P4170 10mg

Adherent cells were harvested by trypsinization. Detached cells and trypsinized cells were pooled, rinsed and fixed in 70% ethanol as previously described [16 (link)]. Fixed cells were treated with propidium iodide (20 µg/mL, P4170-10MG, Sigma Aldrich, Saint-Louis, MO, USA) and RNase (100 µg/mL, 12091-021, Fisher Scientifics SAS, Illkirch, France) as previously described [16 (link)]. DNA content was analyzed using the Cytoflex S Flow Cytometer (Beckman Coulter France SAS, Paris, France). Data were analyzed by Cytexpert^® acquisition software (Beckman Coulter France SAS, Paris, France).

Pluronic^® F-127 (NP) and thiol-terminated Pluronic^® F-127 (NP-S-S) micelles were fabricated using thin-film hydration method described by Caldwell et al. [63 ]. The polymer constituent and 10 wt/wt.% SAHA (Cayman Chemical Company, 10009929, Ann Arbor, MI, USA)/propidium iodide (Sigma-Aldrich, P4170-10MG) were dissolved in acetonitrile (ACN). The acetonitrile was removed by rotary evaporation at 65 °C and under 226 mbar for 1 h, and the flask placed into a desiccator under vacuum overnight, to ensure the film was void of all moisture and residual copolymer matrix acetonitrile removed. Prior to hydration, samples were heated to 65 °C in a water bath under mild rotation for 1 h until the sample resembled a viscous thin film coating the flask. NPs were hydrated in deionized water and returned to heated rotation for 30 min to ensure maximum micelle formation and drug loading. HA-thiol was added in excess (2:1) to NP-S-S and mixed using a magnetic stirrer at 700 rpm for 1 h to form NP-HA; prior to use HA-thiol was sterilized under UV for 1 h. An 0.22 or 0.45 μm filter (Millipore, Burlington, MA, USA) was applied to SAHA-NP and SAHA-NP-HA to remove non-encapsulated drugs agglomeration.
Synthesis of thiolated Pluronic^® F-127 (NP-S-S-Py).

Cells were seeded in 96-well plates at 1 × 10³ per well in 90 µL cell medium. Cell proliferation was assessed by Cell Counting Kit-8 (YEASEN, 40203ES80) according to the manufacturer's instructions. Briefly, 10 µL of CCK-8 solution was added to cell culture and incubated for 3-4 hours. Cell proliferation was evaluated by absorbance at 450 nm wavelength. For analysis of cell cycle, cells were plated in six-well plates at 6 × 10⁵ per well. After staining by propidium iodide (Sigma-Aldrich, P4170-10MG), the cell cycle distribution was analyzed by flow cytometry.

A stock solution of AS-10 was prepared by dissolving in DMSO at a 10 mM concentration, storing at –20 °C. Each stock solution was used within 7 days of preparation. The final DMSO concentration for all treatments was kept at 0.1%. Antibodies were ordered from the following commercial sources: Cell signaling (Danvers, MA, USA) (IκBα (9242s), Bcl-xL (2762s), Mcl-1 (5453s) and PARP (9542s)); Abcam (Cambridge, MA, USA) (P100/P50 (ab31412), GAPDH (5174p) and P65 (ab7970)); Santa Cruz Biotechnology (Dallas, TX, USA) (P21 (sc-397); Jackson Immuno Research (West Grove, PA, USA) (Alexa fluor 680 (711-625-152) and donkey serum (017-000-002)); and Sigma-Aldrich (St. Louis, MO, USA) (β-actin (a-5441)). Rabbit IgG-chip grade protein was ordered from Abcam (ab37415), while propidium iodide (P4170-10MG), and ribonuclease A (RNAse A, R6513-10MG) and crystal violet (C6158-50G) from Sigma-Aldrich. Gemzar (gemcitabine) was obtained from Penn State Hershey Medical Center pharmacy (Hershey, PA, USA).