Sd208

PLGA-based nanoparticles were prepared using single-emulsion evaporation. PLGA (AP041, acid end-capped, 50:50, 10–15 kDa, Akina) was blended with Mal-PEG-PLGA (AI53, diblock copolymer, 50:50, 5–10 kDa, Akina) at 25% w/w. The polymers were dissolved in 1 mL dichloromethane (Sigma) and added to 6 mL of ice-cold 0.25% PVA (30,000–70,000 g mol⁻¹, Sigma) in 50 mM phosphate buffer, pH 5.8. The two phases were emulsified using a sonic probe (Qsonica Q700 with microtip, amplitude 10, 3 s power with 2 s break). SD-208- and R848-loaded nanoparticles were prepared by adding 10% (w/w) SD-208 (Selleckchem) or 40% (w/w) R848 (Sigma) to the solvent/polymer phase. The emulsion was stirred at room temperature for 3 h to evaporate the dichloromethane and afterwards purified by two wash-spin cycles in PBS at 20,000 g for 10 min. Nanoparticles were assessed for size distribution and zeta potential using a Zetasizer Nano series ZS90, and drug encapsulation was determined by absorbance at 370 nm (SD-208) or 280 nm (R848). The morphology of freeze-dried samples was assessed by Scanning Electron Microscopy (SEM; JSM-7800F Prime, Jeol, Japan).

Antibodies for E-cadherin, N-cadherin, vimentin, fibronectin, CREB1, pCREB1, pSMAD2/3, and CD44 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for TGFβ1, TGFβ2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody for SRGN and anti-Rabbit FITC second antibody were purchased from Abcam (Cambridge, MA, USA). The recombinant human TGFβ1 and TGFβ2 protein was obtained from R&D Systems (Minneapolis, MN, USA). The TGFβ receptor I kinase inhibitor SD208 was from Selleckchem (Houston, TX, USA) and MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) was from Promega (Madison, WI, USA). siRNA of CD44 (siRCD44) was purchased from RiboBio (Guangzhou, China) and transformed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

For intracellular staining of phosphorylated SMAD (pSMAD), cells were fixed in 1 mL of Lyse-Fix buffer (BD Bioscience, San Jose, CA, USA) for 10 min at 37 °C and then permeabilized on ice in permbuffer (BD Bioscience) for 30 min. Samples were washed with PBS containing 0.5% BSA and stained with 2 µL antibody per sample. For the assessment of inhibitor function, cells were seeded in 24-well plates at a density of 0.35 × 10⁶/mL and treated with 5 ng/mL TGF-β in the presence or absence of varying concentrations of inhibitors. Apoptosis was measured with a PE-AnnexinV/7AAD apoptosis detection kit (BD Bioscience) according to the manufacturer’s instructions after overnight treatment with TGF-β (5 ng/mL; Immunotools), galunisertib (3.5 µM) or SD208 (0.5 µM) (Selleckchem, Housten, TX, USA) in the presence or absence of 100 µM ARA-C. For cell cycle staining, 0.25 × 10⁶ pelleted cells were resuspended in 250 µL PBS/EDTA (1 mM). Samples were fixed by the addition of 750 µL absolute ethanol and overnight incubation at 4 °C. Prior to staining, cells were washed in PBS and treated with 100 µg/mL RNAse A in a total volume of 475 µL PBS/EDTA for 20 min at 37 °C. Propidium iodide (Sigma) was added to a final concentration of 50 µg/mL to distinguish between cell cycle phases in terms of cellular DNA content.