1.4 na oil objective lens

Microscopic imaging was performed using confocal laser-scanning microscopes (LSM 700 and LSM 800; Carl Zeiss) and a 63× 1.4 NA oil objective lens (Carl Zeiss). Time-lapse microscopy was performed in MCF10A medium at 37°C in a CO₂-humidified incubation chamber (Pecon, CO₂ module S1) using ZEN software (Carl Zeiss).
To induce PM blebbing and entotic invasion, cells were plated on 35-mm glass-bottom dishes (In Vitro Scientific) coated with 12% poly-HEMA wt/wt solution in ethanol to prevent cellular attachment as described previously by Overholtzer et al. (2007) (link).
To quantify the number of entotic events, MCF10A cells were trypsinized and plated on Ultra Low cluster plates (3473; Costar) at densities of 300,000–400,000 cells per well. After 4 h, cells were fixed directly in suspension using 4% formaldehyde/PBS for 10 min. After washing, the cells were rehydrated in PBS and seeded onto 12-mm coverslips on a heating block at 60°C for 5 min. Fixed samples were washed in PBS and permeabilized by 0.05% PBS-Tween for 10 min. Nuclei were stained using DAPI (Sigma-Aldrich) at 1:10,000 for 20 min at RT, and F-actin was labeled by Alexa Fluor 555–phalloidin or Alexa Fluor 488–phalloidin (Invitrogen) at 1:1,000 overnight at 4°C.

HeLa or HeLa HF1–3 cells expressing GFP or A3′SS-T2-CP were grown on glass coverslips for 24 h and transfected with 300 ng TetR-mCherry plasmid using 2 µL of Lipofectamine 2000 (Life Technologies) according to manufacturer instructions. Two hours after transfection, the medium was changed to DMEM with or without 50 µM doxycycline and further incubated at 37°C and 5% CO2 for 24 h. Next, cells were fixed in 3.7% formaldehyde at room temperature (RT) for 10 min followed by permeabilization with 0.5% triton X-100 in 1×PBS for 10 min at RT. Cells were then washed three times with 1× PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) dye (1 µg/mL in water; Thermo Scientific) and washed three times with 1x PBS. Finally, coverslips were mounted with mounting medium (Thermo Scientific). All experiments were carried out on a Zeiss Axiovert 200 M inverted microscope using a 63×/1.4 NA oil objective lens (Zeiss). Excitation was done using the mercury arc lamp (Zeiss). The following filters were used: 350/50 (excitation) and 460/50 (emission) for DAPI, 482/18 (excitation) and 520/28 (emission) for GFP, and 565/30 (excitation) and 620/60 (emission) for mCherry. Images were repeated three times. Data processing was performed using ImageJ.