mammary carcinoma cell lines were serum starved in DMEM containing 0.1% bovine serum albumin (BSA, SIGMA) and then stimulated with 10% FBS or 5% WF for the indicated time points.
The preparation of protein lysates and immunoblotting analysis was performed as previously described [33 (link)], except that membranes were blocked with Odyssey Blocking Buffer (Licor, Biosciences) and, following incubation with primary antibodies overnight at 4°C, incubated 1 hour at RT with IR-conjugated (Alexa Fluor 680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, Licor).
Primary antibodies directed against AKT (sc-1618), ERK1 (sc-94), STAT3 (sc-482), Fibrillarin (sc-25397) and Vinculin (sc-7694) were purchased from Santa Cruz Biotechnology, Inc.; pT202/204 ERK1/2 (#9101), pS473 AKT (#4060), pY705STAT3 (#9131) were purchased from Cell Signaling; Tubulin (#T9026) was purchased from SIGMA; Cyclin D1 (#04-1151) and Bcl2 (#OP60) were purchased from Millipore; Grb2 was purchased from BD Transduction Laboratories (#610112).

Lysates (RIPA buffer) were diluted in NuPAGE LSD Sample buffer and sample reducing agent (Invitrogen) and run on reducing SDS‒PAGE mini-gels (Invitrogen).
Proteins were electrotransferred onto nitrocellulose membranes with a turboblot (30 V, 7 min) and then incubated overnight at 4 °C with the appropriate antibody diluted at the optimal concentration in 5% (w/v) BSA or skim milk. The following antibodies were used for Western blot analysis: BCL2 (Millipore cat. 05-826, Merck), BCL-XL (Cell Signaling Technology, Danvers, MA, USA, 54H6 #2764 1:1000), PARP (CST, cat. 9542, 1:1000), PCNA (CST, cat. 13110, 1:1000), NFKBIZ (CST, cat. 9244 1:1000), NF-κB p65 (CST, cat. 8242 1:1000), Survivin (CST, cat. 2808 1:1000), NRF2 (cat. ARG40635 Arigo, Hsinchu City, Taiwan 1:1000), NQO1 (cat. ARG43340,Arigo 1:1000), HO-1 (cat. ARG43341, Arigo 1:1000), GPX4 (ARG41400, Arigo 1:1000), Laminin B1-HRP conjugate (CST, cat. 15068 1:1000) and anti-β-Actin HRP-conjugated (Sigma 1:20000). The blots were then washed in PBS with 0.2% Tween (PBST) and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at RT. The proteins were visualized using the enhanced chemiluminescence reagent ECL Clarity (Bio-Rad) and detected with a ChemiDoc imaging system (Bio-Rad).

Specific antibodies to p53, HSP70, HSP60, HSP90β, β-tubulin, p21, GAPDH, and HSP90β siRNAs were purchased from Santa Cruz (Santa Cruz, CA, USA). Polyclonal antibodies to Bax, Bcl-2, cytochrome c, and β-actin were obtained from Millipore (Temecula, CA, USA). Polyclonal antibodies to caspase-3, -7, -9, PARP, CDK2, -4, -6, cyclin D, cyclin E, N-cadherin, E-cadherin, claudin, AKT, and phospho-AKT^Ser473 were purchased from Cell Signaling (Beverly, MA, USA). Pan-caspase inhibitor (Z-VAD-FMK) was obtained from Enzo life science (New York, NY, USA).
The overall structure flowchart is shown in Figure 6 as follows:

The whole cell extract was prepared by lysing cells in RIPA lysis buffer with proteinase inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were resolved by SDS-PAGE at different percentages, transferred to PVDF membrane and immunoblotted overnight at 4 °C with antibodies against HOXA13 (rabbit; ab106503 Abcam, Cambridge, UK; 1:1000); RAB11FIP1 (rabbit; 16,778–1-AP Proteintech, Rosemont, IL, USA; 1:1000); N-cadherin (mouse; 14–3259 eBioscience, Waltham, MA, USA; 1:1000); PARP (rabbit; #9542 Cell Signaling Technology, Danvers, MA, USA; 1:1000); Bcl-2 (rabbit; 04–436 Millipore, Burlington, MA, USA; 1:1000); and GAPDH (rabbit; #5174 Cell Signaling Technology; 1:1000). Chemiluminescent signals were developed using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA).