Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line.

<p>Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. <b>a</b>) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). <b>b</b>) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). <b>c</b>) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. <b>d</b>) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).</p

Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins <i>in vitro</i> and downstream effectors, and reduction in cell migration.

<p><b>a</b>) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. <b>b</b>) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x is shown under each band. <b>c</b>) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251^Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x in the absence of doxycycline is shown under each band. <b>d</b>) Scratch width scoring of U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251^Ng2x cells.</p

The combined silencing of Rictor and EGFR <i>in vivo</i> results in a complete inhibition of tumor growth.

<p>U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expression in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. <b>a</b>) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251^EGFR/Rictor group. <b>b</b>) A representative brain section from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). <b>c</b>) A representative tumor section from U251^Ng2x, U251^Rictor and U251^EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). <b>d</b>) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251^Ng2x, U251^Rictor, U251^EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). <b>e</b>) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251^EGFR/Rictor group.</p

Effects of siRNA mediated EGFR silencing on downstream signaling and autophagy.

<p>(<b>A</b>) Following a transfection with EGFR siRNA, SKBR3 cells were treated for 72 h with vehicle or 5 µM gefitinib. (<b>B</b>) Following a double knockdown with the EGFR siRNA MCF7-GFPLC3 cells were treated with vehicle or 4 µM gefitinib for 24 h. Representative experiments are shown.</p

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. </p

3-MA sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death in the presence of gefitinib.

<p>Flow cytometric analysis of apoptosis in SKBR3 (<b>A</b> and <b>B</b>) and MCF7-GFPLC3 (<b>C</b> and <b>D</b>) cells treated for 72 h with vehicle or 10 µM gefitinib (Gef), in the absence or presence of 5 mM 3-MA. Camptothecin (CPT) at 5 µM was used as an inducer of apoptosis (positive control). (<b>A</b> and <b>C</b>) Analysis of the sub-G₀/G₁ apoptotic cell fraction. The inserted histograms show representative DNA profiles of cells treated with the indicated agents where a sub-G₀/G₁ cell fraction is indicated with a marker. Arrows in (<b>C</b>) indicate S-G₂/M cell cycle block. (<b>B</b> and <b>D</b>) Analysis of apoptosis in cells stained with Annexin V-Alexa647 and PI. The inserted representative dot plots show distribution of cell populations treated with the indicated agents where apoptotic Annexin V-positive cells are marked as “A” in a rectangular region. Bar graphs represent the data (mean±SD) from 3 independently stained samples and show fold change relative to the vehicle-treated controls expressed as 1. Asterisks indicate a statistically significant difference (p<0.05) between cells treated with the vehicle and indicated agents. A double asterisk indicates a statistically significant difference (p<0.05) between cells treated with gefitinib in the presence of 3-MA and single-agent treated cells in addition to a statistically significant difference when compared to the vehicle-treated cells. Representative experiments are shown.</p

Gefitinib induces autophagic flux.

<p>(<b>A</b> - <b>D</b>) Autophagic flux assays performed in SKBR3 cells. (<b>A</b>) Western blot analysis of p62 expression in lysates derived from cells treated for 3 h with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) (<b>B</b>) Western blot analysis of LC3 levels in lysates derived from cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of 5 nM bafilomycin A1 (BAF). (<b>C</b>) HCA of TOA in cells treated for 72 h with increasing concentrations of gefitinib in the absence or presence of 10 mM 3-MA added for the last 3 h of treatment. The results are normalized to the vehicle control expressed as 1. Each bar represents the mean±SD from 3 replicate wells. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or 3-MA alone and cells treated with 3-MA in the presence of gefitinib. The results shown are representative of two experiments. (<b>D</b>) Western blot analysis of LC3-I and LC3-II levels in lysates derived from cells treated for 24 h with vehicle, 5 µM gefitinib, 5 mM 3-MA and the combination of gefitinib and 3-MA at the corresponding concentrations. Representative blots in (<b>A</b>), (<b>B</b>) and (<b>D</b>) are shown. (<b>E</b> - <b>G</b>) Autophagic flux assays performed in MCF7-GFPLC3 cells. (<b>E</b>) Western blot analysis of autophagic markers in lysates derived from cells treated for 18 h with increasing concentrations of gefitinib. Cleaved GFP is marked as GFP. (<b>F</b>) Western blot analysis of cleaved GFP levels in lysates derived from cells treated with vehicle or 10 µM gefitinib in the absence or presence of 10 mM 3-MA for 3 h (top panel), in the absence or presence of 50 nM bafilomycin A1 (BAF) for 24 h (middle panel), and in the absence or presence of 10 mg/ml lysosomal inhibitors (LYI; pepstatin A and E-64d) added for the last hour of treatment (bottom panel). Tubulin was used as loading control. Representative blots in (<b>E</b>) and (<b>F</b>) are shown. (<b>G</b>) HCA data showing the average GFPLC3 TOA/cell in MCF7-GFPLC3 cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of indicated autophagy inhibitors added for the last hour of treatment. The results are normalized to vehicle control expressed as 1. Each data point represents the mean±SD from 3 replicate wells and the results shown are representative of two experiments. 3-MA was used at 5 mM, bafilomycin A1 (BAF) was used at 5 nM and LYI were used at 10 µg/ml. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or autophagy inhibitors alone and cells treated with autophagy inhibitors in the presence of gefitinib. </p