Fig. 1: ITSN1-S localized not only in the cytoplasm but also in nuclei of breast cancer tissues and cells.

A Venn diagram showed the DEGs in breast cancer tissues and their paired adjacent tissues by combination analysis of our microarray data from 22 patients’ samples and 34 patients’ samples with the mRNA expression profile of breast cancer patients’ samples (n = 296) downloaded from GEO (ID: GSE70947). Genes with P < 0.05 and |Fold Change | >1.5 were considered as DEGs. B, C Volcano plots showed the DEGs from our microarray analyses of 22 paired breast adjacent normal/tumor tissues (B) and 34 paired breast adjacent normal/tumor tissues (C). The red dots and blue dots represented the upregulated and downregulated genes. X-axes showed log₂ | Fold change| and y-axes showed -log₁₀(P value). D Expression pattern of ITSN1-S in breast cancer tissues (n = 308). Top: representative images of ITSN1-S cytoplasmic expression in IDC specimens. Bottom: representative images of ITSN1-S cytoplasmic and nuclear expression in IDC specimens. Scale bars, 100 μm. E Among 308 IDC cases, 60.7% (187/308) cases showed ITSN1-S cytoplasmic positive expression, 39.3% (121/308) cases showed ITSN1-S cytoplasmic and nuclear positive expression. F Representative immunofluorescence images of endogenous ITSN1-S localization in MDA-MB-231 cells treated with 1 ng/ml LMB for 12 h (+LMB) or without LMB as a control (−LMB). Arrowheads showed the cells with nuclear colocalization. Quantitative results were analyzed in the lower panel. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, ^***P < 0.001). G Western blots analysis of endogenous ITSN1-S expression in the cytoplasm (Cyto) and nuclei (Nuc) of MDA-MB-231 cells. β-actin and histone were used as specific markers for cytoplasm and nuclei, respectively. H Western blot of ITSN1-S expression in 3×flag-ITSN1-S-HA/MDA-MB-231 cells. ITSN1-S expression was detected by anti-ITSN1-S, anti-flag, anti-HA antibodies. I Western blot analysis of exogenous ITSN1-S in the cytosol (Cyto) and nuclear (Nuc) of 3×flag-ITSN1-S-HA/MDA-MB-231 cells. β-actin and histone were used as specific markers for cytoplasm and nuclei, respectively. J Exogenous HA and 3×flag-labeled ITSN1-S protein was detected by immunofluorescence with anti-HA (left panel) and anti-flag (right panel) antibodies in 3×flag-ITSN1-S-HA/MDA-MB-231 cells treated with 5 ng/ml LMB for 5 h (+LMB) or without LMB as a control (−LMB). Scale bars, 25 μm. Quantitative results were analyzed in the lower panel. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, ^***P < 0.001).