Anti p53 antibody

Sub-confluent A549 were seeded in 6 cm diameter Petri dishes. After adherence, cells were treated for the indicated times with Ni(SalPipNONO) (0.5 mM) and specific pathway inhibitors. Protein extraction and Western blot were performed as previously described [58 (link), 61 (link)]. Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [Anti-cytochrome c, anti-phospho-ERK1/2, anti-caspase- 3, anti-ERK antibodies from Cell Signaling (Celbio, Milan, Italy); anti-p53 antibody from Santa Cruz Biotechnology Inc (Dallas TX, USA); anti-COX-2 from Cayman Chemical (Ann Arbor, MI, USA); anti-HIF-1α from BD Biosciences (San Jose, CA, USA); anti-VEGF from Merck KGaA (Darmstadt, Germany)] and then detected by enhanced chemiluminescence system (Biorad, Hercules, CA, USA). Results were normalized to those obtained by using an antibody against beta actin from Merck KGaA (Darmstadt, Germany) or total ERK1/2 (Cell Signaling, Celbio, Milan, Italy), when indicated.

Primary and metastatic tumor tissues from DKO and TKO mice were fixed in 10% (vol/vol) formalin at RT for 24 hours, embedded in paraffin, sectioned at 5 μm, and mounted on slides (Superfrost Plus, Fisher Scientific). Hematoxylin and eosin (H&E) staining was performed on paraffin sections according to standard protocols. Immunohistochemistry was performed as described previously [13 (link)]. The following antibodies were used for immunohistochemical and immunofluorescent analyses: anti-p53 antibody (rabbit; sc-6243; Santa Cruz Biotechnology); anti-KRT14 antibody (rabbit; #PRB-155P; BioLegend, Dedham, MA, USA); anti-Wilms Tumor Protein (WT1) antibody (rabbit; ab89901; Abcam, Cambridge, MA, USA); anti-Ki-67 antibody (mouse; #550609; BD Biosciences, San Jose, CA, USA); anti-phospho-Histone H2AX (Ser139) antibody (clone JBW301; mouse; Millipore Sigma, Kankakee, IL, USA); and anti-CA125 (MUC16) antibody (rabbit; a gift from Dr. Robert Bast Jr.)

DKO and TKO cells were grown to confluency and then lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Protein content was determined using Bradford assay (Sigma). Protein extracts were mixed with 2x Laemmli sample buffer and heated to 95°C for 5 min. Samples were subjected to SDS–PAGE using 4–12% (vol/vol) gradient NuPage gels and subsequently transferred to a nitrocellulose membrane. The membrane was then blocked with TBST-5% skim milk for 1 hour at room temperature (RT) prior to incubation with a primary antibody in TBST-5% BSA (1:500 or 1:1,000 dilution) overnight at 4°C. Next day, the membrane was washed extensively with TBST and incubated with the appropriate peroxidase conjugated secondary antibody in TBST-5% BSA for 1 hour at RT. Proteins were visualized by enhanced chemiluminescence (ECL) using Clarity western ECL substrate (Bio-Rad, Hercules, California, USA). The following primary antibodies were used: anti-p53 antibody (rabbit; sc-6243; Santa Cruz Biotechnology, Dallas, TX, USA), anti-E-cadherin (rabbit; #4065; Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (rabbit; #5741; Cell Signaling Technology), anti-Fibronectin antibody (mouse; F6140; Sigma-Aldrich), and anti-β-actin (mouse; C-4, sc-47778, Santa Cruz Biotechnology).

Endothelial cells were seeded at the density of 50.000/well in 24 multiwell plates in medium with 10% serum. After cell adhesion, NPs or lyophilized biomaterials were dissolved in medium and placed in direct contact with the cells. After 24 h incubation, biomaterials were gently removed and cells were lysed as described in [20 (link)]. Cell lysates were centrifuged at 10000 g for 20 min at 4°C. Following gel electrophoresis, proteins were blotted onto activated nitrocellulose membranes, incubated overnight with the anti caspase-3 antibody (1:1000) (Cell Signaling, Danvers, USA), anti p21 antibody (1:1000, Cell Signaling), anti p53 antibody (1:1000) (Santa Cruz, Heidelberg, Germany), anti inducible nitric oxide synthase (iNOS, 1:1000, Santa Cruz) or anti cyclooxygenase-2 (COX-2) antibody (1:1000) (Cayman, Ann Arbor, USA). The primary antibody was detected by incubating the membranes for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Promega, Madison, USA)) diluted 1:2500 in PBS, followed by enhanced chemiluminescence detection system (Bio-Rad, Hercules, USA). Images were digitalized with CHEMI DOC Quantity One. Results were normalized to those obtained by using an antibody anti β-actin (1:10000) (Sigma). Original blots are reported as Supplemental Information S1 Fig.

Active p70S6K recombinant protein, anti-β-actin antibody, bafilomycin and sodium selenite were purchased from Sigma-Aldrich (St Louis, MO, USA). Pifithrin-α and MnTMPyP were purchased from Merck Calbiochem (San Diego, CA, USA). Anti-p53 antibody and MnTBAP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ULK1 and anti-LC3 antibodies (for immunofluorescence) were purchased from Abgent (San Diego, CA, USA). Anti-p-p53 (Ser392) antibody was purchased from Nanjing EnoGene Biotechnology (Nanjing, China). Anti-p70S6K antibody was obtained from Proteintech Group, Inc. (Chicago, IL, USA). The HRP-conjugated anti-mouse (ZB-2305) and anti-rabbit (ZB-2301) antibodies were obtained from ZSGB-BIO (Beijing, China). Anti-p-p70S6K, anti-LC3 and DyLight 488-conjugated anti-rabbit secondary antibody were purchased from Cell Signalling Technology (Danvers, MA, USA). The Cy3-conjugated anti-rabbit (89856) and FITC-conjugated anti-mouse (89750) antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). p53 recombinant protein was obtained from Boston Biochem (Cambridge, MA, USA).