Insulin like growth factor 1 igf 1

CD34⁺ cells were cultured using a single-phase ex vivo expansion and differentiation protocol [53 (link)] with minor modifications. Briefly, cells were cultured in IMDM supplemented with 2 U/ml erythropoietin (EPO) (Sigma-Aldrich, Chemie GmbH Germany), 1 μM glucocorticoid dexamethasone (Sigma-Aldrich, Chemie GmbH Germany), 40 ng/ml insulin-like growth factor 1 (IGF-1) (Sigma-Aldrich, South Africa), 100 ng/ml stem cell factor (SCF) (Sigma-Aldrich, Chemie GmbH Germany) and 400 μg/ml holo-human transferrin (Sigma-Aldrich, Germany). The initial seeding density of 0.5−1 × 10⁶ cells/ml were expanded and differentiated to erythroid progenitors for 15 days, with ad hoc demi-population. Post-differentiation, flow cytometry was used to determine expression of CD71 and CD235a (Glycophorin-A). The human erythroleukaemia cell line K562 was cultured in IMDM initially supplemented with 10 % FBS and then 0.5 % in all subsequent experiments.

Recombinant human TNF-α, IL-1β, TGF-β, HGF, insulin-like growth factor 1 (IGF-1), EGF, and bFGF were obtained from Sigma-Aldrich (St. Louis, MO, USA). To evaluate EMT-dependent RFP induction after exposure to inflammatory mediators, HCT116 and RKO cells were treated with TNF-α^{29 (link)} (20 ng/ml), IL-1β^{30 (link)} (1 ng/ml), TGF-β⁵⁴ (10 ng/ml), HGF^{55 (link)} (50 ng/ml), IGF-1^{56 (link)} (20 ng/ml), EGF^{57 (link)} (20 ng/ml), or bFGF^{57 (link)} (10 ng/ml) for 48 h. The plasticity of EMT-dependent RFP induction was assessed by replacing with fresh medium after treatment with TNF-α or IL-1β.

Insulin-like growth factor-1 (IGF-1) and PI3K inhibitor Wortmannin (WM) were purchased from Sigma-Aldrich (Milan, Italy). The dual insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) inhibitor OSI-906 (Linsitinib) was obtained from Tocris Bioscience (Space, Milan, Italy). Focal Adhesion Kinase selective inhibitor VS-4718 (PND-1186) was bought from Santa Cruz Biotechnology (DBA, Milan, Italy). The YAP/TEAD complex suppressor Verteporfin was purchased from Med Chem Express (DBA, Milan, Italy). All the aforementioned compounds were dissolved in dimethyl-sulfoxide (DMSO) except IGF-1 which was dissolved in water.

ERα+ human breast cancer epithelial cell lines MCF-7, ZR-75-1 (ZR-75), and T-47D, as well as MDA-MB-231 triple-negative breast cancer (TNBC), were purchased from ATCC (LGC Standards S.r.l., Milan, Italy) and authenticated as previously described [26 (link)]. MCF-7 and MDA-MB-231 cells were cultured in DMEM/F-12 medium, ZR-75 in RPMI 1640 plus 10 mM HEPES, 1 mM Na-pyruvate, and 0.24% glucose (all from Sigma-Aldrich, Merck, Milan, Italy) and T-47D in RPMI 1640 plus 0.2 U/mL insulin (Sigma-Aldrich, Merck). All media were supplemented with 10% (5% for MCF-7) fetal bovine serum (FBS), 100 IU/mL penicillin, 100 ng/mL streptomycin and 0.2 mM L-glutamine. All media and reagents were purchased from Gibco (Thermo Fisher Scientific Inc., Monza, Italy). insulin-like growth factor-1 (IGF-1) was purchased from Sigma-Aldrich/Merck, Italy, and the PI3K inhibitor alpelisib hydrochloride was obtained from MedChemExpress (MCE, Princeton, NJ, USA).

The mouse livers were perfused with Hank's solution containing collagenase, and viable HSCs were isolated by percoll isodensity centrifugation as described previously 8. HSCs were observed in UV light of microscope at day 1, 3, 7 after isolation. Naringin (20 ng/ml, NIFDCC, China) was added into day 1 HSCs for 24 hrs and day 3 activating or day 7 fully activated HSCs for 48 hrs. 3‐methyladenine (3‐MA, 750 ng/ml, Sigma‐Aldrich, St. Louis, MO, USA) was added into day 7 fully activated HSCs for 2 hrs. Insulin‐like growth factor‐1 (IGF‐1, 500 ng/ml, Sigma‐Aldrich) was added into day 7 fully activated HSCs for 2 hrs.

The basic chondrogenic differentiation medium (CDM) was DMEM/F12 GlutaMAX
supplemented with 10 ng/mL transforming growth factor β1 (TGFβ1; R&D
systems), 1% insulin-transferrin-sodium selenite media supplement
(Sigma-Aldrich), 0.1 µM dexamethasone (DexaGalen, GALENpharma), 0.1 mM ascorbic
acid 2-phosphate (Sigma-Aldrich), 1.25 mg/mL human serum albumin (Octapharma),
4.5 g/L glucose (B. Braun), 40 µg/mL proline (Sigma-Aldrich), 1 mM sodium
pyruvate (Gibco), and P/S. Growth factors tested in CDM were 500 ng/mL bone
morphogenetic protein 2 (BMP2; InductOs), 100 ng/mL insulin-like growth factor 1
(IGF1; Sigma-Aldrich), 100 ng/mL growth/differentiation factor 5 (GDF5;
PeproTech), and 100 ng/mL fibroblast growth factor 18 (FGF18; PeproTech).