Reagents and antibodies

Citalopram, dimethyl sulfoxide (DMSO), foetal bovine serum (FBS), as well as trypan blue and MTT, were obtained from Sigma (St. Louis, USA). Dulbecco’s (DMEM) Medium as well as penicillin/streptomycin. HEPES buffer solution, gentamicin, and 0.25% trypsin–EDTA were also provided by Sigma (St. Louis, USA). The primary antibodies for B-actin, Bcl-2, BAX, cleaved caspase-9, cleaved caspase-3, Cytochrome c, and p21 were brought from Santa Cruz (Dallas, TX, USA). For Secondary antibodies, we used horseradish peroxidase-labelled immunoglobulin G (IgG) from DAKO (Darmstadt, Germany).

Cell culture

The HEP-2 cell line was purchased from ATCC, Rockville, MD, USA. The cells were allowed to grow in DMEM with heat inactivated FBS (10%). HEPES buffer, penicillin (100 U/ml), streptomycin (100 g/ml), l-glutamine (1%), and gentamycin (50 g/ml), were added to cells, then they were kept in a humid environment at 5% CO2 and 37 °C.

Cell viability assessment

Cells were seeded in a 96-well plate with a density of 1 × 104 cells per well. Fresh media containing various citalopram concentrations was added 24 h after plating. The MTT test was used to calculate the viable cell yield after incubation. After replacing the medium with 100 ml of fresh medium. 10 ml of the 12 mM 3-1-2,5-diphenyltetrazolium bromide (MTT) solution was added to the well, then the plate was incubated for 4 h. After removing the medium, the formazan salt was dissolved in DMSO. Finally, the optical density was obtained at a wavelength of 550 nm. GraphPad Prism software. (San Diego, CA, USA) was used to calculate the 50% inhibitory concentration (IC50) of the drug.

Apoptosis assay using annexin V and PI

The proportion of the apoptotic cells was further determined by annexin V and PI. HEP-2 cells were treated with citalopram, then collected after 48 h and washed twice in PBS for 20 min. HEP-2 cells were re-suspended in 100 µl of the binding buffer (10 mm HEPES/NaOH (pH 7.4), with 1 µl of FITC-annexin V added, followed by 40 min of incubation at 4 °C with propodium iodide (PI) (1 µg/ml in PBS). The cells were then examined with the BD FACS (BD Biosciences, CA).

Cell cycle analysis

First, after seeding HEP-2 cells, the cells were synchronized before measuring the cell cycle distribution using the Cycle TESTTM PLUS DNA reagent kit. Cell synchronization achieved by serum starvation through depriving cells of growth factors that can arrest them in G1 phase. After treatment with various doses of citalopram for 48 h, the cells were harvested and collected after washing with 10 ml PBS and centrifuged 5 min at 200 × g. Then the supernatant removed and cells resuspended in 0.5 ml PBS and 4.5 ml pre-chilled 70% cold ethanol (−20 °C) were added in a drop wise manner to the cell suspension while vortexing to ensure fixation for all cells and avoid cell clumping. After fixation, cells were washed by PBS, then treated with RNase (DNase free). Finally, the HEP-2 cells were stained using PI dye. PI binds to DNA, and during flow cytometry, the DNA will emit a fluorescent signal which is vary depending on the amount of DNA in the cell. The cells that are in the S-phase would have more DNA than cells in G1. Cell Quest software was used to compute the cell cycle distribution.

Western blot analysis

SDS Page was carried out as instructed. Briefly, cells were lysed in cold lysis buffer NP40 with 1:300 protease and phosphatase inhibitor cocktail tablets (Sigma/Roche, respectively) for 10 min on ice. Then, the Bradford method was used to measure the concentration of proteins in the supernatant. SDS loading buffer was mixed with 20 µg of total protein and heated to 95 °C for 10 min. After cooling, the mixture was electrophoresed by SDS PAGE using a Cleaver electrophoresis unit (BioRad). Before the incubation with primary antibodies, the membrane was blocked with 5% non-fat dried milk in TBS-T. The horseradish peroxidase-labelled secondary antibodies (Dako) were then applied to the membrane. The band intensities were evaluated after the chemiluminescent signals were recorded by a camera-based imager.

Light microscope examination

The cells were fixed on the plate through 15 min incubation in 10% formalin at room temperature. After that, crystal violet was used to dye the cells for 20 min. After removing the stain and rinsing the panels with deionized water, they were air-dried. The morphological alterations in treated cells as compared to the control cells were captured by an inverted microscope at a 400× magnification.

Electron microscope examination

After 48 h of treatment, the cells were harvested and the cell pellets were first fixed for 1 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature, and post-fixed for 1 h in osmium tetroxide (1%) in the same solution. After that, the samples were washed three times (15 min each) in cacodylate buffer, dehydrated in 10% increments, and finally, ethanol series up to absolute ethanol. Using the programmable tissue processor model LEICA EM TP, fixation and dehydration processes were carried out through a graded sequence of infiltrations until the samples were lying in clean epoxy resin. The samples were then moved to a 60 °C oven for 72 h to polymerize before being allowed to reach 25 °C desiccators for 24 h. Blocks that were mounted were cut with razor blades to create a trapezoidal shape that was less than 1 mm wide and height. Using an Ultracut UCT Ultramicrotome (Leica, Austria) and a freshly prepared glass knife to assure cleanliness and sharpness, ultrathin slices (60 nm) were cut. The incredibly tiny parts were placed on 2.05 mm copper hex grids. After that, sections were doubly stained by uranyl acetate and lead citrate. A JEOL 1010 Transmission Electron Microscopy (Tokyo, Japan) was used to read stained sections at 80 kV, and a Hamamatsu digital camera C4742-57-12NR (Hamamatsu, Japan) was used to take digital pictures.

Statistical analysis

At least three different experiments were conducted and the mean and standard deviation (SD) for each piece of data were displayed. To examine the statistical differences, the Student’s t-test was used. The analyses were carried out using the SPSS 21.0 software package (SPSS Inc., Chicago, IL, USA, version 21.0). The differences between the studied groups were considered to be significant at P ≤ 0.05.