Igf 1

Intestine tissues frozen at −80°C were thawed and fully grinded into homogenate in lysis buffer containing protease inhibitors and phosphatase inhibitors in glass grinder. After lysing on ice for 30 min, the samples were centrifuged at 12,000 r/min for 15 min and supernatants were collected whose protein concentrations were detected by BCA Kit. Thirty microgram proteins of each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were then blocked with 5% nonfat milk in TBST at room temperature for 1 h and followed by incubation in primary antibody IGF1 (dilution in 1:1000, Cell Signaling Technology, Inc., U.S.A.), PI3K (dilution in 1:1000, Cell Signaling Technology, Inc., U.S.A.), Akt (dilution in 1:1000, Cell Signaling Technology, Inc., U.S.A.), mTOR (dilution in 1:1000, Cell Signaling Technology, Inc., U.S.A.), or GADPH (dilution in 1:5000, Cell Signaling Technology, Inc., U.S.A.) at 4°C overnight. For secondary antibody incubation, the membranes were first washed with TBST three times, 10 min each time, and then incubated with goat anti-rabbit secondary antibody (1:2000) at room temperature for 1 h. Target bands were illuminated by ECL developer and captured by gel imaging system. Absorbance value was analyzed by ImageJ (NIH, U.S.A.).

Colon cancer MC38 cell line was originally derived from C57BL/6 mice treated with the carcinogen 1,2-dimethylhydrazine [21 (link)], it was purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM medium supplemented with 10% fetal bovine serum, 1% glutamine and 1% of penicillin/streptomycin. The cells were maintained at 37°C and 5% CO₂ in a humid environment. Medium was replaced every 48 hours and cells were trypsinized when reached 80% confluence. The cells were serum-starved overnight and then treated with 5 ng/ml or 50 ng/ml concentrations of insulin (Sigma, St. Louis, MO) and IGF-1 (Cell Signaling Technology, Beverly, MA) dissolved in phosphate buffered saline (PBS). In ERK1/2 or JNK inhibiting assays, 40 μM ERK1/2 inhibitor PD98059 (Sigma, St. Louis, MO) or 20 μM JNK inhibitor SP600125 (Sigma, St. Louis, MO) dissolved in dimethyl sulfoxide (DMSO) were added into cultured cells.

The liver tissues from rats were lysed using Tissue Lysis Buffer (Cell Biologics, Chicago, IL, USA). Equal amounts (80 μg/lane) of total protein were dissolved in NuPAGE^® LDS sample buffer (Life Technologies, Carlsbad, CA, USA) and were separated in a 10% SDS-polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA), which was then blocked for 1 h in a blocking buffer. The membrane was then incubated for 12 h at 4 °C with antibodies recognizing IGF-1, IGFBP-3, JAK2, p-JAK2, STAT5, p-STAT5, and β-actin (Cell Signaling Technology, Danvers, MA, USA, 1:1000). This was followed by incubating the membrane with a secondary antibody (anti-rabbit IgG HRP-linked antibody, 1:5000, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The immunoreactive protein bands were detected using chemiluminescence (ECL, Bardhaman, India) Western blotting detection reagents (Bio-Rad, Hercules, CA, USA).

Cells were plated in muscle growth media (fetal bovine serum 0.05 mL/mL, fetuin 50 μg/mL, epidermal growth factor 10 ng/mL, basic fibroblast growth factor 1 ng/mL, insulin 10 μg/mL, and dexamethasone 0.4 μg/mL) for 48 hours before changing to the skeletal muscle differentiation media, which is a proprietary serum-free medium containing 10 μg/mL insulin (PromoCell, Heidelberg, Germany) [26 (link)], with or without 100 ng/mL of either IGF-1 or IGF-2 (Cell Signaling Technology, Danvers, MA). IGFs were added every 3 days at the time of media change. Cells were grown in six-well plates in a standard tissue culture incubator at 37°C in 5% CO₂.

Imiquimod was purchased from InvivoGen (San Diego, CA, USA). Rapamycin, 3-MA, and 4-HNE were purchased from Santa Cruz Biotech (Dallas, TX, USA). Doxycycline (DOX) was purchased from Clontech (Mountain View, CA, USA). MDA, arachidonic acid, oleic acid, and other standard reagents were purchased from Sigma (St. Louis, MO, USA). Primary antibodies for TLR7, Myd88, LC3A/B, IGF-1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies (i.e., anti-rabbit, anti-goat, or anti-mouse IgG antibody conjugated with horseradish peroxidase; anti-rabbit IgG antibody conjugated with Alexa Fluor 488) were obtained from Millipore (Temecula, CA, USA). Hoechst staining kit was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were of analytic grade.

Tissue samples (15–20 mg) were placed in the centrifuge tube, ground 50–60 times with a plastic stick, added with 200 μL of natural cell lysate and ground 30–60 times. The samples were incubated on ice for 5 min and centrifuged at 14,000–16,000 rpm at 4 °C for 1–2 min. Then, the tube was placed on ice, and protein was collected. Protein concentration was determined by a bicinchoninic acid kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted protein was separated by 10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked by 5% skim milk and incubated with primary antibodies IGF1 (1:1000), caspase-3 (1:500), Cleaved caspase-3, Bax, Bcl-2, and GAPDH (1:1000, Cell Signaling Technology, Beverly, MA, USA). After that, the membrane and the secondary antibody (1:5000; Thermo Fisher Scientific) were incubated for 1 h and the protein bands were visualized by the enhanced chemiluminescence kits (Pierce, Rockford, IL, USA). GAPDH was the internal control and protein band images were analyzed by ImageJ2x V2.1.4.7 (Rawak Software, Inc., Germany). This method is also suitable for cell experiments.