Rabbit anti p21

Immunofluorescence staining was performed as we previously described^{8 (link)}. Briefly, after washing twice with PBS, HepG2 cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, at indicated times. Then, cells permeabilization with 0,1% Triton X-100 twice for 10 min, and blocking with 2% BSA were performed. Cells were incubated overnight with primary antibodies, including rabbit anti-p21 (1:100, Santa Cruz), rabbit anti-Ki-67 (1:100 Millipore) and mouse anti-Mdm-2 (1:100, Santa Cruz). Secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100, Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The samples were incubated at room temperature for 1 h and washed 3 times with PBS. Nuclei were counterstained with DAPI, and samples were mounting with prolong Gold antifading solution (Thermoscientific). Primary antibody was omitted in negative controls (data not shown). Cells were visualized and photomicrographed under an inverted fluorescence microscope (Nikon). Positive Alexa 488 cells were analyzed and quantified using FIJI-Image J software (Bethesda, MD, USA).

Hydrogen peroxide (H₂O₂) was obtained from Junsei (Tokyo, Japan). FK866 was purchased from Adipogen (Switzerland). The antibodies used in the present study were as follows—rabbit anti-p21 (Santa Cruz Biotech, Dallas, TX, USA), mouse monoclonal anti-p53 (Calbiochem, San Diego, CA, USA), mouse monoclonal anti-β-Actin (Abcam, Cambridge, MA, USA), mouse monoclonal anti-TRF1 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-phospho (Ser139)-histone H2AX (γH2AX) (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-NF-κB p65 (Santa Cruz Biotech, Dallas, TX, USA), horseradish peroxidase-conjugated goat anti-rabbit, and anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor^® 488-conjugated goat anti-mouse IgG, and Alexa Fluor^® 594-conjugated goat anti-rabbit IgG (Invitrogen, Camarillo, CA, USA).

Recombinant human IL-17A was purchased from PeproTech (USA). Rat PE-conjugated
anti-mouse CD86 and rat FITC-conjugated anti-mouse CD206 were purchased from
BioLegend (USA). Rabbit anti-GSK-3β, Arg1, β-catenin, active-β-catenin (ABC),
phospho-STAT1 (signal transducers and activators of transcription 1), and
phospho-STAT3 antibodies were products of Cell Signaling Technology (USA).
Rabbit anti-STAT6 and phosphor-STAT6 antibodies were purchased from Affinity
Biosciences (USA). Rabbit anti-iNOS antibody was purchased from Abcam (USA),
rabbit anti-p21 was a product of Santa Cruz Biotech (USA). Rabbit anti-STAT3,
SOCS3, BCL-XL, c-Myc, TCF-4, β-actin, and mouse anti-Cyclin D1 antibodies were
purchased from Proteintech (China). The Wnt signaling inhibitor XAV939 was
purchased from Santa Cruz Biotech.

H₂O₂ was obtained from Junsei (Tokyo, Japan). Visfatin and FK866 were purchased from Adipogen (San Diego, CA, USA). Primary antibodies used in this study were as follows: rabbit anti-Visfatin (Adipogen, San Diego, CA, USA), rabbit anti-p21 (Santa Cruz Biotech, Dallas, TX, USA), mouse monoclonal anti-p53 (Calbiochem, San Diego, CA, USA), rabbit anti-α-Tubulin (Bioworld, Minneapolis, MN, USA), mouse monoclonal anti-β-Actin (Abcam, Cambridge, MA, USA), mouse monoclonal anti-c-Myc (Santa Cruz Biotech, Dallas, TX, USA), mouse monoclonal anti-TRF-1 (Santa Cruz Biotech, Dallas, TX, USA), rabbit anti-phospho (Ser139)-histone H2AX (γH2AX) (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-NF-κB p65 (Santa Cruz Biotech, Dallas, TX, USA). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Alexa Fluor^® 488-conjugated goat anti-mouse IgG, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, Alexa Fluor^® 594-conjugated goat anti-mouse IgG, and Alexa Fluor^® 594-conjugated goat anti-rabbit IgG were purchased from Invitrogen (Camarillo, CA, USA).

For immunostaining of γ-H2AX, after 30 minutes, 24 hours, 72 hours and 7 days of exposure to ⁹⁰SrCl₂, cells were washed twice with 1x PBS and fixed for 15 minutes at room temperature with a 4% PFA solution. After washing, cells were incubated with lysing solution for 3 minutes at room temperature then with primary rabbit anti γ-H2AX antibody (Millipore, Guyancourt, France) diluted into 2% PBS-BSA (bovine serum albumin) solution for 40 minutes at 37 °C. After washing, cells were incubated for 20 minutes at 37 °C with anti-rabbit IgG coupled to FITC (Life Technologies). After washing, slides were mounted with Vectashield mounting medium containing DAPI (Life Technologies) for nucleus staining. Images were automatically acquired onto fluorescent microscope connected to the Metafer system (Metasystem, Alltussheim, Germany). At least 250 nuclei were captured and the number of γ-H2AX foci/nuclei was assessed using Histolab software (Microvision Instruments, Evry, France).
For p21 and p53 detection, cells were stained following the same protocol using a rabbit anti-p21 (Santa Cruz Biotechnologies, Heidelberg, Germany) or a rabbit anti-p53 (R&D Systems, Lille, France) and a secondary anti-rabbit IgG coupled to FITC (Life Technologies). Images were acquired on a fluorescent microscope using Histolab software (Microvision Instruments).

ARO cells were plated in 10-cm plates (8x10⁵ cells) and grown for 24 h. Cells were then treated either with 0.35% MCP, 75 μM FTS, 0.35% MCP plus 75 μM FTS or as a control with 0.35% D-lactose for 48 h. In a second set of experiments, cells were treated either with 0.35% MCP, 75 μM FTS, 0.35% MCP plus 75 μM FTS or as a control with 0.35% D-lactose for 72 h. Next, cells were lysed in 300 μl homogenization buffer (50 mmol/l Tris-HCl—pH 7.6, 20 mM MgCl₂, 200 mM NaCl, 0.5% NP40, 1 mM DTT, and protease inhibitors), centrifuged for 10 min at 14 000 r.p.m. at 4 °C and the supernatant was collected. Equal amounts of proteins (40 μg per lane) were subjected to SDS-PAGE, followed by immunoblotting with mouse anti-pan-Ras antibody (Ab, Calbiochem, San Diego, CA, USA), rabbit anti-β tubulin Ab (Sigma Aldrich, Rehovot, IL, USA), mouse anti- K-Ras (Calbiochem), rat anti-Galectin-3 (Mac2), rabbit anti-total ERK (Santa Cruz, Dallas, TX, USA), mouse anti-p-ERK (Sigma Aldrich), rabbit anti-p21 (Santa Cruz), mouse anti-p53 (Calbiochem) and mouse anti-actin (MP Biomedicals, Santa Ana, CA, USA). Blots were then exposed to the appropriate secondary peroxidase-coupled IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and subjected to enhanced chemiluminescence. Protein bands were quantified by densitometry with Image EZQuant-Gel software (EZQuant Ltd, Tel-Aviv, Israel).