β actin clone ac 74

For immunoblots, cells were harvested in modified RIPA buffer (50 mM tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-630 [v/v], 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM AEBSF, protease inhibitor cocktail) and incubated on ice for 30 min prior to SDS–PAGE and transfer to nitrocellulose. The following antibodies were used in immunoblots: β-actin (clone AC-74, Sigma-Aldrich; dilution 1:2000), E2F1 (3742S, Cell Signaling Technology, dilution 1:1000), symmetric di-methyl arginine (SDMe) (13222S, Cell Signaling Technology, dilution 1:1000), FLAG (clone M2, F1804, Sigma, 1: 1000), GAPDH (clone 6C5, MAB374, Millipore, 1:2000). Uncropped versions of immunoblots are presented in the supplementary figure 11.

Total cellular proteins were extracted by MSCs using lysis buffer (0.1 M KH₂PO₄, pH 7.5, 1% NP-40, 0.1 mM β-glycerolphosphate, supplemented with protease inhibitor cocktail; Sigma-Aldrich) and quantified spectrophometrically by the Bio-Rad Protein Assay (Bio-Rad Laboratories). Thirty micrograms of proteins were subjected to 8–12% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare Life Sciences) at 30 mA for 2 hours and 30 min. Membranes were blocked with 5% non-fat dry milk in TBS-Tween for 1 hour at room temperature and incubated with primary antibodies against OPN (1:500; Millipore), BMP-2 (1:1000; Santa Cruz), PPAR-γ (1:200; Santa Cruz) and β-actin (clone AC-74; Sigma-Aldrich) at 4 °C o/n. Incubation with secondary antibody human anti-rabbit/mouse horseradish peroxidase-conjugated (GE Healthcare) was performed for 1 hour at room temperature. The protein signal was detected using Westar ηC chemiluminescent substrate (Cyanagen) and band intensities were quantified by densitometry analysis by ImageJ software (NIH, USA). Similarly, equal volumes (18 μl) of MSC supernatants, previously cultured in serum-free DMEM for 24 hours and stored at −80 °C, were loaded into 8% SDS-PAGE and transferred to nitrocellulose membrane as already described, using primary MMP-9 antibody (1:1000; Cell Signaling).

All lysates were boiled in 1x Laemmli sample buffer with β-mercaptoethanol for 5 min followed by electrophoresis on 4–20% Mini-PROTEAN TGX precast Tris-Glycine-SDS gels (Bio-Rad, Hercules, CA). Proteins were transferred to low-fluorescent PVDF (Bio-Rad) and incubated overnight in primary antibody at 1:1000 dilution. Blots were incubated in IRDye-conjugated secondary antibodies at room temperature for 1 hr and imaged/quantitated by an Odyssey CLx imaging system (Li-Cor, Lincoln, NE). For western blotting, HNF1A (clone GT4110) and KRAS (ab55391) from Abcam (Cambridge, MA), β-Actin (clone AC-74) from Sigma-Aldrich, Cadherin-17 (CDH17) from Proteintech (Rosemont, IL), β-Galactosidase from Promega (Madison, WI) and RAS^G12D, CD44, EPCAM, DPP4, Cleaved Caspase-3 (D175), Cleaved Caspase-6 (D162), Cleaved Caspase-7 (D198), Cleaved Caspase-9 (D315), Cleaved Caspase-9 (D330), phospho-ERK1/2, phospho-AKT S473, OCT4A and GFP from Cell Signaling Technology (Danvers, MA). For flow cytometry, mouse anti-human EPCAM (CD326) clone HEA-125 was purchased from Miltenyi Biotec (San Diego, CA). Mouse anti-human CD44 clone G44-26, CD24 clone ML5 and APC-Cy7 Streptavidin were purchased from BD Biosciences (San Jose, CA). Biotinylated mouse anti-mouse H-2Kd/H-2Dd clone 34-1-2S was purchased from SouthernBiotech (Birmingham, AL).

Cell lysates were prepared using ice-cold cell-lysis buffer (50 mmol/L HEPES, pH 7.5; 150 mmol/L NaCl; 1% triton X-100; 2% aprotinin; 2 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0; 50 mmol/L sodium fluoride; 10 mmol/L sodium pyrophosphate; 10% glycerol; 1 mmol/L sodium vanadate; and 2 mmol/L Pefabloc SC) for 30 min. Subsequently, samples were centrifuged at 13,000 rpm for 10 min in a table top centrifuge. Supernatants were transferred to new tubes and stored at −80 °C till use. A total of 10 µL of virus sample was mixed with loading dye. SDS-PAGE of proteins was performed on a 10% polyacrylamide gel (for detection of RSV F and VSV N: heated loading buffer without β-Mercaptoethanol added to samples; for detection of LCMV GP: samples boiled in loading buffer with β-Mercaptoethanol). Subsequently, proteins were transferred to 0.45 µm nitrocellulose membranes (Whatman, Dassel, Germany) and membranes were blocked with MPBST (PBS containing 5% skim milk and 0.1% Tween-20). Proteins were detected using primary antibodies (RSV F (18F12) [36 (link)], VSV N (Kerafast, Boston, USA), β-actin (clone AC-74, Sigma-Aldrich, St. Louis, MO, USA)) or hybridoma supernatant (LCMV GP (KL25)) and appropriate peroxidase-conjugated secondary antibodies.

Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN). Secondary antibodies used were: goat anti-mouse IgG horse radish peroxidase (HRP) conjugate and goat anti-rabbit IgG HRP conjugate, both from Calbiochem (EMD Biosciences, La Jolla, CA). Recombinant human VEGF₁₆₅ was purchased from R&D Systems (Minneapolis, MN). Recombinant Human TGFβ2 (100-35B) was from Peprotech (Quebec, QC). Avastin® (DIN 02270994) was from Roche (Mississauga, ON) and was used at a concentration of 1 μg/ml.