Gefitinib inhibits cell growth and induces appearance of MDC-labeled organelles in breast cancer cells.

<p>(<b>A</b>) Example images obtained with IN Cell 1000 showing SKBR3 cells treated for 72 h with 10 µM gefitinib and stained <i>in </i><i>situ</i> with DRAQ5 (stains nuclei in viable and dead cells), ETH (stains nuclei in dead cells with compromised plasma membrane) and MDC (stains acidic organelles). Based on differential staining and morphological features image recognition software identifies different cell populations. Left image: nuclear imaging masks shown in blue and red indicate viable and dead cells, respectively; cytoplasm of viable cells with MDC-labeled organelles is shown in yellow and cytoplasm of cells without MDC-labeled organelles is shown in light blue. Middle image: magnification of an area, as marked in the left image, showing transparent imaging masks outlining nuclei, cytoplasm and MDC-labeled organelles, as recognized by the HCA Investigator software; DRAQ5 staining is shown in blue, ETH staining is shown in red and MDC staining is shown in green. Right image: a high magnification image showing MDC-labeled organelles outlined in yellow within the cells’ cytoplasm; some of the MDC-labeled structures, indicated by the red arrows, represent multiple closely grouped organelles. (<b>B</b>) Representative images of indicated cells cultured in the presence of 0.5% DMSO (vehicle) or 10 µM gefitinib for 24 h (BT474) or 72 h (SKBR3, JIMT-1, MCF7-GFPLC3) stained <i>in </i><i>situ</i> with DRAQ5 (blue), ETH (red) and MDC (green). Images in (<b>A</b>) and (<b>B</b>) were pseudo-colored and overlaid using the Investigator software. (<b>C</b>) Quantitation of different cell populations and autophagic organelles in BT474, SKBR3, JIMT-1 and MCF7-GFPLC3 cells by HCA. Cells were treated with vehicle or increasing concentrations of gefitinib for the indicated time. Numbers of viable cells in culture are normalized to vehicle-treated controls. Dead cells are shown as a percent difference in the content of dead (ETH-positive) cells between drug-treated minus vehicle-treated cultures. The proportion of viable cells with MDC-labeled organelles (puncta) in culture is shown as a percent difference in cells with >1 MDC-labeled organelle between drug-treated minus vehicle-treated cultures. Each data point represents a mean±SD from 3 replicate wells. HCA screenings were repeated 2 - 3 times for each cell type with consistent results; representative experiments are shown.