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Additional Supporting Information may be found in the online version of this article.

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Supplementary Figure 1. Effect of Rab27B overexpression in MCF-7 breast cancer cells on the expression of V-ATPase subunits V0a1 and V0d1. Lysates were subjected to Western blotting with V-ATPase V0a1, V0d1 and Rab27B-specific antibodies. Tubulin was used as loading control.

Supplementary Figure 2. Effect of V-ATPase inhibitor bafilomycin A1 on matrix invasion of MCF-7 breast cancer cells expressing GFP-Rab27B Q78L, a constitutive active mutant defective in GTP hydrolysis. Upper part, phase contrast images showing morphology of MCF-7 GFP-Rab27B Q78L cells on collagen type I gels under control condition or reversibly treated with V-ATPase inhibitor bafilomycin A1 (1 nM, 24h). Scale bar, 50 µm. Bottom part, quantification of collagen type I invasion by MCF-7 GFP-Rab27B Q78L cells untreated (con) or reversibly treated with bafilomycin A1 (0.1, 1 or 5 nM) for 24h. Data are presented as the mean ± s.e.m. of three independent experiments performed in triplicate.

Supplementary Figure 3. Effect of V-ATPase inhibitor concanamycin A on matrix invasion of GFP-Rab27B wild type and Q78L expressing MCF-7 breast cancer cells. Quantification of collagen type I invasion by MCF-7 GFP-Rab27B (wild type and Q78L) cells untreated or reversibly treated with concanamycin A (0.1, 1 or 5 nM) for 24h. Data are presented as the mean ± s.e.m. of three independent experiments performed in triplicate.

Supplementary Figure 4. Effect of pharmacological inhibition and siRNA targeting of V-ATPase on EMT-dependent matrix invasion by MCF-7 Snail-6SA cells. Upper part, phase contrast images showing morphology of MCF-7 Snail-6SA cells on collagen type I gels under control condition or treated with V-ATPase inhibitor bafilomycin A1 (1 nM, 24h). Scale bar, 50 µm. Bottom part, quantification of collagen type I invasion by MCF-7 Snail-6SA cells untreated (con), reversibly treated with bafilomycin A1 (1 or 5 nM) for 24h, or transiently transfected with control siRNA (siCON) or siV-ATPase V0a1 or V0d1 siRNAs. Data are presented as the mean ± s.e.m. of three independent experiments performed in triplicate.

Supplementary Figure 5. Effect of siRNA targeting of V-ATPase on proliferation of MCF-7 Snail-6SA cells. A total of 104 cells were plated into each well of a total of 12 wells on day 1 to establish one growth curve under each condition in triplicate. The total number of cells per well was automatically counted every day until day 4.

Supplementary Figure 6. Effect of V-ATPase targeting by siRNA and bafilomycin A1 inhibitor (1 nM, 24h) on HSP90α and β expression in MCF-7 GFP-Rab27B breast cancer cells. Lysates were subjected to Western blotting with HSP90α and β antibodies. Tubulin was used as loading control.

Supplementary Figure 7. Epithelial tissue was microdissected from frozen fresh primary human breast cancer tissues (patient samples 1 and 2 are poor prognosis ERα-breast cancers with high Rab27B expression). Lysates were subjected to Western blotting with V-ATPase V0a1, V0d1 and Rab27B-specific antibodies. Tubulin was used as loading control.

Supplementary Table 1. Immunohistochemical analysis of V-ATPase in primary breast tumors

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