Caspase 3

MDA-MB-231 and MCF-7 cells were lysed in RIPA lysis buffer on ice for 30 min, then centrifuged (12000 g/min; 30 min) at 4°C. A bicinchoninic acid (BCA) assay was used to detected protein concentrations. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (millipore, USA), and then incubated with primary antibodies overnight at 4°C after the membranes were blocked (5% skim milk in PBS with 0.1% Tween 20) for 4 h. The next day, the membranes were imaged with gel imaging equipment (Bio-Rad, USA) after the membranes were incubated with secondary antibodies for 2 h. β-actin was used as a loading control. The following antibodies were used: Bcl-2 and Bax (Cell Signaling technology, USA); anti-RIP1, anti-RIP3, and p-RIP3 (Santa Cruz Biotechnology, USA); TNF-α (Abcam, USA); Caspase 3 (Enzo, USA); Ppm1b (BETHYL, USA); anti-β-actin (Biosharp, China) All reagents were dissolved according to the manufacturer’s instructions.

Cell lines were lysed in RIPA buffer (100 mM Tris–HCL pH 8, 150 mM NaCl, 1 % Triton X-100, 1 mM MgCl, 25 mM NaVO₄) in the presence of complete protease-inhibitor mixture (Sigma). Immunoblotting was performed with antibodies to: caspase-9 (rabbit polyclonal antibody, Enzo Life Sciences); caspase-8 (mouse monoclonal antibody, Transduction Laboratories); caspase-3 (mouse monoclonal antibody, Enzo Life Sciences); cathepsin B (rabbit polyclonal antibody, Calbiochem). As a control, the membranes were incubated with specific antibodies of anti-α-tubulin (mouse monoclonal antibody, Sigma). The intensities of bands of active fragments were quantified normalizing to Tubulin. The optical density of the bands [integrated area in arbitrary units (AU)] was measured by using the National Institutes of Health Image J software (rsb.info.nih.gov/ij).

RA-FLSs were treated with different concentrations of shikonin for 24 h and then collected. Total cell lysates were prepared using RIPA buffer supplemented with protease inhibitors (Roche, Shanghai, China) and PMSF (Sigma, USA). Protein concentrations were determined with BCA kits (Beyotime, Shanghai, China). The cells were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the PVDF membranes were incubated with their specific primary antibodies in TBST at 4 °C with primary antibodies recognizing PKM2 (1:1000, Cell Signaling Technology, USA), GULT1, HK2, PI3K, p-PI3K (1:1000, Santa Cruz Biotechnology, USA), AKT, p-AKT (1:1000, Abcam, USA), mTOR, BAX (1:1000, Cell Signaling Technology, USA), Bcl-2 (1:1000, Cell Signaling Technology, USA), caspase 3 (1:1000, Enzo, USA), LC3 (1:1000, Santa Cruz Biotechnology, USA), and anti-β-actin (1:1000, Biosharp, China). All reagents were dissolved according to the manufacturer’s instructions.
After three washes with TPBS, the secondary antibody (1:5000) was added followed by incubation at room temperature for 1 h. Proteins were visualized and detected by enhanced chemiluminescence detection reagents (Pierce, Thermo Fisher Scientific) and analyzed with an Image Quant LAS 4000 imaging system (GE Healthcare, Pittsburgh, PA, USA).

Dewaxing and rehydration of paraffin-embedded paw sections were performed using xylene and then degreased with gradient alcohol. Hydrogen peroxide (3%) at room temperature was used to fix the samples for 10 min and block them with blocking solution for 30 min. The primary antibody (1:50) was incubated at 37 °C for 60 min and stained with the secondary antibody. Samples were incubated for 10 min at room temperature and developed with DAB. Secondary staining of the nucleus was performed with 1% hematoxylin. Antibodies against the following proteins were used: Bcl-2 and Bax (Cell Signaling Technology, USA) and caspase 3 (Enzo, USA). All reagents were dissolved according to the manufacturer’s instructions.

Chemicals and reagents were obtained as follows: CZM from, 8-TQ from, Bz from Selleckchem (Houston, TX, USA); TRAIL from KOMABIBIOTECH (Seoul, South Korea); Ru360 from Calbiochem (Millipore Corp., Billerica, MA, USA); MitoTracker-Red (MTR), propidium iodide (PI), Fluo-3-AM, Rhod-2-AM, and 4′,6-diamidino–2-phenylindole (DAPI) from Molecular Probes (Eugene, OR, USA); z-VAD-fmk from R&D Systems (Minneapolis, MN, USA); necrostatin-1, 3-methyladenine (3-MA), bafilomycin A1, 2-bis(o-amino phenoxy)ethane-N, N, N’N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), celastrol, and cycloheximide (CHX) from Sigma-Aldrich (St. Louis, MO, USA). The following primary antibodies were used: Ub from Santa Cruz (Dallas, TX, USA), p62 from BD Biosciences (San Jose, CA, USA); p-eIF2α, eIF2α, ATF4, Nrf1, and CHOP/GADD153 from Cell Signaling Technology (Danvers, MA, USA); caspase-3 from Enzo Life Science (Farmingdale, NY, USA); PARP from Abcam (Cambridge, UK). The secondary antibodies, including rabbit IgG HRP, mouse IgG HRP, rabbit Alexa Fluor 488, and mouse Alexa Fluor 594, were obtained from Molecular Probes.

WB was performed as described [49 (link)]. Antibodies used were the following: CK1α, PARP, Mcl1, Ser45 β-catenin, Ser33/37/Thr41 β-catenin, total β-catenin, Ser473 AKT, total AKT (Cell signaling Technology, MA, USA); Mdm2 (Millipore, Italy), GAPDH (Ambion, USA); α-tubulin (Sigma-Aldrich, Italy); Bak (Merck, MA, USA); Bax, p21, p53 (Becton Dickinson, Italy); Caspase 3 (Enzo Life Science, UK). Images were acquired using the Image Quant LAS 500 chemiluminescence detection system (GE Healthcare, USA). Densitometric analysis was performed with Quantity One software (Bio-Rad, Italy).

WB was performed as described (27 (link)). Antibodies used were the following: CK1α, PARP, Mcl1, total β-catenin, Ser 473 AKT, total AKT, Ser 176/180, Ser 177/181 IKKα/α, total IKKα, IKKβ, Ser 536 NF-κB p65, Ser 652 CARD11, Tyr 223 BTK, total BTK, Ser 32 Ikbα, BCL10 (Cell signaling Technology, MA, USA); GAPDH (Ambion, USA), β-actin (Sigma-Aldrich, Italy); p21 (Becton Dickinson, Italy); Caspase 3 (Enzo Life Science, UK); total p65 (abcam, UK), DEPTOR (Millipore, Itlay); CARD11 and BTK for immunoprecipitation (Santa Cruz Biotechnology, Inc; Italy). Images were acquired using the Image Quant LAS 500 chemiluminescence detection system (GE Healthcare, USA).