Recombinant human insulin like growth factor 1 igf 1

The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

PBMCs were collected and isolated following an adapted version of the protocol previously established [38 (link)]. The PBMCs were collected from a superficial vein of the arm thought Vacutainer tubes with Ficoll monolayer (cat.# 362761; BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. Approximately 1–2 × 10⁶ of PBMCs were cultured in StemSpanTM medium (cat.# 09655, Stem Cell Technologies, Vancouver, BC, Canada) supplemented with the following cytokines: 100 ng/mL Recombinant Human Stem Cell Factor (SCF) (cat.# 255-SC-01M, R&D SYSTEM), 10 ng/mL Recombinant Human IL-3 (cat.# 203-IL-010, R&D SYSTEM), 2 U/mL Recombinant Human Erythropoietin (EPO) (cat.# 2877-TC-500, R&D SYSTEM), 40 ng/mL Recombinant Human Insulin-like Growth Factor 1 (IGF-1) (cat.# 291-G1-200, R&D SYSTEM), and 1 μg/mL dexamethasone (cat.# D2915 Sigma-Aldrich Corp., St. Louis, MO, USA). The PBMCs were maintained and cultured at concentration of 2 × 10⁶ cell/mL 12 days at 37 °C, 5% CO₂. Approximately at D12-D14 days the PBMCs enriched with erythroblastic population were used for reprogramming.

IMC-RON8 [33 (link)] and IMC-A12 [34 (link)] monoclonal antibodies were provided by ImClone Systems Corporation (Branchburg, NJ, USA) through a Material Transfer Agreement. Recombinant human MSP (Cat-No. 352-MS) was from R&D Systems (Minneapolis, MN, USA) and recombinant human insulin-like growth factor 1 (IGF1; Cat-No. I3769) and 5-Aza-2’-deoxycytidine (5-Aza-CdR, Decitabine; Cat-No. A3656) were from Sigma-Aldrich (St. Louis, MO, USA).