Effect of nutrient deprivation and growth factor inhibitors on autophagy in human breast cancer cells.

<p>(<b>A</b>) MCF-7 cells were treated with DPBS for the indicated times. At the end of treatment, formation of the autophagy marker LC3-II was detected by immunoblotting with an anti-MAP-LC3 antibody. (<b>B</b>) MCF-7 cells transfected with 3 µg of GFP-LC3 plasmid were treated or untreated with DPBS for 1 h, and then observed under a fluorescent microscope. A representation of GFP-LC3 positive cells was shown. (<b>C, D</b>) MCF-7 (C) or MDA-MB-468 (D) cells cultured in medium containing 10% FBS were treated with 0.5, 1, 2.5, 5 or 10 µM gefitinib or lapatinib for 24 h, and the LC3-II level was examined by immunoblotting. (<b>E</b>) MDA-MB-468 cells were transfected with a GFP-LC3-expressing vector, and then treated with the indicated concentration of gefitinib or lapatinib in the presence of the lysosomal protease inhibitors E64d (10 µg/ml) and pepstain A (10 µg/ml). At the end of treatments, cells were fixed with 4% formaldehyde for 15 min. To determine the autophagic response, cells were inspected at 60× magnification for numbers of GFP-LC3 puncta.</p

CompuSyn analysis of the combinations of growth factor inhibitors and eEF-2 kinase siRNA.

<p>Combination index (CI) was calculated using the computer program, CompuSyn.</p

Effect of nutrient deprivation on protein synthesis and cellular ATP in MCF-7 cells.

<p>(<b>A</b>) MCF-7 cells were treated with DPBS for the indicated times and the rate of protein synthesis was measured by labeling the cells with 25 µCi/ml of EasyTag EXPRESS [³⁵S] protein labeling mix and liquid scintillation counting, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009715#s4" target="_blank">Material and Methods</a>”. The specific activity of protein synthesis was determined by the amount of incorporated ³⁵S-methionine/cysteine per mg of total protein per min, and relative activities at indicated times of starvation were calculated as percent of control. Results shown are the mean ± SD of quadruplicate determinations from one of three identical experiments; *<i>p</i><0.05, <i>t</i>-test. (<b>B</b>) MCF-7 cells were treated with DPBS for the indicated times and ATP content was measured using the ATPlite™ Luminescence Assay Kit. (<b>C</b>) AMPK activity was determined by Western blot analysis of phospho-AMPK using an anti-phospho-AMPK antibody, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009715#s4" target="_blank">Material and Methods</a>”. Actin was used as a loading control. Results shown are the representative of three similar experiments; each bar or point represents mean ± SD of quadruplicate determinations. Results shown are the mean ± SD of quadruplicate determinations from one of three identical experiments; **<i>p</i><0.01, <i>t</i>-test.</p

Effects of autophagy suppression on growth and survival of human breast cancer cells.

<p>(<b>A</b>) MCF-7 cells were transfected with a non-targeting RNA or a siRNA targeting eEF-2 kinase, beclin1, or ATG5. Expressions of eEF-2 kinase, beclin1, or ATG5 were determined by Western blot using the respective antibodies. β-actin or Ran was used as a loading control. (<b>B</b>) MCF-7 cells treated with 3-MA or with siRNA targeting eEF-2 kinase, beclin1, ATG5 or a non-targeting RNA were seeded with 10% FBS RPMI 1640 medium in 96-well culture plates (3×10³ cells per well). After overnight incubation, medium was changed to serum-free medium. Cell viability was determined at the indicated times using MTT assay. Results shown are the representative of three similar experiments; each point represents mean ± SD of quadruplicate determinations. (<b>C</b>) MCF-7 cells treated with 3-MA or with siRNA targeting eEF-2 kinase, beclin1, ATG5 or a non-targeting RNA were seeded with 10% FBS RPMI 1640 medium in 96-well culture plates (3×10³ cells per well). After overnight incubation, medium was changed to HBSS. Cell viability was determined at the indicated times using MTT assay. Results shown are the representative of three similar experiments; each point represents mean ± SD of quadruplicate determinations.</p

Effect of nutrient deprivation on eEF-2 kinase activity and the associated signaling molecules.

<p>(<b>A</b>) MCF-7 cells were treated with DPBS for the indicated times, and the levels of eEF-2 kinase, p-EF2, EF2, p-eEF2 kinase (S398) and p-eEF2 kinase (S366) were examined by Western blot using the respective antibodies. (<b>B</b>) MCF-7 cells were treated with DPBS for the indicated times, and p-S6 kinase, S6 kinase, p-4EBP1, and 4EBP1 were examined by Western blot using the respective antibodies. β-actin was used as a loading control. Results shown are the representative of three similar experiments.</p

Effects of eEF-2 kinase silencing on sensitivity of human breast cancer cells to growth factor inhibitors.

<p>MCF-7 (<b>A, B</b>) and MDA-MB-468 (<b>C, D</b>) cells transfected with an siRNA targeting eEF-2 kinase or a non-targeting RNA were cultured in RPMI 1640 or DMEM media supplemented with 10% fetal bovine serum at 37°C in a humidified incubator (5% CO₂ and 95% air), and then treated with a series of concentrations of gefitinib (<b>A, C</b>) or lapatinib (<b>B, D</b>) for 48 h. At the end of treatment, cell viability was determined using MTT assay. Results shown are the representative of three similar experiments; each point represents mean ± SD of quadruplicate determinations; *<i>p</i><0.05, **<i>p</i><0.01, <i>t</i>-test.</p

Silencing of Cav-1 expression sensitizes cells to IR and bleomycin.

<p>MDA-MB-468 cells with or without silencing of Cav-1 were treated with varying doses of γ-radiation, and colony formation assay was performed to compare cell survival. Results shown are the representative of three similar experiments; each point represents mean ± SD of quadruplicate determinations of the experiment.</p

Treatment with IR stimulates the expression of Cav-1 protein.

<p>(<b>A</b>) MDA-MB-468 cells were irradiated (5 Gy) for the indicated period of time, and then the treated cells were collected for Western blot analysis of Cav-1. β-actin was used as a loading control. Expression of Cav-1 and β-actin were quantified using imageJ software, and Cav-1 level was normalized to that of β-actin. The normalized Cav-1 at the zero time point was arbitrarily set as 1. Bar represent mean ± S.D. of three separate experiments. (<b>B</b>) MCF-7, NCI/ADR-RES, PC-3, T98G and MCF-10A cells were treated or untreated with 5 Gy ionizing radiation, and Cav-1 expression was analyzed by Western blot. Results shown are the representative of three identical experiments.</p

Silencing of Cav-1 expression reduces the DSB repair by HR.

<p>(<b>A</b>) HT-1080 cells were irradiated (5 Gy) for the indicated period of time, and then cell lysates were prepared for Western blot analysis of Cav-1 and γ-H₂AX. β-actin was used as a loading control. (<b>B</b>) To determine the turnover of the silencing effect of Cav-1 siRNA in HT-1080 cells, we performed Western blot of analysis of Cav-1 at the indicated time after siRNA transfection. (<b>C</b>) HT-1080 cells were transfected with a non-targeting RNA or Cav-1 siRNA. Twenty-four hours after transfection, the cells were transfected with an HA tagged I-SceI endonuclease expressing vector (HA-I-SceI) or empty vector (HA) by electroporation, followed by Western blot analysis of Cav-1 and HA-I-SceI (upper panels), and by puromycin screening for HR frequency (lower panels). HR frequency was calculated as follows: the average numbers of colonies/dish were divided by the plating efficiency of transfection and divided by 85,000 (the total number of cells plated). The results shown are the representative of three similar experiments; each bar represents the mean±S.D. of triplicate determinations.</p

Silencing of Cav-1 expression by siRNA increases the IR-induced accumulation of ssDNA and γ-H2AX.

<p>(<b>A</b>) MDA-MB-468 cells were transfected with a non-targeting RNA (NT) or either Cav-1-targeted siRNA sequence 1 or sequence 2. At the indicated time following transfection, the cells were collected for Western blot analysis of Cav-1. β-actin was used as a loading control. (<b>B</b>) MDA-MB-468 cells were transfected with a Cav-1 siRNA or a non-targeting RNA, followed by IR (5 Gy). The cells were collected at the indicated time points and fixed for immunofluorescent detection of ssDNA. The signals of ssDNA and total DNA were quantified using imageJ software, and ssDNA signal was normalized to total DNA signal at each time point. The results shown were mean±S.E. of five similar experiments. * <i>p<0.05</i>; ** <i>p<0.01</i>. (<b>C</b>) MDA-MB-468 cells were transfected with a Cav-1-targeted siRNA or a non-targeting control (NT). Forty-eight hours later, the transfected cells were irradiated (5 Gy) for the indicated period of time followed by Western blot analysis of γ-H₂AX. Levels of γ-H₂AX and H₂AX were quantified using imageJ software. γ-H₂AX/H₂AX ratios of untreated samples (zero time) were arbitrarily set at 100 as controls, and the treated samples were normalized to the controls. Results shown are the representative of three similar experiments; each point represents mean ± SD of triplicate determinations. * <i>p<0.05</i>, ** <i>p<0.01</i>.</p