96 well clear bottom plates

Each of the five LIHC cell lines was seeded in 96-well, clear bottom plates (BD Biosciences, Franklin Lakes, NJ), with 5,000 cells in 200 μl of growth media per well. The cells were then treated with respective compounds in fivefold serial dilutions ranging from 100 to 0.0064 μM. After treatment period of 72 h, the media and compounds were removed and replaced with 100 μl of fresh growth media and 20 μl of CellTiter-96 AQueous One Solution Reagent (Promega, Madison, WI). After incubation for 2–4 h, absorbance was measured at 490 nm using the Powerwave XS microplate spectrophotometer (BioTek, Winooski, VT). IC₅₀s were calculated as an estimate of each compound’s efficacy. Three independent experiments were done, each in triplicates.

Each of the HCC cell lines was seeded in 96-well, clear bottom plates (BD Biosciences, Franklin Lakes, NJ), with 500 cells in 200 μL of growth media per well. The cells were then treated with either 0.5% DMSO alone (used as vehicle control) or with XAV939 or WXL-8 in a 2-fold dilution range (from 100 μM to 1.562 μM). After 7 days of incubation, the media and compounds were removed and replaced with 100 μL of fresh growth media and 20 μL of CellTiter-96 AQueous One Solution Reagent (Promega, Madison, WI). After incubation for 2 to 4 hours, absorbance was measured at 490 nm using the Powerwave XS microplate spectrophotometer (BioTek, Winooski, VT).

Cells were seeded onto 96-well clear bottom plates (BD Biosciences, Prague, Czech) at a density of 5000 cells per well. After cells were incubated with cell culture media (EMEM, 10% FBS) containing different concentrations of nanoparticles for 24 h. For lysosomal stability assessment, we utilized an acridine orange (AO) assay. The AO assay was performed in accordance with our previously verified protocol [6 (link),34 (link)]. Briefly, cells with incorporated nanoparticles were labeled with 5 µg mL⁻¹ AO in culture medium for 15 min at 37 °C. Following nanoparticle treatment, cells were cultured at 37 °C for indicated periods of time and the intensity of orange fluorescence was then measured using a microplate reader SpectraFluor Plus (TECAN, Mannedorf, Switzerland). Readings were done in quadruplicates. Three independent experiments were performed for each measurement. Normalized fluorescence data are presented as means ± SEM.
Additionally, we evaluated the lysosomal integrity microscopically. Cells were seeded in 6-channel µ-Slides (Ibidi, Martinsried) at density 15,000 cells per well. Then cells were stimulated with NPs for 24 h. After, cells were labeled with 5 µg mL⁻¹ AO in culture medium for 15 min at 37 °C and imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). As positive control treatment with 20 % ethanol for 10 min was used.