Trypan blue reagent

Cells were seeded (2.0 × 10⁴/6-well plates) and induced with DOX (1 μg/ml). After 48 h, in a time-course-dependent manner, both floating and adherent cells were collected, stained with 0.4% Trypan blue reagent (Sigma, St. Louis, MO, USA), and counted to determine cell proliferation and viability with hemocytometer. All the experiments were performed in triplicate.
To determine the percentage of apoptotic cell death along with ATG5 depletion, 25 μl of cell suspensions were mixed with 1 μl of dye mix (DAPI, 1 μg/ml+ethidium bromide, 100 μg/ml). The mixture was placed on a microscope slide, covered with a 22-mm² coverslip and slides examined with ‘Zeiss Axioskop 2 plus' fluorescent microscope. For each sample, 200 cells were counted and recorded as V (viable cells), NVN (non-viable cells with normal nuclei), and NVA (non-viable cells with apoptotic nuclei) characterized by highly condensed or fragmented nuclei. The % of apoptotic dead cells was then calculated as follow: % apoptotic cell=100 × NVA/(VA+NVN+NVA).

BCSCs, SDACs and BCSCs infected with shmiR-205-5p were seeded into six-well plate at 5 × 10⁴ cells per well. Viable cell count was performed with Trypan Blue reagent (Sigma-Aldrich) at the indicated time points. When indicated, cells were treated with 0.5 μM Lapatinib (Biovision, Milpitas, CA, USA).

Viability of Hep3B cells was assessed using Trypan Blue exclusion assays. In brief, cells were collected by trypsinization after transfection with either control vector or pMS2-lincRNA-p21 plasmids and stained with 0.4% trypan blue reagent (Sigma). The number of live cells was determined using a hemocytometer. All experiments were performed in triplicate.

Trypan blue, soft agar growth, and MTT assays were used to determine growth and survival. For trypan blue assay, cells were plated at a density of 1 × 10⁴ cells/well in 24-well plates for 2–3 days, and then trypsinized, combined with the Trypan blue reagent (Sigma, St. Louis, MO, USA), and cell numbers were counted. For soft agar assay, cells were seeded at a density of 10,000 cells/well in 96 well plate containing semisolid agar media. Transformation ability of these cells was measured using CytoSelect^TM 96-well Cell Transformation Assay (Cell Biolabs, San Diego, CA, USA) following 6–8 days incubation. Fluorescence was read on SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) using 485/520 nm. Briefly, cells growing in semisolid agar media were solubilized, lysed, and incubated with CyQuant GR Dye (Cell Biolabs) for measuring fluorescence. For cell proliferation, cells were seeded in triplicate at a density of 4 × 10³ per well in 96-well plate. Cell proliferation was detected following 72 h incubation essentially as described previously [20 (link)] by measuring absorbance at 570/650 nm.

After 72 h of doxycycline induction, 2 × 10⁵ OCI-AML 3 cells/well were seeded onto 12-well plates (Eppendorf, MI, Italy). Viable and dead cell counts were performed with Trypan Blue reagent (Sigma-Aldrich, St. Louis, MI, USA) at the indicated time points, considering 72 h post-induction with doxycycline as the starting point. For cell proliferation curves, only viable cells were considered as part of the total number counted for each well considered. The same assays were performed under treatments with different concentrations of etoposide for the time points indicated. For dose–response cell viability assays, 2 × 10⁴ cells per well were seeded onto 96-well plates (Eppendorf, MI, Italy) once incubated with etoposide treatments at indicated concentrations or vehicle solutions at 72 h post-induction with doxycycline, considered as a starting point. At the indicated time points, cells were incubated for 2 h with CellTiter-Blue Cell Viability assay (Promega, Madison, WI, USA), according to manufacturer’s instructions, and then the plates were acquired with the microplate reader Synergy H1 Gen5 (Biotek, Agilent, Santa Clara, CA, USA).

Fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Biowest and Gibco, respectively. Acetic acid, dimethyl sulfoxide (DMSO), ethylene diamine tetraAcetic acid (EDTA), sulforhodamine B (SRB), trypan blue reagent, doxorubicin, trichloroAcetic acid (TCA), Tris base and bromodeoxyuridine (BrdU) were purchased from Sigma-Aldrich. Stock solutions of the synthesized compounds were prepared with the solvent DMSO as vehicle, at stock concentration of 60 mM and kept at −20 °C. The other working stock solutions of 40 mM, 20 mM and 5 mM were also prepared in DMSO and kept at −20 °C.