Filipin

The antibodies used in this study were Alexa Fluor 546 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, v5 (Life Technologies), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), FLAG (Sigma-Aldrich, St. Louis, MO, USA), STIP1 mouse (Abnova, Taipei, Taiwan), and STIP1 rabbit (Gentex, Taipei, Taiwan). The procedures used for the production and purification of the v5-tagged rhSTIP1 proteins have been previously described in detail.³¹ (link) The FLAG-Rev peptide consists of a FLAG tag and the Rev peptide, which is a 13-amino-acid fragment of the HIV and has high affinity with the B23 protein.⁵⁰ (link) The commercial sources of the chemicals were as follows. Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada). CellLight Early Endosomes-GFP BacMan 2.0 (C10586) and CellLight Late Endosome-RFP BacMan 2.0 (C10589) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cells were grown on glass coverslips, and fixed in 10% formalin, quenched with 100 mM glycine. A total of 0.05 mg/mL filipin (Santa Cruz, Dallas, TX, USA, #480-49-9) was used in 10% normalized goat serum in PBS and stained for 1.5 h, mounted with Prolong Diamond Antifade Mountant, and imaged using an Olympus BX40 microscope and a 40× objective.

To enrich membrane cholesterol, cancer cells were exposed to methyl-β-cyclodextrin (MβCD, Santa Cruz Biotechnology) solution saturated with cholesterol^{61 (link)} for 30 min before addition of trastuzumab. To inhibit caveolae-mediated pathways, cells were incubated with 2.5 mM MβCD or 0.5 μg mL⁻¹ filipin (Santa Cruz Biotechnology) for 30 min before addition of trastuzumab. For in vitro experiments with lovastatin, cells were incubated with 25 μM of the active form of lovastatin (Millipore) for 4 h prior addition of trastuzumab^{40 (link)}. To determine whether mevalonic acid treatment rescued the lovastatin effect, the cell lines were treated with 25 μM lovastatin and 200 μM of R-Mevalonic Acid (Santa Cruz Biotechnology) for 4 h^{62 (link)}. Cells were then treated with 1 μM [⁸⁹Zr]Zr-DFO-trastuzumab for 1.5 h.