Results: 4

1.
Fig. 1.

Fig. 1. From: Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.

Pharmacological blockade of FAS activity down-regulates expression and activity of p185HER2 oncoprotein. (A) SK-Br3 cells were cultured in the presence of cerulenin for 48 h. Twenty micrograms of protein was subjected to Western blot analyses with the p185HER2 Ab-3. Activation status of HER2 was analyzed by assessing tyrosine phosphorylation of p185HER2 using the anti-phosphotyrosine Ab 4G10. (B) HER2 concentration in cell lysates from cerulenin-treated SK-Br3 cells was quantified by using the Human neu Quantitative ELISA System per the manufacturer's instructions. Data are presented are means ± SD (bars) (n = 3, in duplicate). (C) SK-Ov3 cells was cultured in the presence of C75 for 48 h. Twenty micrograms of protein was subjected to Western blot analyses for HER2 expression as described in A. (D) BT-474, MDA-MB-453, and T47-D breast cancer cells were cultured in the presence of 10 μg/ml cerulenin for 48 h. Twenty micrograms of protein was subjected to Western blot analyses for HER2 expression as described in A.

Javier A. Menendez, et al. Proc Natl Acad Sci U S A. 2004 July 20;101(29):10715-10720.
2.
Fig. 2.

Fig. 2. From: Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.

Accumulation of PEA3 and inhibition of HER2 gene transcription after pharmacological blockade of FAS activity. (A)(Upper) SK-Br3 cells were cultured either with graded concentrations of cerulenin for 48 h (Left) or exposed to 5 μg/ml cerulenin for 96 h (Right). Fifty micrograms of protein was subjected to Western blot analyses with anti-PEA3 and Cyclin D1 Abs. (Lower) SK-Br3 cells were treated with 2.5 μg/ml cerulenin for 48 h, and subcellular localization of immunofluorescent PEA3 was assessed with a Leica DMIRE2 confocal microscope. PEA3-stained cell nuclei are presented at two magnifications (control, i and ii; and cerulenin-treated cells, iii and iv). (B) Total RNA from cerulenin- and C75-treated SK-Br3 cells was isolated and RT-PCR analyses for HER2 and GADPH transcripts and expression were performed as described in Materials and Methods.

Javier A. Menendez, et al. Proc Natl Acad Sci U S A. 2004 July 20;101(29):10715-10720.
3.
Fig. 4.

Fig. 4. From: Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.

Concurrent inhibition of FAS and HER2 signalings synergistically enhances apoptotic cell death. (A) SK-Br3 cells were treated with 10 μg/ml trastuzumab, 2 μg/ml cerulenin, or a combination of trastuzumab and cerulenin. Seventy-two hours later, quantification of apoptotic cell death was determined by Cell Death ELISA (Roche Molecular Biochemicals) per the manufacturer's instructions. Data are expressed as absorbance by using the formula [A405 - A490]TREATED/[A405 - A490]UNTREATED. (B) SK-Br3 cells were grown in eight-well chamber slides and treated with 5 μg/ml trastuzumab, 1 μg/ml cerulenin, or a combination of 5 μg/ml trastuzumab plus 1 μg/ml cerulenin. Seventy-two hours later, TUNEL analyses were performed by using the DeadEnd Fluorometric TUNEL System per the manufacturer's protocol. The immunofluorescence photomicrographs (iiii) of cells undergoing apoptosis (green staining) and the corresponding 4′,6-diamidino-2-phenylindole-counterstained cells are shown. The number in the lower right of each panel represents the percentage of TUNEL-positive cells. (C) (Upper) SK-Br3 cells were grown in eight-well chambers and transfected with 50 nM FAS siRNA, 50 nM HER2 siRNA, or a combination of 50 nM FAS siRNA plus 50 nM HER2 siRNA. Seventy-two hours later, TUNEL analyses were performed by using the DeadEnd Fluorometric TUNEL System. The number in the upper right of each panel represents the percentage of TUNEL-positive cells. (Lower) The amount of p185HER2 in FAS RNAi- and/or HER2 RNAi-transfected SK-Br3 cells was quantified by flow cytometry with the anti-p185HER2 Ab-5. The mean fluorescence signal ± SD (n = 3) was quantified by using the Geo Mean fluorescence parameter provided with cellquest software.

Javier A. Menendez, et al. Proc Natl Acad Sci U S A. 2004 July 20;101(29):10715-10720.
4.
Fig. 3.

Fig. 3. From: Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.

(AC) RNAi-mediated silencing of the FAS gene suppresses HER2 expression. (A) SK-Br3 cells were transfected with siRNA targeting FAS gene or with a nonspecific control siRNA pool for 72 h. Twenty micrograms of protein was subjected to Western blot analyses with specific Abs against FAS, p185HER2, or β-actin. (B) The amount of p185HER2 in FAS RNAi-transfected cells was quantified by flow cytometry using the p185HER2 Ab-5. The mean fluorescence signal ± SD (n = 3) was quantified by using the Geo Mean fluorescence parameter provided with cellquest software. (C)(Upper) Fifty micrograms of protein from FAS RNAi-transfected SK-Br3 cells was subjected to Western blot analyses for PEA3. (Lower) Total RNA from FAS RNAi-transfected SK-Br3 cells was isolated and RT-PCR analyses for HER2 and β-actin transcripts and expression were performed as described in Materials and Methods. (D) Impact of FAS RNAi and pharmacological blockade of FAS activity on p185HER2 cellular localization. Nonspecific RNAi- and FAS RNAi-transfected cells were fixed and labeled with the p185HER2 Ab-5 (ad) or the p185HER2 Ab-3 (a′–d′). SK-Br3 cells were treated for 72 h with DMSO vol/vol (a″), 2.5 μg/ml cerulenin (b″), or 2.5 μg/ml C75 (c″), and then fixed and labeled with the p185HER2 Ab-3. Cellular localization of p185HER2 was detected by indirect immunofluorescence by incubating with TRITC (ad)- or FITC (a′–d′)-conjugated anti-mouse IgG. After counterstaining with 4′,6-diamidino-2-phenylindole, cells were examined and photographed by using a Zeiss fluorescent microscope. A representative immunostaining analysis (n = 3) is shown.

Javier A. Menendez, et al. Proc Natl Acad Sci U S A. 2004 July 20;101(29):10715-10720.

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