Ly294002 hydrochloride

Recombinant human CX3CL1 was obtained from Peprotech. Recombinant human IL-6 protein was obtained from R&D systems. Acriflavine (HIF-1 inhibitor), KN-93 (CaMKII inhibitor), GSK690693 (pan-Akt inhibitor), BAY 11-7082 (NF-κB signaling inhibitor), U73122 hydrate (pan-PLC inhibitor), Stattic (STAT3 inhibitor), LY-294002 hydrochloride (PI3K inhibitor), Sodium oxamate (LDHA inhibitor), pertussis toxin (PTx,Gα_i/0 inhibitor), H-89 (PKA/ROCK inhibitor), Y-27632 (ROCK inhibitor) and CoCl₂ were all obtained from Sigma-Aldrich. Rapamycin (mTORC1 inhibitor) was purchased from Cayman Chemical. 17-AAG (HSP90 inhibitor) and P6 (pan-JAK inhibitor), Bisindolylmaleimide I (PKC inhibitor) were purchased from Calbiochem. The Gα_q-inhibitor UBO-QIC was purchased from the Institute of Pharmaceutical Biology, University of Bonn. Gallein (βγ-subunit inhibitor) was obtained from Tocris.

PBLs, PBMCs, or tumor cell suspension obtained as previously described were incubated with LNCs at the final DiD concentration of 50 ng/ml for 90′, 3 h, or overnight at 37 °C. As control, Blank-LNC formulations were used. 10 µg/ml Cytochalasin B (Sigma-Aldrich), 100 µg/ml Colchicine (Sigma-Aldrich), 50 µM LY294002 hydrochloride (Sigma-Aldrich), 100 nM Wortmannin (Sigma-Aldrich), and 100 U/ml Nystatin were used to test the mechanism of internalization. At the end of the incubation, cells were stained for flow cytometry analysis. Further details are reported in Additional file 1: Supplementary methods.

Primary antibodies used in this work were β-actin (Abcam, ab8226), SNAI1 EC3 (ref^{20 (link)}.), PY99 (Santa Cruz, sc7020), PDGFR-β (Cell Signaling, 3169), ERK1/2 (Cell Signaling, 9102), p-ERK1/2 (Cell Signaling, 9109), Akt (Cell Signaling, 4691), p-Akt (Cell Signaling, 4060), FAK (Cell Signaling, 3285), p-FAK (Cell Signaling, 3281), VE-Cadherin (Abcam, ab33168), VCAM-1 (Abcam, ab98954), MMP-9 (Abcam, ab38898), Snail (ref^{20 (link)}.), MT1-MMP (Abcam, ab51074), CD31/PCAM-1 (1:10, SC-506) Fibronectin (DAKO, A0245), Beta 1 integrin (Cell Signaling, 34971 S), Beta 3 integrin (Cell signaling, 13166 S), NF-KB p65 (Cell signaling, 8242 S), Phospho-NF-KBp65 (Cell signaling 3033 S) and Collagen I (Abcam, ab34710). Secondary antibodies used were anti-Mouse or anti-Rabbit IgG Antibody DyLight™ 680 or 800 Conjugate.
Other compounds used were PDGF-BB (Peprotech, 100-14B), BAPN: 3-Aminopropionitrile fumarate salt (Sigma, A3134), FAK inhibitor, PF-573228 (PZ0117, Sigma), ERK inhibitor, U0126-monoethanolate (Sigma, U120) and PI3K/Akt inhibitor LY294002 hydrochloride (Sigma, L9908).

Aripiprazole, sertindole, and ketamine were purchased from Chemipharm Pharmaceutical Industries (Sixth October City, Egypt), H. Lundbeck A/S (Copenhagen, Denmark), and Trokiaa Pharmaceuticals Ltd. (Ahmedabad, India), respectively. Aripiprazole and sertindole were dissolved in a minimum amount of 0.1-M hydrochloric acid and diluted with saline, and then administered (1 ml/200 g of body weight) orally (p.o.). Ketamine was diluted in normal saline and administered (0.1 ml/200 g of body weight) intraperitoneally (i.p.). PI3K inhibitor, LY294002 hydrochloride, was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and dissolved it in 1% DMSO. All other chemicals were of the highest commercially available purity grade.

GLP1 was obtained from Novo Nordisk A/S (Bagsvaerd, Denmark). Insulin and the C-peptide assay (enzyme-linked immunosorbent assay (ELISA)) kit were from Mercodia (Uppsala, Sweden). The anti-insulin mouse monoclonal antibody (SAB4200691) and anti-forkhead box O 1 (FOXO1) antibody (AV32107) were from Sigma-Aldrich. The anti-glucagon antibody (2760S) was from CST Biological Reagents Company Limited (Shanghai, China). The anti-mast cell function-associated antigen B (MAFB) antibody (DF8895) and anti-PDX1 antibody (DF7170) were from Affinity Biosciences (Cincinnati, OH, USA). The anti-neurogenin 3 (NGN3) antibody (GTX60254) was from GeneTex Inc. (Irvine, CA, USA). The primary antibodies against MAFA (sc-390491), pan-Cytokeratin (sc-8018), AKT1/2/3 (sc-81434), exendin (9-39) (SC-364387), and amylase were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The PI3K inhibitor LY294002 hydrochloride was also from Sigma-Aldrich.