Alexa fluro 488

The following antibodies were utilized for western blot and immunofluorescence studies. Mouse anti-PI3,4P2 mAb and mouse anti-PI3,4,5P3 mAb conjugated with FITC from Echelon Bioscience (Salt Lake City, UT). Rabbit anti-EEA1 mAb from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit Ig-HRP conjugate, goat anti-mouse Ig-HRP conjugate, and goat anti-mouse pAb conjugated with Alexa Fluro 488 from Invitrogen (Carlsbad, CA). Rabbit anti-LAMP1 pAb, and rabbit anti-Rab5 pAb from Abcam (Cambridge, England). Mouse anti-Rab7 mAb from Sigma Aldrich (St. Louis, MO). For phagocytosis and phagosome maturation assays, pHrodo™ Red E. coli BioParticles™ Conjugate and DQ™-BSA Red (used in 96 well plate assay) and DQ™-BSA -Green (used for live cell assay) was purchased from Invitrogen.

All chemicals were purchased from Sigma-Aldrich unless noted otherwise. Antibodies against c-myc, cleaved caspase3, cleaved PARP, and GST were purchased from Cell Signaling Technology (Beverly, MA). Anti-GAPDH, γ-H2A.X, β-actin, CDK5, p35, GFP, HA, p-S/T/Y, and Thiophosphate ester antibodies were from Abcam (Cambridge, MA). Anti-CLIC4 antibody was from Novagen (Madison, WI). Fluorescent anti-mouse or anti-rabbit IgG antibodies conjugated with Alexa Fluro 488 or Alexa Fluro 594 were from Invitrogen. Protein G Dynabeads were from Life technologies, and Glutathione Sepharose beads were from GE. ATP-γ-S and p-nitrobenzyl mesylate (PNBM) were from Abcam.

The frozen primary or PDX breast cancer tissues were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, blocked with 3% BSA, and stained with an anti-TRIB3 (1:100, Abcam, ab137526), anti-CD68 (1:100, Abcam, ab955), anti-FOXO1 (1:100, Abcam, ab52857), or anti-SOX2 (1:100, Abcam, ab171380) primary antibody followed by Alexa Fluro 488 and/or Alexa Fluro594 secondary antibodies (1:200, Life Technologies). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained by using a laser scanning confocal imaging system (Olympus Microsystems).

Immunofluorescence assays were performed on paraffin-embedded sections prepared as described above. Sections were dried for 1 hour at 55°C, deparaffinized and rehydrated. After antigen retrieval (Vector, H-3300), sections were blocked for 1 hour at room temperature in blocking solution (PerkinElmer, FP1020), and then incubated with primary antibodies diluted in blocking solution at 4°C overnight. After washing three times with PBST (0.1% Tween20 in 1xPBS), sections were incubated with secondary antibodies at room temperature for 1 hour. For periostin and Lhx6 protein detection, fluorescent signal amplification Tyramide SuperBoost Kits, including goat anti-rabbit Alexa Fluor-647 (B40926, Thermo Fisher Scientific) and goat anti-mouse Alexa Fluro-488 (B40912, Thermo Fisher Scientific) were used according to the supplier’s protocols. DAPI (Sigma, D9542) was used for nuclear staining. Detailed information on antibodies used in this study is available in S2 Table.

Invasive ductal carcinoma and adjacent normal breast tissue samples were obtained from US Biomax, BC08118a. The slide was baked at 60°C for 2h followed by incubation in Neo-clear (Sigma 109843) for 5min. The slide was re-hydrated in serial dilutions of Ethanol and then boiled in Tris/EDTA (pH 9,0) for 15min. After washing with PBS for 5min the slide was permeabilized using triton X-100 (0,25%) for 8min and then blocked in 5% BSA (in PBST) for 1h at RT. To stain the Golgi, the slide was incubated with anti-Giantin antibody (Nordic BioSite, bs-13356) in 1% BSA (PBST) for 2h at RT. After three times washing with PBST for 5min the slide was incubated for another 1h in goat anti-mouse secondary antibody, Alexa Fluro 488 (Thermo Fisher Scientific, A-11001). After several washing steps, the Hoechst 33342 dye (Thermo Fisher Scientific, H3570) was used to label the nuclei. Finally, the slide was rinsed with Milli-Q water and embedded in polyvinyl alcohol mounting medium (Sigma Aldrich, 10981). The fluorescence images were acquired and analyzed as previously described (section 2.6).