Fig 2: PTP1B is highly expressed in normal human breast tissue. (a) PTP1B is expressed both in the epithelial and stromal compartments of the breast. Immunohistochemical stainings show prominent co-expression of PTP1B (red) and K14 (green) in myoepithelial cells (top row), PTP1B (red) and K18 (green) in luminal epithelial cells (middle row), PTP1B (red) and vimentin (green) stains myoepithelial and fibroblasts (bottom row). Bar 100 µm. (b) PTP1B is predominantly expressed in myoepithelial cells and fibroblasts in the breast gland. qPCR mRNA analysis shows that PTP1B expression is higher in myoepithelial cells (MEP) and fibroblasts than in luminal epithelial cells (LEP) and endothelial cells (ENDO). Expression levels were normalized to GAPDH (upper). Western blot confirmed high expression of PTP1B in myoepithelial cells (lower). Actin was used as a loading control

Fig 3: Inhibition of PTP1B affects branching morphogenesis and induces anoikis-like effects in D492 cells cultured in 3D. (a) Expression of PTP1B in TDLU-like structures generated by D492 cells in 3D culture. (A) D492 cells form branching structures when cultured in 3D rBM matrix. (B) Expression of F-actin (green), PTP1B (red) and (C) expression of ß4-integrin (green) and PTP1B in D492 cells cultured in 3D rBM. Note, the strong expression of PTP1B at the lobular ends. Bar 50 µm. (b) Inhibition of PTP1B induces loss of cell adhesion in 3D structures. D492 cells cultured in 3D rBM were treated with 10 µM of CPT, 32 µM of the PTP1B inhibitor or DMSO as control for 48 h. (c) D492 cells treated with PTP1B inhibitor die by anoikis. D492 cells treated with the PTP1B inhibitor do not show staining for cleaved caspase 3 in 3D culture (green). CPT was used as a positive control for apoptosis, arrows point to cells positive for cleaved caspase 3. Phalloidin stains F-actin (red). DAPI was used for nuclear staining (blue)

Fig 4: PTP1B inhibition affects expression of cell adhesion molecules and actin polymerization. (a) D492 cells treated with PTP1B inhibitor show loose surface attachment. D492 cells were treated with 8 or 16 µM of the PTP1B inhibitor, 10 µM of CPT or DMSO as control for 24 h, and assayed for sensitivity to trypsinization. (b) Expression of various cell adhesion molecules is reduced in D492 cells after PTP1B inhibition for 48 h. D492 cells were treated with various concentrations of the PTP1B inhibitor for 48 h followed by protein isolation. Claudin-1, FAK and E-cadherin expression is lost when treated with 16 µM of the inhibitor. PTP1B inhibition does not affect occludin, ß-catenin and actin expression. (c) F-actin polymerization is lost in D492 cells treated with inhibitor for 5 h. Phalloidin stains F-actin (green), Concavalin A stains endoplasmic reticulum (red)

Fig 5: PTP1B inhibitor decreases Src activation in D492 cells. (a) Treatment with PTP1B inhibitor results in increased phosphorylation of the inhibitory SrcY529. Left, western blot analysis of pSrcY529 shows that PTP1B inhibitor increases phosphorylation of pSrcY529. Protein extracts from D492 cells after treatment with DMSO and various concentrations of PTP1B inhibitor for 48 h. GAPDH as loading control. Right, Immunofluorescence staining of pSrc in D492 cells treated with the PTP1B inhibitor confirms increased phosphorylation of pSrcY529 after PTP1B inhibition. pSrc (green), PTP1B (red), TOPRO-3 nuclear staining (blue). Bar 20 µm. (b) Inhibition of PTP1B disrupts PTP1B protein interactions. PLA shows that PTP1B complexes with Src (left) and EGFR (right) (green arrows) are disassembled (left). DAPI nuclear stain (blue). Cell profiler was used to quantify frequency of positive puncta per cell (right)