Ecn 631 1577

HEK293T cells (7 × 10⁴) were seeded on glass coverslips (ECN 631–1577; VWR International, Radnor, PA, USA). Cells were washed in PBS, fixed with ice-cold 100% methanol for 10 min, and washed again. Coverslips were blocked for 30 min with PBS buffer with 0.1% Tween-20 containing 3% bovine serum albumin (A 9647; MilliporeSigma) and then incubated overnight at 4°C with primary antibody (LC3 A/B, 4108S; Cell Signaling Technology, Danvers, MA, USA) at 1:200 dilution in 1% bovine serum albumin containing PBS buffer with 0.1% Tween-20. Samples were washed with PBS and then incubated for 1 h at room temperature with Alexa Fluor 488 (4412S; Cell Signaling Technology) secondary antibody at 1:600 dilution. Cells were washed with PBS and treated with DAPI at 1:1000 dilution in PBS for 10 min, followed by wash with PBS. Coverslips were mounted by using FluorSave Reagent (345789; MilliporeSigma) and visualized by using a Zeiss LSM 710 laser confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Three images per given conditions were taken. All data are expressed as means ± sem. Comparisons between groups were made by using ANOVA with Tukey’s multiple comparison post hoc test. Statistical significance was evaluated, and values of P < 0.05 were considered significant.

CCD-1137Sk human fibroblasts were kept in Iscove’s Modified Dulbecco’s Medium (Capricorn; IMDM-A; Lot #CP18-2245) supplemented with 10% fetal bovine serum (Capricorn; FBS-12B; Lot #CP16-1422), and 1% penicillin/streptomycin (Capricorn; PS-B; Lot #CP18-2207). HT-29 human colon cancer cells were cultivated in McCoy’s 5a media (Capricorn; MCC-A; Lot #CP19-2689), supplemented with 10% FBS-12B and 1% PS-B. The media for MDA-MB-231 human breast cancer cells consisted of Dulbecco’s Modified Eagle Medium (Capricorn; DMEM-HPA; Lot #CP18-2096) supplemented with 10% FBS-12B, 1% PS-B, and 1% Minimum Essential Medium Nonessential Amino Acids (Capricorn; NEAA-B; Lot #CP17-1726). All cell lines were passaged twice per week with a seeding density of 1 × 10⁶ cells/T75 flask. Two-dimensional cell culture analysis was based on cover slips (12 mm; VWR; ECN 631-1577; Lot #43395 819) placed in cell culture dishes (Greiner; PS; 664 160) before seeding a total of 1.2 × 10⁶ cells in appropriate ratios and culturing for four days.