8 well chamber slide

Primary BECs were characterised by positive staining of E-Cadherin (Abcam 15148, Abcam, Cambridge, UK), pan-cytokeratin (sc-8018, Santa Cruz Bio technology, Santa Cruz, CA, USA), or cytokeratin-14 (Abcam 9220) and by negative staining for fibronectin (Abcam 23751) as described earlier [19 (link)]. In brief, cells were grown on 8-well chamber slides (Sarstedt, Sevelen, Switzerland) until 80% confluence and fixed for 2 × 5 minutes in 4% formalin. Cells were permeabilised for 5 minutes in PBS containing 0.1% TWEEN-20 and 0.05% Triton-X100 before being washed 3x with PBS-T (PBS + 0.1% Tween-20), and unspecific binding was blocked by 30-minute incubation in 2% skim milk in PBS-T and overnight incubation with one primary antibody. The slides were then washed 3x with PBS and incubated for 30 minutes at room temperature with a fluorescence labelled antibody specific for the species of the primary antibody (anti-mouse cat# R37114, anti-rabbit cat# R37119, Thermo Fisher). Slides were washed 3x with PBS before being monitored for fluorescence on an EVOS live-cell imaging station (Thermo Fisher).

Cells from mammospheres, attached to 8-well chamber slides (Sarstedt, Newton, NC, USA) and incubated or not with 1 mM melatonin at 37°C for 24 h, were fixed immediately in 4% paraformaldehyde and permeabilized 0.4% Triton X-100 for 20 minutes. Cells were blocked with 10% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA) and then incubated with the antibodies E-cadherin (1:400 Abcam), N-cadherin (1:400 Abcam), Vimentin (1:100 Sigma Aldrich, St. Louis, MO, USA) and OCT-4 (1:1000 Abcam) at 4°C overnight, followed by incubation with secondary Alexa Fluor 488 anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) per 1 hour at room temperature. Nuclear staining was performed by 4',6-diamidino-2-phenylindole (DAPI, Life Technologies, Eugene, OR, USA) and mounted with Prolong Gold (Life Technologies, Eugene, OR, USA). Images were captured on a confocal microscopy (ZEISS, modelo LSM 710, software ZEN 2010, Thornwood, NY, USA). The spheroids were measured at 405 nm for nuclei staining and 488 nm for cytoplasmic staining.

The RV-16 strain used had been described earlier, and the same RV-16 stock was used [19 (link)]. Based on preliminary experiments, epithelial cells were infected with 0.1, 0.5, or 1 MOI (multiplicity of infection) of RV-16 for up to 3 days. The infection rate was determined by immunofluorescence staining using an anti-RV-16 antibody (cat# 18758, QED-Bioscience Inc. San Diego, USA) as described earlier [19 (link)]. In brief, epithelial cells were seeded into 8-well chamber slides (Sarstedt) and allowed to adhere overnight. The cells were then treated for 24 hours with OM-85 or Pulmonarom before being infected with 0.1, 0.5, and 1.0 MOI of RV-16 for up to 72 hours. The cells were fixed in 4% formalin (in PBS, 5 min), washed 2x with PBS, and permeabilised by 0.05% Triton X-100 in PBS (15 min). After blocking with 2% bovine serum albumin (30 min), cells were incubated overnight (4°C) with anti-RV-16 antibody (1 : 100 dilution). Following 3x washes (PBS), the slides were incubated with a second FITC labelled antimouse antibody (1 hour, room temperature) and then washed 3x with PBS. The number of RV16 positive cells was counted by immunofluorescence microscopy (EVOS FLoid cell imaging station, Thermo Fisher Scientific).

Mouse neuroblastoma cells (N2a) were grown at 37C in a humidified incubator chamber under 5% CO₂ in OptiMEM (Gibco), supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin, and 1% streptomycin (Gibco). Cells were plated into 8-well chamber slides (Sarstedt) at a density of 30,000 cells/well and transiently transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions and imaged 24 h later.
Primary cortical neurons were prepared as previously described [8 (link)]. Briefly, neurons were obtained from embryonic day 14 (E14) CD1 (Charles Rives Laboratories) mouse embryos. Neurons were dissociated using Papain dissociation system (Worthington Biochemical Corporation, Lakewood, NJ, USA). Cells were plated in 8-well chamber slides previously coated with poly-D-lysine at a density of 30,000 cells/well and were maintained for 10–14 days in vitro (DIV) in Neurobasal medium supplemented with 2% B27 (Gibco), 1% penicillin/streptomycin (Gibco) and 1% glutamax (Gibco) in a humidified 37 C incubator with 5% CO₂ without further media exchange. Neurons were either transfected using Lipofectamine 2000 (Life Technologies) or infected with AAV.hSyn.mt-roGFP after 12–14 DIV, and imaged 1 or 3 days later respectively. Experiments were performed after a culturing period of 12–14 DIV.

Freshly isolated acinar cells were incubated for 30 min with supplemented Medium-199 in 48-well plates (Greiner Bio-One, Hungary), then AZA containing (0.1–1-10–100-1000 μg/mL) media was added, and cells were incubated for another 60 min at 37 °C. Cerulein (100–1000 nM) was applied as positive control. We determined the intracellular ATP content, which is proportional to the number of viable cells, using the CellTitre-Glo 3D (Promega) luminometric assay on a CLARIOStar plate reader. The total protein amount was determined using by Bradford assay in Spectrophotometer (Thermo Scientific™ NanoDrop™ One). Blank-corrected data were normalized to the total protein amount.
In addition, living, apoptotic, and necrotic cells were stained in 8-well chamber slides (Sarstedt, Germany) with Apoptosis-Necrosis Kit (Abcam) and were visualized under a Zeiss LSM880 confocal microscope using a 40× oil immersion objective (Zeiss, NA: 1.4). The total number of cells was calculated in FIJI (NIH, USA) using the built-in Cell Counter plugin and the ratio of the live, apoptotic and necrotic cells were given as the % of the total cell number.

PNT1A cells were seeded at a density of 2.5 × 10⁴/well in 8-well chamber slides (Sarstedt, Nürnbrecht, Germany). After three different times of treatments (30 min, 2 h, and 4 h), cells were fixed with ice-cold methanol for 5 min at RT, washed in PBS, and blocked in 1% bovine serum albumin (BSA) in PBS–Tween20 for 30 min. Afterwards, cells were incubated with the following primary antibodies overnight at 4 °C: ERα (Santacruz, Dallas, TX, United States, sc8005, mouse monoclonal, 1:100) and AR (Abcam, Cambridge, United Kingdom, Ab74,272, rabbit polyclonal, 1:300). The following day, cells were incubated with secondary antibody diluted in PBS. Secondary antibodies were Goat anti-Mouse IgG (H&L) DyLight^® 488 Conjugate (Immunoreagent Raleigh, North Carolina NC, United States, GtxMu-003-D488NHSX, 1:250) and Goat anti-Rabbit IgG (H&L) DyLight^® 594 Conjugate (Immunoreagent Raleigh, North Carolina NC, United States, GtxRb-003-D594NHSX, 1:250). Nuclei were stained with Höechst (Thermo Fisher Scientific, Waltham, MA, United States) [98 (link)]. All morphological observations were carried out using a Zeiss Axioskop microscope, and images were acquired by using an Axiovision 4.7 Software (Zeiss, Oberkochen, Germany) epifluorescence microscope.