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1.
Figure 3

Figure 3. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

A, quantitative RT-PCR of ATG5 and BNIP3 mRNA expression in MDA-MB-231 cells cultured at pH 6.7 for 72 hours relative to cells cultured at pH 7.4. The fold change was calculated using β-actin as the internal control. The data represent the mean ± SD of 3 experiments. B, whole-cell lysates from MDA-MB-231 cells cultured at pH 7.4 or at pH 6.7 for 72 hours were analyzed for the expression of ATG5 and BNIP3.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
2.
Figure 4

Figure 4. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

Transmission electron microscopy of double-membrane autophagic vacuoles in MDA-MB-231 cells cultured at pH 7.4 (A), pH 6.7 for 48 hours (B), or at low pH (6.7) for approximately 3 months (C). Autophagic vacuoles were detectable in MDA-MB-231 cells transiently or chronically exposed to pH 6.7. AV, autophagic vacuole; LY, lysosome; M, mitochondria; N, nucleus.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
3.
Figure 1

Figure 1. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

A and B, proliferation and viability of MDA-MB-231 cells cultured transiently at low pH (6.7) for 72 hours or (C) chronically at pH (6.7) for approximately 3 months were determined. Data represent the mean × SD of 3 independent experiments. D, cell-cycle profiles were also determined using FACS analysis. Data are representative of 3 independent experiments.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
4.
Figure 2

Figure 2. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

Morphologic analysis of MDA-MB-231 cells cultured at pH 7.4 (A) or pH 6.7 for 48 hours (B) or for approximately 3 months (C). Subcellular localization of LC3 in MDA-MB-231 cells cultured at pH 7.4 (D) or pH 6.7 (E) for 48 hours. Images were captured by confocal microscopy and are representative of 3 independent experiments. Size bars = 20 μmol/L. F, the number of LC3 puncta was quantified using Definiens Developer XD. Data represent the average LC3 puncta per cell ± SD.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
5.
Figure 7

Figure 7. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

A, immunohistochemical staining of LC3 in representative tap and NaHCO3 treated MDA-MB-231 tumor cross-sections. Positive pixel analysis of LC3 staining was carried out using Aperio positive Pixel Count v9 (red, strong positive). B and C, positive pixel analysis was completed for LC3 and carbonic anhydrase IX (CA9) staining on whole tumor cross-sections. An overall significant decrease in the percentage of strong positive LC3 pixels was observed in NaHCO3 treated samples with no significant change in CA9 expression. The data are plotted as the mean ± SD of 3 tumor cross-sections from each treatment group. D, LC3 and CA9 expression in tumor lysates from tap and NaHCO3-treated tumors.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
6.
Figure 5

Figure 5. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

A and B, whole-cell lysates from MDA-MB-231 cells cultured at pH 7.4 or at pH 6.7 for 72 hours were analyzed for expression of phospho-Akt (Ser473) and phospho-ribosomal protein S6 (Ser235/236) using a fluorescent-based phospho-protein array and by Western blot. C, whole-cell lysates from MDA-MB-231 cells cultured at pH 7.4 or at pH 6.7 for approximately 3 months were analyzed for the expression of the autophagy marker LC3-II. Elevated levels of LC3-II were observed in MDA-MB-231/6.7ext cells in comparison to MDA-MB-231/7.4 cells. D, cultures were treated with 10 mmol/L 3-methyladenine (3-MA) for 48 hours and CyQuant, a nonmetabolic indicator of viable cells, was used to determine cell number. The data represent the mean ± SD of 3 experiments.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.
7.
Figure 6

Figure 6. From: Chronic Autophagy Is a Cellular Adaptation to Tumor Acidic pH Microenvironments.

Histologic analysis of in vivo LC3 expression in MDA-MB-231 MFP tumors. A, H&E staining of a representative region from an MDA-MB-231 tumor cross-section. A 20× magnification shows a single vessel encapsulated by viable cells (dark purple) surrounded further by necrotic tissue (pink). The same vascular region from sequential cross-sections was used for immunohistochemical analysis. B–D, pimonidazole hydrochloride was injected 1 hour before tumor removal to detect hypoxic tissue. Additional staining shows similar spatial expression patterns for BNIP-3 (C) and LC3 (D) to pimonidazole hydrochloride. E, positive pixel analysis of LC3 and pimonidazole hydrochloride in the outer region (black region) and inner region (white region) of a vascular cross-section using Aperio™ Positive Pixel Count v9 (blue, negative; yellow, weak positive; orange, positive; red, strong positive). Each region of interest is of the same size containing the same number of pixels. The number of strong positive pixels in the outer and inner region of 5 separate vascular cross-sections is plotted for LC3 and pimonidazole hydrochloride.

Jonathan W. Wojtkowiak, et al. Cancer Res. 2012 August 15;72(16):3938-3947.

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