Filipin 3

A total of 3 × 10⁴ cultured cells were preseeded in 24-well plates. Then, the cells were harvested, fixed with 4% paraformaldehyde and incubated with 0.05 mg/ml filipin III (Sigma, F4767) working solution for 2 h at room temperature. Then, the cells were sealed with SYTOX Deep Red stain (Invitrogen, P36990). This dye is excited by red light at 660 nm when bound to DNA and has an emission maximum at 682 nm; we detected these signals using a Cy5/deep red traditional filter. filipin III staining of cells was visualized with a Leica DMI6000 B microscope at excitation wavelengths of 340–380 nm and emission wavelengths of 385–470 nm. The quantification of Filipin staining was performed by ImageJ (v1.8.0).

Cells were transfected and cultured as described above then fixed with 3% paraformaldehyde (PFA) at room temperature for 20 min, washed with PBS, incubated with 30 mM glycine for 5 min and washed again with PBS. For LDLR staining cells were permeabilized with 0.05% Filipin III (Sigma #F4767) in 10% FCS for 30 min at room temperature. Primary antibody: rabbit monoclonal anti-LDLR (Fitzgerald #20R-LR002) was diluted 1:100 in 5% FCS overnight at 4 °C. Secondary antibody: goat polyclonal goat anti-rabbit IgG Alexa 568 (Invitrogen #A11011) was diluted 1:400 in 5% FCS. Fixed cells were imaged using a Zeiss LSM 780 confocal microscope using a 63×/1.4NA oil immersion objective. For plasma membrane LDLR staining, cells were not permeabilized, but directly stained for 1 hour at 4 °C with antibody recognising the extracellular part of LDLR, namely, mouse anti-LDLR-C7 antibody (Progen #61087) diluted 1:100 in PBS. As a secondary antibody, we used chicken anti-mouse IgG Alexa 488 (Invitrogen #A-21200) diluted 1:400. Cells were imaged on an Olympus FluoView 3000 confocal microscope using 60×1.3NA silicon oil immersion objective.

Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).

Cell culture media and reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). These include Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin. A CellTiter 96 Aqueous One Solution Cell Proliferation Assay System was purchased from Promega (Madison, WI, USA). U18666A, Filipin III, 2-hydroxypropyl-β-cyclodextrin (HPβCD) and 2-hydroxypropyl-γ-cyclodextrin (HPγCD) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies used in this study are as follows: rabbit polyclonal antibody for mCherry (Abcam ab183628); rabbit monoclonal antibody for GAPDH (Cell Signaling #5174).

Curcumin (95% purity) was obtained from Alfa Aesar (Ward Hill, MA, USA); PLGA (Resomer RG 503 H) was supplied by Evonik (Essen, Germany); poly(vinyl alcohol) (PVA, Mowiol 4-88) was purchased from Kuraray (Hattersheim, Germany); polysorbate 80 (Tween 80), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2’,7’-dichlorofluorescin diacetate (DCFDA) and tert-butyl hydroperoxide (TBHP) were obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany). Inhibitors of cellular uptake, viz. Filipin III, chlorpromazine and dynasore were also obtained from Sigma Aldrich. Ultrapure water, generated by a PURELAB flex 4 device (ELGA LabWater, High Wycombe, UK) was used for all experiments. For cell culture studies, ultrapure water was additionally autoclaved and filter-sterilised using 0.2 μm polyethersulphone membrane filters (Sarstedt, Nümbrecht, Germany) prior to use. HPLC-grade ethyl acetate (VWR, Darmstadt, Germany) was used to prepare the organic phases. All other chemicals used were of analytical grade. All buffers used in this study were prepared in the laboratory, unless stated otherwise.