Data on SVCT2 transporter expression and localization in cancer cell lines and tissues

The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer”, available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.

and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.
© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Data
Here we report experimental data on SVCT2 transporter expression and localization in human cancer cell lines and tissues. Analysis of SVCT2 expression by RT-PCR and Western blot in four breast cancer cell lines are shown (Fig. 1). Immunofluorescence colocalization assays for SVCT2 with various organelle markers in the breast cancer cells MCF-7, MDA-231 and MDA-468 revealed mitochondrial localization of SVCT2 (Fig. 2). This was confirmed with cellular fractionation experiments followed by Western blot for various subcellular fractions (Fig. 3). We finally analyzed the SVCT2 expression in the Human Protein Atlas, a public database containing a tissue-based map of the human proteome. Examination of numerous tumor samples of different origins (Fig. 4A-U) revealed a mainly intracellular Specifications

Data format
Analyzed. Experimental factors Breast cancer cell lines were cultured in standard conditions.

Experimental features
RT-PCR and western blot were performed to analyze SVCT2 expression. Immunofluorescence and cellular fractionation followed by western blot were performed to analyze SVCT2 subcellular localization. Several cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas were analyzed. Data source location Concepci on, Chile.

Data accessibility
Data is provided in this article.

RT-PCR analysis
PCR experiments were performed using the protocol described in Ref. [2]. Briefly, total RNA was extracted from different cell lines using an RNeasy mini kit (Qiagen). cDNA was synthesized with the AffinityScript Multi-Temp RT (Agilent) plus oligo(dT) and random primers. PCR reaction mixture included specific primers for SVCT2 (or Actin) amplification, Taq 2 Master Mix (New England BioLabs) and 100 ng of cDNA. Amplification products were examined by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining.

Western blot analysis
Immunoblotting experiments were performed using the protocol described in Ref. [1]. Briefly, total extracts were prepared from different cell lines, quantified, separated by SDS-PAGE and transferred to PVDF membranes. Detection was performed using anti-SVCT2 and anti-GAPDH primary antibodies (Santa Cruz Biotechnology, Inc) followed by Alexa 680 and Alexa 790 secondary antibodies (Jackson    Immunoresearch). Membranes were scanned and analyzed with an Odyssey CLx Imaging system (LI-COR Biosiences).

Immunolocalization
Immunofluorescence experiments were performed using the protocol described in Ref. [1]. Briefly, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, blocked with bovine serum albumin, incubated with primary antibodies and incubated with secondary antibodies, before observation by confocal microscopy. The following primary antibodies were used: anti-SVCT2 and anti-RCAS1 (golgi protein) from Santa Cruz Biotechnology, Inc.; anti-COXIV (inner mitochondrial membrane), anti-LAMP2 (lysosome), anti-PDI (endoplasmic reticulum membrane) and anti-GLUT1 from Abcam, Inc. Three different secondary antibodies were used: fluorescein-labeled anti-mouse IgG and Cy3-labeled anti goat-IgG from Jackson Immunoresearch; fluorescein-labeled anti-rabbit IgG from Dako. Samples were observed using an Olympus IX81 fluorescence microscope with a DSU (Disk Scanning Unit) spinning disk confocal system and images were obtained by a HAMAMATSU ORCA-R2 camera controlled by Olympus Xcellence R software. After deconvolution, images were subjected to Pearson colocalization analysis. Statistical correlation of the colocalization index among the different organelle markers was established by one-way Anova, followed by Tukey test for multiple comparison.

Mitochondrial isolation
Cellular fractionation and mitochondria purification were performed using the protocol described in Ref. [1]. Briefly, mitochondria were isolated from different cell lines by differential centrifugation with all steps carried out at 4 C [3]. The final pellet containing mitochondria and different fractions obtained (T: total homogenate; N: nuclear fraction; C: cytoplasmic fraction; R: endoplasmic reticulum) were separated by SDS-PAGE, transferred to PVDF membranes and immunodetected with anti-SVCT2 and anti-COXIV antibodies.