Chx41 microscope

The cells were seeded in 24- or 12-well plates and grown overnight. Treatments were performed in triplicate with different concentrations of nerolidol (25, 50, 60, 75, and 100 mg/L) indicated for each experiment. Incubation times were 0.5, 2, 4, 24, 48, or 72 h of treatment, as indicated for each experiment. For experiments involving inhibitors, cells were pre-treated with inhibitors for 1 h before nerolidol administration. Cell counting was performed using an Olympus CHX41 microscope (Olympus Corporation) using a Neubauer chamber. Cell viability was assessed by staining cells with trypan blue stain after obtaining a single cell suspension. Cells were counted as described before. Cells stained blue were considered not viable. Data are presented as a share of trypan blue-stained cells in a total cell count (100%) for each measurement.

T24 and TCCSUP cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (D6429, Sigma-Aldrich) with 10% fetal bovine serum (F0804, Sigma-Aldrich) and 100 U of penicillin/streptomycin (P0781, Sigma-Aldrich) in a humidified incubator at 37 °C in a 5% CO₂ atmosphere. Trypsin-EDTA (T4049; Sigma-Aldrich) was used for cell passage and division. For nerolidol treatment, nerolidol was mixed with the complete growth medium to obtain the indicated concentrations. For experiments involving the use of inhibitors, cells were treated with inhibitors dissolved in complete growth medium at the indicated concentrations for 1 h before nerolidol administration: H-89 (50 nM), z-Vad (20 or 40 μM), SB203580 (0.5, or 4 μM), DRN (10 μM), XSC (380 nM), KH7 (3 and 10 μM), and L755507 (50 pM or 600 nM). All chemicals were mixed with complete growth medium and treatments were applied by replacing the existing growth medium to ensure homogenous administration. For each treatment, an appropriate control was used, which was an equivalent amount of vehicle (usually DMSO). Microscopy was performed using an Olympus CHX41 microscope (Olympus Corporation, Tokyo, Japan) and cell photographs were taken using an SC50 camera (Olympus Corporation).

Immunofluorescence was performed on HeLa cells grown in a complete growth medium on sterilized coverslips. Cells were either untreated or treated with 400 μM of FA for 15, 30 and 60 min. After treatment coverslips were washed with cold PBS. Pre-extraction was performed twice with cold CSK buffer (10 mM HEPES–KOH pH 7, 300 mM sucrose, 100 mM NaCl and 3 mM MgCl₂) supplemented with 0.2% Triton X-100 for 10 min. Cells were washed 2 times with cold PBS followed by fixation in 2% paraformaldehyde in PBS for 20 min at room temperature. After 3 washes with PBS, permeabilization was performed with 0.15% Triton X-100 for 20 min. Coverslips were washed 3 times with cold PBS and then blocking was done in 3% BSA in PBS for 1 h. Coverslips were then incubated for 2 h with the SPRTN antibody (in-house) diluted in 3% BSA in PBS followed by washing 4 times with 0.05% Tween 20 in PBS. Secondary antibody (AlexaFluor 488 donkey anti-rabbit IgG (H + L), Invitrogen A21206) was diluted in 3% BSA in PBS, and coverslips were incubated for 1 h in the dark. After four washes with 0.05% Tween 20 in PBS, mounting medium (Mowiol) with DAPI (Sigma-Aldrich) was applied. Images were obtained using an Olympus CHX41 microscope (Olympus Corporation).