Volume 33, Issue 1, Pages (February 2014)

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Volume 33, Issue 1, Pages 136-142 (February 2014) Calcitriol supplementation effects on Ki67 expression and transcriptional profile of breast cancer specimens from post-menopausal patients Yuri Nagamine Urata, Eduardo Carneiro de Lyra, Maria Lucia Hirata Katayama, Ricardo Alves Basso, Paulo Eduardo Zuccolotto de Assis, Ana Paula Torres Cardoso, Rosimeire Aparecida Roela, Suely Nonogaki, João Carlos Guedes Sampaio Góes, M. Mitzi Brentani, Maria Aparecida Azevedo Koike Folgueira Clinical Nutrition Volume 33, Issue 1, Pages 136-142 (February 2014) DOI: 10.1016/j.clnu.2013.04.001 Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 1 Ki67 expression in breast cancer samples. Post-menopausal patients were supplemented with calcitriol for a median period of 30 days. Tumor samples were collected before (through biopsy, left hand side) or after (during breast surgery, right hand side) supplementation and processed for Ki67 immunodetection. Tumors included in the control group were collected from post-menopausal breast cancer patients, who were not supplemented with calcitriol. Bar inside figure: 10 μm. Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 2 Ki67 expression in paired individual tumors from patients supplemented or not (control) with calcitriol. Tumor samples were collected before (through biopsy) or after (during breast surgery) supplementation and processed for Ki67 immunodetection. Tumors included in the control group were collected from post-menopausal breast cancer patients, who were not supplemented with calcitriol. Each line represents matched tumor samples from a single patient [pre & post-supplementation or biopsy (bio) & surgery (sur)]. Interrupted lines: Ln (%Ki67+) >1; Continuous line: Ln (%Ki67+): <1, in samples collected during surgery. Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 3 Age of patients, duration of calcitriol (D3) supplementation (days), tumor dimension evaluated by ultrasonography (mm) and 25(OH)D3 serum concentration (ng/mL) pre- and post-calcitriol supplementation, grouped according to lnKi67 (≥1 or <1) in tumor samples after supplementation. No significant differences were found between groups (p > 0.05, Mann Whitney or Student t test, as appropriate). Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 4 Hierarchical clustering of breast cancer samples from post-menopausal patients supplemented with calcitriol. Matched samples (identified by numbers 15–43) were collected before (pre) and after (post) calcitriol supplementation and gene expression was analyzed by microarray. Each column represents a tumor sample. Colored lines of the dendrogram stand for the support for each clustering, black and gray, more reliable; yellow and red, less reliable. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Fig. 5 Expression of ATF3, FOS, EGR1, PBEF1 in tumor samples before and after calcitriol supplementation. RT-qPCR was performed in paired tumor samples from another 4 patients supplemented with calcitriol. Relative expression values are shown on the Y axis. Pre-supplementation samples on the left; post-supplementation samples on the right. There were no expression differences between groups (p > 0.05, Wilcoxon signed ranks test). Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions

Supplementary Fig. 1. Correlation between expression of genes evaluated through microarray and RT-PCR. Tumor samples from 14 patients were collected before and after calcitriol supplementation (n = 28); gene expression was evaluated through microarray and RT-PCR. Values shown are log2 relative expression (p < 0.05; Pearson correlation). Clinical Nutrition 2014 33, 136-142DOI: (10.1016/j.clnu.2013.04.001) Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism Terms and Conditions