Fig 2: Msi is associated with the epithelial-luminal state in breast cancer.(A) mRNA expression of Msi2 across different breast tumor types in TCGA RNA-Seq. (B) Immunofluorescence staining for Ecadherin (ECAD, red) and Msi1 (MSI1, green). Top: luminal human breast tumor with high number of ECAD-positive cells. MSI1 shows primarily cytoplasmic localization (white arrowheads). Inset shows magnified version of ECAD and MSI staining. Bottom: triple negative, basal-like tumor. ECAD-positive cells showed strong cytoplasmic MSI1 stain (blue arrowheads) while ECAD-negative cells were MSI1-negative (red). Single confocal stacks shown, 10 μm scale. (C) mRNA expression of Msi1, Msi2, Ecad, Fn1, Vim, and Jag1 in breast cancer cell lines by RNA-Seq (datasets are listed in Supplementary file 1). (D) Western blot for MSI1/2 (MSI1/2 cross react. antibody), MSI2, phosphorylated HER2 (p-HER2) and HER2 in panel of breast cell lines. ‘HMLE + pB’ indicates HMLE cells infected with pB empty vector, ‘HMLE + Twist’ indicates HMLE cells infected with Twist transcription factor to induce EMT. MDAMB231-derived metastatic lines (231-Brain, 231-Bone) and Sum159 are basal, HER2-negative cancer cell lines. BT474 and SKBR3 are HER2-positive, epithelial-luminal cancer cell lines. Epithelial-luminal (HER2-positive) lines show increased expression of Msi proteins compared with basal lines, and Twist-induced EMT reduces Msi expression. (E) mRNA expression of Msi1, Msi2, Ecad, Fn1, Vim, and Twist1 in GBM tumors classified as mesenchymal (n = 20) or epithelial (n = 20) using an EMT gene signature.DOI: http://dx.doi.org/10.7554/eLife.03915.005

Fig 3: Knockdown of Msi1/2 in breast cancer cell lines.(A) Western blot for BT474 cells with control (shLuc) or Msi1/2 targeting hairpins. (B) Morphology of MCF7-Ras cells upon Musashi knockdown.DOI: http://dx.doi.org/10.7554/eLife.03915.018

Fig 4: Effect of Msi1 gain and loss of function on Jag1 mRNA levels and protein expression.Fold-change in Jag1 expression in Msi1 overexpression and double knockout samples for Ribo-Seq and RNA-Seq experiments.DOI: http://dx.doi.org/10.7554/eLife.03915.012

Fig 5: Genetic system for studying effects of Msi loss/gain of function on gene expression.(A) Experimental setup and use of Msi1/2 inducible overexpression and conditional double knockout mice for derivation of neural stem cells, which were then used for ribosome profiling (Ribo-Seq) and mRNA sequencing (RNA-Seq). (B) Western blot analysis of Musashi overexpression and knockout in neural stem cells. Overexpression and conditional knockout cells were exposed to Dox and 4-OHT for 72 hr, respectively. (C) mRNA-Seq expression values (RPKM) scatters between Msi1 overexpressing cells and controls (left), Msi2 overexpressing cells and controls right (72 hr Dox). Msi1/2 each robustly overexpressed with similar magnitude following Dox. (D) Comparison of translational efficiency (TE) values using Ribo-Seq on Msi1 overexpressing cells on Dox (72 hr) vs controls (left) and conditional knockout cells following 4-OHT for 48 hr (right). Colored points indicate select genes with large changes in TE.DOI: http://dx.doi.org/10.7554/eLife.03915.008