SUPPLEMENTARY DATA

Preclinical study

Molecular and Cellular Biochemistry

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase pump regulation

Naira Fernanda Zanchett Schneider1, Izabella Thais Silva1, Lara Persich1, Annelise de Carvalho1,

Sayonarah C. Rocha2, Lucas Marostica1, Ana Carolina Pacheco Ramos2, Alex G. Taranto3, Rodrigo M.

Pádua4, Wolfgang Kreis5, Leandro A. Barbosa2, Fernão C. Braga4, Cláudia M. O. Simões1*,

Affiliation

1 Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de

Santa Catarina, Florianópolis, SC, Brazil;

2 Laboratório de Bioquímica Celular, Universidade Federal de São João del Rei, Campus Centro-Oeste

Dona Lindu , Divinopolis, MG, Brazil;

3 Laboratório de Bioinformática, Universidade Federal de São João Del Rei, Campus Centro Oeste Dona

Lindu, Divinópolis, MG, Brazil;

4 Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas

Gerais, Belo Horizonte, MG, Brazil;

5Department of Biology, Friedrich-Alexander Universität, Erlangen-Nürnberg, Germany.

*Corresponding author: Cláudia M. O. Simões, Mailing address: LVA, CIF, CCS, Universidade Federal

de Santa Catarina (UFSC), Florianópolis, 88040-900, SC, Brasil. Tel.: +55-48-3721-5207; fax: +55-48-

3721-9258.

E-mail address: claudia.simoes@ufsc.br (Cláudia M. O. Simões).

MATERIALS AND METHODS

1. Evaluation of autophagic vacuoles by acridine orange.

A549 cells were grown in a 12-well plate and incubated in medium containing 10 and 100 nM of CON

for 24 and 48 h. Then, cells were washed with PBS and incubated for 15 min with 1 µg/mL acridine

orange (AO) (Sigma-Aldrich) at room temperature. Subsequently, cells were analyzed under an inverted

fluorescence microscope (Olympus BX41, Hicksville, NY, USA) and quantified by flow cytometry

(FACS Canto II cytometer).

2. Cytotoxic effect of CON in the presence of autophagy inhibitor

A549 cells were grown in a 96-well plate and after 24 h treated with CON (10, 50 and 100 nM) for 48 h

in the absence or presence of chloroquine (CQ), an inhibitor of autophagy, for 4 and 24 h. After

incubation, 10% trichloroacetic acid (TCA) was added to each well to fix the cells and then washed and

stained with sulforhodamine B assay (SRB). The protein-bound dye was dissolved in 10 mM Tris-Base

[(tris(hydroxymethyl) aminomethane] solution and optical densities (OD) were read at 510 nm on a

spectrophotometer Spectra Max M2 (Molecular Devices, USA).

RESULTS

Figure 1S: Positive control PAC effects in A549 cells. Cells were treated with PAC (100 nM) for 48 h

and stained with DAPI in order to detect nuclear morphology. Brightness contrast images were captured

by a phase contrast microscope to detect cell morphological changes. Red arrows indicate shrinked and

fragmented nucleus. Magnification 400x. Images showed are representative of three independent

experiments.

Figure 2S: Autophagy determination (a) Visualization of AVOs (Acidic Vesicular Organelles) by

acridine orange (AO) staining. A549 cells were treated with CON 10 and 100 nM for 48 h, stained with

AO for 15 min and then observed and photographed under a fluorescent microscope. Magnification ×

400. The image shown is representative of three independent experiments. (b) Dot plots of AVOs

(red:green fluorescence ratio) in A549 cells determined by flow cytometry. (c) Percentage of AVOS

determined by flow cytometry. The data shown is represented as scatterplot (+ bars representing the mean

of three independent experiments). (d) Effects of CON on LC3B-II expression. The image shown is

representative of two independent experiments. (e) A549 cells were treated with CON for 48 h in the

absence or presence of chloroquine (CQ) for 4 and 24 h. Cell viability was evaluated by sulforhodamine

B assay (SRB). The data shown is represented as scatterplot (+ bars representing the mean of three

independent experiments). Statistical analysis performed by ANOVA followed by Dunnett’s test when

compared to the untreated control (CC) showed no statistical significance.