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1.
Figure 5

Figure 5. Dynamics of Autophagy Flux in Tzb-refractory cells.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Since increased LC3-II levels together with a reduction of p62 protein levels characterize the occurrence of increased autophagic flux, SKBR3 parental cells and TzbR cells (untreated and treated with 100 µg/mL Tzb) were triple stained with antibodies against LC3 and p62 and with Hoechst 33258 for nuclear counterstaining. Tzb-refractory cells exhibit an exacerbated autophagic clearance compared to Tzb-sensitive SKBR3 parental cells when considering overlapping fluorescence of LC3 and p62 as an indirect marker of p62 presenting ubiquitinated protein bodies to the autophagic machinery in a LC3-dependent manner. Images show representative portions of SKBR3 and TzbR cell cultures that were captured using different channels for LC3 (red), p62 (green) and Hoechst 33258 (blue) with a 20× objective and merged on BD Pathway™ 855 Bioimager System using BD Attovision™ software. Scale bar = 25 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
2.
Figure 1

Figure 1. Characterization of Tzb-refractory HER2-gene Amplified SKBR3 Breast Cancer Cells.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Left. The metabolic status of Tzb-naive SKBR3 parental cells and Tzb-refractory TzbR POOLs treated with graded concentrations of Tzb was evaluated using a MTT-based cell viability assays and constructing dose-response graphs as % of untreated cells (dashed line = 100% cell viability). Results are means (circles) and 95% confidence intervals (bars) of three independent experiments performed in triplicate. Treatment with Tzb concentrations as high as 100 µg/mL Tzb failed to significantly decrease cell viability in TzbR POOLs whereas a 10 times lower concentration of Tzb drastically reduced cell viability (>50%) in SKBR3 parental cells. Right: Representative microphotographs of untreated and experimental cell cultures following 72 hours treatment with different concentrations of Tzb, as specified. Examination of microphotographs reveals the smaller size of Tzb-refractory TzbR POOLs when compared to Tzb-naive SKBR3 parental morphology. Scale bar = 10 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
3.
Figure 4

Figure 4. Dynamics of Autophagic Degradation in Tzb-refractory Cells.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Immunoblotting. Autophagic degradation in whole cell lysates of Tzb-naive SKBR3 parental cells and Tzb-refractory TzbR POOLs was detected with Western blot analysis using a p62 antibody. Immunoblotting bands (64 kDa) represent p62/SQSTM1, a selective substrate of autophagy. Autophagic degradation (i.e. down-regulation of endogenous p62 protein expression) is significantly increased in Tzb-refractory TzbR cells when compared to Tzb-sensitive SKBR3 parental cells. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments. Immunofluorescence. After fixation and permeabilization, cellular distribution of p62 was assessed following staining with a p62 antibody and Hoechst 33258 for nuclear counterstaining. SKBR3 parental cells display a homogenous and strong cytoplasmic p62 staining, which is typical of absent or low-level of autophagic degradation. TzbR POOLs show a significant decrease in the cytoplasmic distribution of p62 that appears somewhat vesiculated, which is typical of enhanced autophagic degradation. Images show representative whole populations of SKBR3 and TzbR cells growing in individual wells that were captured using different channels for p62 (green) and Hoechst 33258 (blue) as a 4×4 montage with a 20× objective on BD Pathway™ 855 Bioimager System, and merged using BD Attovision™ software. Scale bar = 200 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
4.
Figure 2

Figure 2. Dynamics of Autophagosome Formation in SKBR3 cells-derived Tzb-Refractory POOLs.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Immunoblotting. Autophagosome formation in whole cell lysates of Tzb-naive SKBR3 parental cells and Tzb-refractory TzbR POOLs was detected with Western blot analysis using a LC3 antibody. Top band (18 kDa) represents LC3-I. Bottom band (16 kDa) represents LC3-II, a typical marker of autophagosomes. Autophagosome formation is robust in Tzb-refractory TzbR cells when compared to low to undetectable levels of LC3-I/LC3-II expression in Tzb-sensitive SKBR3 parental cells. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments. Immunofluorescence. After fixation and permeabilization, cellular distribution of autophagosome marker LC3 was assessed following staining with a LC3 antibody and Hoechst 33258 for nuclear counterstaining. SKBR3 parental cells display a homogenous but weak cytoplasmic LC3 staining, which is typical of absent or low-level autophagosome formation. TzbR POOLs show a marked contrast enhancement in the punctated pattern of endogenous LC3 expression, which is characteristic of autophagosome formation. Of note, Tzb exposure further increases autophagosome LC3 pattern. Images show representative portions of SKBR3 and TzbR cell cultures captured in different channels for LC3 (red) and Hoechst 33258 (blue) with a 20× objective, and merged on BD Pathway™ 855 Bioimager System using BD Attovision™ software. Scale bar = 25 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
5.
Figure 8

Figure 8. Dynamics of Autophagosome Formation and Autophagic Flux in Tzb-naive SKBR3 Cells Treated with Tzb.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Tzb-naive SKBR3 parental cells were exposed to graded concentrations of Tzb (0, 10, 100 and 200 µg/mL Tzb) for 72 hours. Immunofluorescence. After fixation and permeabilization, cells were triple stained with LC3 and p62 antibodies and counterstained with Hoechst 33342 to visualize cell nuclei. Untreated control SKBR3 cells likewise show homogenous cytoplasmatic staining of LC3 and p62 (i.e. absent or low-level autophagosome formation). Surviving SKBR3 cells following exposure to graded concentrations of Tzb notably exhibit a marked contrast enhancement in the punctated pattern of LC3 (i.e. autophagosome formation) concurrently accompanied by p62 down-regulation (i.e. enhanced autophagic flux). Images show representative portions of SKBR3 and TzbR cell cultures captured in different channels for LC3 (red) and Hoechst 33258 (blue) with a 20× objective, and merged on BD Pathway™ 855 Bioimager System using BD Attovision™ software. Scale bar = 25 µm. Immunoblotting. Autophagosome formation and autophagic degradation in whole cell lysates of Tzb-treated Tzb-naive SKBR3 cells was confirmed with Western blot analyses using LC3 and p62 antibodies. High-dose Tzb notably increases LC3-II while reducing p62 expression, thus revealing an enhanced autophagic flux in the surviving fraction of Tzb-treated SKBR3 cells. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
6.
Figure 7

Figure 7. Changes in the Cell Proliferation of Tzb-refractory Cells Upon siRNA-induced Knock Down of the Autophagosome Marker LC3.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Tzb-refractory TzbR POOLs were mock transfected, transfected with a non-specific control siRNA Pool (negative control) or transfected with siRNA-targeting LC3. 72 h after transfection, one set of mock-, non-specific negative control-, and RNAi LC3-transfected cells were used for immunoblotting analyses of LC3 expression. A second set of cells were harvested, re-cultured in 24-well plates at a density of 10,000 cells/well and treated with regular medium in the absence or presence of Tzb (1 µg/mL Tzb in SKBR3 parental cells and 10 µg/mL Tzb in TzbR POOLs). There were no significant changes in cell numbers when TzbR cells were treated with Tzb as single agent (omitted). siRNA-induced blockade of LC3 expression significantly reduces cell proliferation rates in TzbR POOLs. More importantly, supra-additive growth inhibitory interactions occur in LC3-depleted Tzb-treated TzbR POOLs. The data presented are mean of number cells ×105/well (circles) and 95% confidence intervals (bars) of three independent experiments made in duplicate after 3, 6 and 9 days. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions (i.e. LC3 siRNA±Tzb) and control cells (i.e. siRNA [-]) are shown. All statistical tests were two-sided.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
7.
Figure 6

Figure 6. Changes in the Cell Viability of Tzb-refractory Cells Upon Pharmacological Modulation of Autophagosomes Formation/Function.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

The metabolic status of SKBR3 parental cells and Tzb-refractory TzbR POOLs treated with autophagy inhibitors including 3-MA, LY294002 and bafilomycin A1 was evaluated using MTT-based cell viability assays and constructing dose-response graphs as % of untreated cells (dashed line = 100% cell viability). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Figure panels display results obtained with representative doses of each autophagy inhibitor, as specified. Pharmacologically-induced loss of autophagosome formation/function is highly cytotoxic to TzbR cells (A, B) compared to cell viability effects in Tzb-naive SKBR3 parental cells (C). Statistically significant differences (one-factor ANOVA analysis) between experimental conditions (i.e. treatment with autophagy inhibitors) and unsupplemented control cells are shown. All statistical tests were two-sided. To confirm that 3-MA-induced changes in cell viability related to 3-MA-induced changes in the number of autophagosomes in TzbR cells, cellular distribution of autophagosome marker LC3 was assessed following 72 hours exposure to 5 mM 3-MA. The total number of LC3-positive autophagosomes per cell is notably decreased in 3-MA-treated TzbR cells. Images show representative portions of SKBR3 and TzbR cell cultures captured in different channels for LC3 (red) and Hoechst 33258 (blue) with a 20× objective, and merged on BD Pathway™ 855 Bioimager System using BD Attovision™ software. Scale bar = 25 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.
8.
Figure 3

Figure 3. Correlation Between Proliferative Profiles and Dynamics of Autophagosomal Formation in TzbR-refractory Cells.. From: Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab.

Top panels. SKBR3 parental cells and SKBR3-derived Tzb-refractory POOLs were plated in 24-well plates at a density of 10,000 cells/well and cultured with regular medium in the absence or presence of Tzb (10 µg/mL Tzb in SKBR3 parental cells; 100 µg/mL Tzb in TzbR POOLs). The data presented are mean of number cells ×105/well (circles) and 95% confidence intervals (bars) of three independent experiments made in duplicate obtained after 0, 3, 6 and 9 days. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown. All statistical tests were two-sided. No statistically significant differences were observed in the number of TzbR cells growing in the presence of 100 µg/mL Tzb up to 9 days whereas 10 µg/mL Tzb significantly reduced cell proliferation in SKBR3 parental cells as early as 3 days after Tzb exposure. Slope of the growth curves clearly denotes a faster proliferation of TzbR POOLs regardless Tzb exposure. Bottom panels. Images show representative whole populations of SKBR3 cells and TzbR cells growing in individual wells that were captured using different channels for LC3 (red) and Hoechst 33258 (blue) as a 4×4 montage with a 20× objective on BD Pathway™ 855 Bioimager System, and merged using BD Attovision™ software. As discussed in Figure 2, TzbR POOLs notably exhibit a marked contrast enhancement in the punctated pattern of endogenous LC3 expression, which is typical for autophagosomal formation. Scale bar = 200 µm.

Alejandro Vazquez-Martin, et al. PLoS One. 2009;4(7):e6251.

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