Relationship between autophagy marker expression and dynein in the mechanism of action of Amblyomin-X.

<p>Representative western blots of whole-cell lysates of cultured cells treated with vehicle (PBS), 0.5 µM Ambly for 2 h, 4 h or 24 h, 5 µM MG-132 for 24 h or 0.2 µM rapamycin for 16 h. Images are representative of three independent experiments containing the autophagic markers (mTOR, AMBRA1, LC3-I and LC3-II) in <b>A</b>) SK-MEL-28 cells and (<b>B</b>) MIA PaCa-2 cells and (<b>C</b>) autophagic/apoptosis marker (Bim) in SK-MEL-28 cells and (<b>D</b>) MIA PaCa-2 cells. Confocal microscopy analysis of cultured cells treated with vehicle (PBS) or 0.5 µM Ambly for 24 h. The final overlay image represents five fields of three independent experiments in which (<b>E</b>) the red fluorescence represents HC1, while the green fluorescence represents mTOR and the merging of the two is in yellow; or (<b>F</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents AMBRA1 (originally, the yellow fluorescence was artificially colored by the microscope software) and the merging of the two is in yellow; or (<b>G</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents Bim and the merging of the two is in yellow.</p

Gene expression of dynein and targets related to intracellular protein quality control induced by Amblyomin-X.

<p>qPCR analysis of dynein chains and two chains of dynactin (p150Glued and dynamitin) with Ambly induction (<b>A</b>) 2 h, (<b>B</b>) 4 h and (<b>C</b>) 24 h. qPCR analysis of targets related to intracellular protein quality control with Ambly induction (<b>D</b>) for 2 h, 4 h and 24 h. Cultured cells were treated with vehicle (phosphate buffered saline, PBS) or 0.5 µM Ambly for 2 h, 4 h or 24 h. The results were calculated related to the control (vehicle) and are expressed as the means ± standard error of fold increase over control (considered as 1) in arbitrary units. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p

Protein expression of dynein, NFKB1 and β-actin induced by Amblyomin-X.

<p>Representative western blots of whole-cell lysates of (<b>A</b>) dynein chains in SK-MEL-28 cells, (<b>B</b>) dynein chains in MIA PaCa-2 cells, (<b>C</b>) NFKB1 and β-actin in SK-MEL-28 cells and (<b>D</b>) NFKB1 and β-actin in MIA PaCa-2 cells. Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 2 h, 4 h or 24 h or 5 µM MG-132 (NFKB1 and β-actin). Images are representative of three independent experiments.</p

K-linkage profile and visualization of autophagy steps in the mechanism of action of Amblyomin-X.

<p>(<b>A</b>) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of K48 and K63 linkage. Cultured cells were treated with vehicle (PBS) or 0.5 µM Ambly for 24 h. Results are expressed as the means ± standard error in arbitrary units of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (<b>B</b>) Immunofluorescence analysis of autophagic membrane formation. Cultured cells were treated with vehicle (PBS), 5 µM MG-132 for 24 h, 0.2 µM rapamycin for 16 h or 0.5 µM Ambly for 24 h or 48 h. LC3 was stained with FITC and is represented in diffused green fluorescence in the cytoplasm, while the nucleus was stained with DAPI and is represented in blue. Images are representative of five fields from each experiment (n = 3). (<b>C</b>) Fluorescence microscopy analysis using acridine orange stain. Cultured cells were treated with vehicle (PBS), 5 µM MG-132 for 24 h, 0.2 µM rapamycin for 16 h, 0.5 µM Ambly for 24 h pretreated with 0.2 µM rapamycin for 16 h (rapa/Ambly) or 0.5 µM Ambly for 24 h, 48 h or 72 h. Bright red fluorescence indicates acidic vesicles, while green fluorescence indicates the cytoplasm and nucleus. White arrows indicate the zoomed area located in the upper right position of the image. Images are representative of five fields from each experiment (n = 3). (<b>D</b>) Cell viability assay of tumor cells treated with the compounds used in autophagy visualization. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p

Aggresome formation induced by Amblyomin-X.

<p>Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (<b>A</b>) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (<b>B</b>) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (<b>C</b>) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p