Hela cells

HeLa cells (RIKEN BRC, Tsukuba, Japan) plated on a chambered glass slide (Matsunami, Osaka, Japan) were transfected with each GFP-MCD construct in the presence or absence of an MLO marker using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. Twenty-four hours after transfection, the cells were fixed with formalin-PBS, and coverslips were mounted using ProLong Gold-DAPI antifade reagent (Thermo Fisher Scientific). For ActD treatment, the transfected HeLa cells were treated with 100 nM of ActD (Sigma-Aldrich, St. Louis, MO, USA) for the indicated period. For the digitonin permeabilization assay, the transfected HeLa cells were treated with a buffer containing 10 mM HEPES (pH 7.4), 0.1% digitonin (Nacalai, Kyoto, Japan), 300 mM sucrose, and 100 mM or 200 mM NaCl for 10 min before fixation. The cells were imaged with FV10i confocal microscopy (Olympus, Tokyo, Japan). Image analyses were performed using the ImageJ Fiji software (http://rsbweb.nih.gov/ij, accessed on 6 July 2022).

HeLa cells (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; MP bio, Ringmer, UK), 50 mg/ml penicillin and streptomycin (regular medium) in a 5% CO₂ incubator at 37 °C. Tetracycline-on (Tet-on) cells were generated by lentiviral transduction with a pCW57.1 vector (Addgene plasmid 41393, David Root lab) containing a single-vector Tet-on component and cultured in the presence of 1 µg/ml doxycycline (Clontech, Mountain View, CA, USA) during induction. For starvation treatment, cells were washed with PBS and incubated in amino acid-free DMEM without serum (starvation medium (Wako)). The cells were treated with 0.03 µM bafilomycin A₁ (LC Laboratories, Woburn, MA, USA) to inhibit lysosomal degradation. The cells were incubated with 1 µM or 10 µM cytochalasin D (Cayman Chemical, Ann Arbor, MI, USA), 100 µM arsenite (Sigma-Aldrich, St. Louis, MO, USA), 20 µM CCCP (Nacalai Tesque), 2 µg/ml tunicamycin (Sigma), 5 µg/ml nocodazole (Cayman Chemical), 0.1 µg/ml latrunculin A (Cayman Chemical), and 1 µM cucurbitacin E (Cayman Chemical) to induce actin stress.

The SKG-I CC cell line was established by our group and deposited in the cell bank of the National Institutes of Biomedical Innovation (Japan). HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in our laboratory. These two cell lines are both HPV18-positive. To confirm the identity of the analyzed cell lines, we performed short terminal repeat (STR) genotyping that revealed a correspondence of >80% of the tested markers. SKG-I cells were cultured in Ham’s F-12 medium (Sigma-Aldrich, St. Louis, MI, USA) supplemented with 10% FBS and 1% penicillin–streptomycin. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS and 1% penicillin–streptomycin.