Insulin like growth factor 1

Dissociated PDX cells were cultured in neurobasal media-A supplemented with N2 (×1/2; Life Technologies, Carlsbad, CA, USA), B27 (×1/2; GIBCO, San Diego, CA, USA), basic fibroblast growth factor (bFGF; 25 ng/mL; R&D Systems, Minneapolis, MN, USA), epidermal growth factor (EGF; 25 ng/mL; R&D Systems), neuregulin 1 (NRG; 10 ng/mL; R&D Systems), and insulin-like growth factor 1(IGF1; 100 ng/mL; R&D Systems). The cells grown in these serum-free sphere culture conditions were seeded in 384-well plates (500 cells/well), and treated with a drug library (Selleck, Houston, TX, USA). The drug library was composed of targeted agents and cytotoxic chemotherapeutics, which were included in the clinical guideline or current clinical trial for the treatment of non-small cell lung cancer. After 3 days of incubation at 37 °C in a 5 % CO₂ humidified incubator, cell viability was analyzed using an adenosine triphosphate monitoring system based on firefly luciferase (ATPlite™ 1step; PerkinElmer, Waltham, CA, USA). Test concentrations for each drug were empirically determined to produce a clinically relevant spectrum of drug activity. Dose response curves and corresponding half maximal (50 %) inhibitory concentration values (IC₅₀) were calculated using the S+ Chip Analyzer (Samsung Electro-Mechanics, Suwon, Korea) [51 (link)].

Whole organs of Corti were separated from the stria vascularis and from the modiolus by micro-dissection from postnatal day (P) 2-4 mice, into Hank’s Balanced Salt Solution with no calcium and no magnesium (Life Technologies), and digested with 0.125% trypsin-EDTA for 10 minutes. Enzymatic digestion was halted with the addition of 10 mg/mL soybean trypsin inhibitor (Life Technologies) and 1 mg/mL DNase I (Sigma Aldrich). Cells were triturated gently 20 times using a 1000 μL pipette tip to be single cell suspension, and passed through a 40-μm cell strainer. The cells were grown in renewal media consisting of: DMEM/F12 (Life Technologies) with supplement of 1× N2 (Life Technologies), 1 × B27 (Life Technologies), 50 ng/mL Ampicillin (Fisher Scientific), mouse epidermal growth factor (20 ng/ml), mouse basic fibroblast growth factor (10 ng/ml), insulin-like growth factor-1 (50 ng/mL; all growth factors purchased from R&D Systems) and heparin sulfate (50 ng/mL; Sigma). After 1–3 days, solid spheres were identified and harvested.

In order to evaluate the EPC colony forming unit (EPC-CFU), 1 × 10⁶ peripheral blood mononuclear cells of each group of mice were cultured in methylcellulose-containing medium M3236 (StemCell Technologies). Stem cell-derived factors are 20 ng/mL, 50 ng/mL vascular endothelial growth factor, 20 ng/mL IL-3, 50 ng/mL basic fibroblast growth factor, 50 ng/mL epidermal growth factor, 50 ng/mL insulin-like growth factor-1 (all from R&D Systems), 2 U/mL heparin (Sigma-Aldrich), and 10% FBS (Gibco), which were added. After culturing for 10–12 days, the EPC-CFU culture was treated with 0.4 mg/mL DiI-LDL (Biomedical Technologies) for 2 hours and fixed by applying 1 mL of 2% paraformaldehyde (PFA, Sigma-Aldrich) for 1 hour at room temperature. After washing the methylcellulose-containing medium with PBS, the culture was reacted with FITCBS-lectin 1 (Vector Laboratories) at room temperature for 1 hour. After washing with PBS, the culture was observed under a fluorescence microscope (IX70; Olympus). The number of EPC-CFU reflects the number of original EPCs in the initial classified cell fraction.

Cartilage sheets were constructed and cultured in accordance with our previous study (Li et al., 2017 (link)). Briefly, harvested P3 AUCs were seeded in six-well plates (9.6 cm² area) at a density of 2 × 10⁷ cells per well in 12 ml of regular medium, and cultured for 3 days in a 5% CO₂ incubator at 37°C. Next, the regular medium was replaced with the chondrogenic medium comprising DMEM with 10 ng/ml transforming growth factor beta-1 (R&D Systems, Minneapolis, MN, United States), 40 ng/ml dexamethasone (Sigma-Aldrich, St. Louis, MO, United States), and 100 ng/ml insulin-like growth factor 1 (R&D Systems). Additionally, the medium was changed daily to meet the nutritional needs of a large number of chondrocytes. The two species of AUC sheets were cultured for 2 weeks (2 w), 4 weeks (4 w), or 8 weeks (8 w) for detection of regenerated cartilage in vitro and subcutaneous implantation in nude mice.

Suberoylanilide hydroxamic acid (SAHA, vorinostat), LBH589 (panobinostat), MS275 (entinostat), lenalidomide (Len), pomalidomide (Pom) and bortezomib (BTZ) were purchased from Selleck Chemicals (Houston, TX, USA). HDAC6 inhibitor ACY1215 (ricolinostat) was obtained from ChemieTek (Indianapolis, IN, USA). Anti-acetylated α-tubulin antibody (Ab) and -cereblon Abs were purchased from Sigma (St. Louis, MO, USA). Anti-c-Myc, anti-acetylated lysine, anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-caspase-8, anti-caspase-9, anti-cleaved-caspase-3, anti-poly (ADP-ribose) polymerase (PARP), anti-X-linked inhibitor of apoptosis protein (XIAP), Bcl2, anti-cIAP2, anti-α-tubulin, phospho-STAT3, HDAC6, IKZF1, IKZF3 and IRF4 Abs were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IKZF1 Abs were purchased from Cell Signaling Technology or R&D Systems (Minneapolis, MN, USA) Figure 4d. Human interleukin-6, insulin-like growth factor 1, vascular endothelial growth factor and tumor necrosis factor α were obtained from R&D Systems.