Polyethylenimine (pei)

Each culture dish was first coated with a solution of branched polyethylenimine (PEI, 25,000 g/mol, Sigma-Aldrich) (15 mg/L, in autoclaved water) for 30 min at room temperature; then the PEI solution was removed and the culture dish coated with PEI was washed with autoclaved water three times; and finally a microgels dispersion (5 mg/mL) was added to coat the surface for 6 h in an incubator under a humidified atmosphere of 5% CO₂ in air at 37 °C. Afterwards, the microgels dispersion was removed and the microgels-coated culture dish was washed three times using phosphate-buffered saline (PBS).

HEK293 cells were transiently or stably transfected and MDCK-C7 cells were stably transfected with WT or mutated CLDN10b vectors containing a puromycin or neomycin resistance using PEI (Polyethylenimine, Sigma-Aldrich). Transiently transfected HEK293 cells were used for confocal laser microscopy after 24 or 48 hours. For stable transfection, MDCK-C7 cells were treated with puromycin (10 μg/ml, Sigma-Aldrich) or G418 (1000 μg/ml, Biochrom, Berlin, Germany), respectively. After 2 weeks, G418 resistant clones were picked with cloning-cylinders and the cells were further cultured with 600 μg/ml G418. puromycin-resistant MDCK-C7 cells were pooled after 7 days, grown for further 10 days, seeded onto Millicell cell culture inserts (pore size 0.45 μm, effective area 0.6 cm²; Millicell-HA) and grown for further 5 days before they were mounted in an Ussing chamber. In contrast, HEK293 cells were treated with 600 U/ml G418 (Biochrom, Berlin, Germany) for 4 weeks and the resistant cells were further cultured in the presence of 150 U/ml G418.

The cell lines HL-60 VASP-EGFP and HL-60 EGFP were generated through retroviral transduction using a pMSCV-EGFP-VASP plasmid kindly donated by Matthias Krause (King’s College, London, UK). To generate the pMSCV-EGFP plasmid, pMSCV-EGFP-VASP was cut and ligated to remove the VASP sequence. Correct ligation was assessed by DNA sequencing.
Retroviral particles were produced in the packaging HEK 293T cell line through transfection using 9 µg of polyethylenimine or PEI (Sigma) complexes with 3 µg of total DNA per 200,000 cells. Packaging, envelope and vector plasmid proportions were maintained as per the manufacturer’s recommendations (2:1:3; pCMV-GP/pCMV-VSV-G/vector). The supernatants containing retroviral particles were harvested and added to HL-60 cells grown to log phase and treated with polybrene (Sigma) to a final concentration of 8 μg/mL Cells were then centrifuged (2200 rpm, 90 min), grown overnight, and afterwards, cultured normally. Transfection was monitored through fluorescence microscopy. Afterwards, cells were sorted according to fluorescence at the cell sorting service at CBMSO (CSIC, Madrid, Spain) and cultured normally until a stable cell line had been established. The characterization of these cells was done via Western blotting.

HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Waltham, MA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% Penicillin–Streptomycin (Gibco, Waltham, MA) in a 37 °C incubator with 5% CO₂. Cells were transiently transfected using polyethylenimine (Sigma-Aldrich, Saint Louis, MO) in a 1:5 ratio (DNA:polyethylenimine) for 48 h prior to performing the experiments. To induce mitochondrial depolarization, HeLa cells were treated with 10 µM CCCP (Sigma-Aldrich) for the indicated times, prior to cell harvesting or fixation. DMSO was used as a control treatment.

Tetraethylorthosilicate (TEOS), tris(2,2′-bipyridine)dichlororuthenium(II) hexahydrate (RuBpy), hexadecyltrimethylammonium bromide (CTAB), di(N-succinimidyl) 3,3′-dithiodipropionate (DSP), dithiothreitol (DTT), polyethylenimine (25 kDa branched PEI, Mw ~25,000 Da), polyethylenimine (800 Da branched PEI, Mw ~800 Da), penicillin, and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin hydrochloride (Dox) was obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, China). Cell counting kit-8 was purchased from Dojindo Molecular Technologies (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco BRL (Grand Island, NY, USA). Survivin siRNA and Cy3-labeled siRNA were synthesized by Ribo Biochemistry (Guangzhou, China). Anti-survivin antibody and goat anti-rabbit IgG (H + L) HRP were purchased from Bioworld Technology, Co., Ltd. (Nanjing, China). Deionised water (H₂O) was purified by a Millipore system (Milli-Q, 18.2 MΩ·cm, NANOPure, Barnstead, NH, USA). All other chemicals were from commercial sources and of analytical reagent grade, unless indicated otherwise.