Bc08013a

Immunofluorescence staining was performed as described previously (Rosales et al. 2015 (link)). Cells were starved of glutamine for 24 h prior to staining with 1:75 diluted anti-IKKβ (Novus Biologicals 10AG2) and 1:300 diluted anti-PFKFB3 (OriGene TA590185) antibodies. Nuclei were visualized by staining with 1 µg/mL DAPI. Images were captured using a Zeiss LSM 700 confocal (immunofluorescence) or Zeiss Observer II (immunohistochemistry) microscope at 20× objective and analyzed using Zen 2012 software. Immunohistochemistry was performed as described previously (Fong et al. 2015 (link)) using 10 mM Tris-HCl, 1 mM EDTA, and 0.05% Tween-20 (pH 9.0) retrieval buffer. p-PFKFB3 Ser 269 antibody was used at 1:100 dilution. Tissue arrays were purchased from US Biomax (brain BS17016a, breast BC08013a, and skin BC21014)

Human breast carcinoma tissue microarray, containing 60 samples of invasive ductal carcinoma, 9 samples of medullary carcinoma, and 3 samples of cancer adjacent normal tissue, was obtained from US Biomax Corporation (BC08013a); all tissue cores were obtained from diagnostic or surgical samples as serial sections according to the supplier (Table S1). RHBDF1 expression and EGFR phosphorylation status were determined by immunohistochemical staining with DAB kit (Invitrogen, USA) according to the manufacturer's instructions. The sections were incubated with anti-phospho-EGFR antibody (Tyr1068) (#3777, 1: 50 dilution, CST) or anti-RHBDF1 antibody (LS-C81438, 1: 100 dilution, LSBio) at 4 °C overnight, followed by incubation with biotinylated secondary antibody and streptavidin-peroxidase. Sections were then visualized with DAB Chromogen Solution, counterstained with haematoxylin, and dehydrated in ethanol. Images were acquired at 20× magnification using an Aperio Digital Pathology Microsystems scanner (Leica, GER). Staining intensities of each section were scored (grades 0–3) using Image Pro Plus. Statistical analysis was performed using SPSS 19.0 software.