104778-14-5 br Flow cytometry br Cells were seeded
Cells were seeded in 6-well plates and once 70% confluency was reached, detached with 0.05% trypsin, washed with FACS buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0. 02% NaN3), and incubated for 1 h with the appropriate antibody (Table S1) in FACS buffer at room temperature. After washing with FACS buffer, cells were analyzed using CyFlow space flow cytometer (Sysmex-Partec). For the determination of ALDH activity, the ALDEFLUOR kit (STEMCELL Technologies) was used following the manufacturer's instructions and analyzed with Cytoflex flow cytometer (Beckman).
Immunoprecipitation & immunodetection of proteins
Recombinant, his-tagged COMP was purified from Free-style 293-F cell supernatants using Ni-column (Thermo Fisher Scientific) . Total protein lysates were obtained with RIPA buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.2], 0.1% SDS, 1% Triton X-100, 1% deoxycholate) or NP40 buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP40) containing Pierce prote-ase inhibitor mini tablet EDTA-free and HALT phos-phatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations in lysates were estimated with Pierce BCA Protein Assay Kit, with equal amounts separated by SDS-PAGE, and transferred onto PVDF membranes with Trans-Blot Turbo Transfer Pack (Bio-Rad). Nuclear and cytoplasmic cellular fraction-ation was achieved utilizing the NE-PER kit (Thermo Fisher Scientific). For immunoprecipitation, 1 mg of total protein was used with 50 μl of Dynabeads Protein G pre-coated with 5 μg of capturing antibody, after overnight incubation, beads were extensively washed with NP40 buffer, and finally immunoprecipitated proteins eluted with 40 μl of Laemmli buffer. Concen-tration of COMP in cells supernatants measured utilizing commercially available ELISA (R&D systems)
following manufacturer instructions. For ELISA binding assays, Maxisorp 96 well microtiter plates (Thermo Fisher Scientific) were coated with 5 μg/ml of anti-Notch3 or anti-Jagged1 antibody. The cells were lysed with NP40 buffer and the same amounts of total protein as determined using the BCA method (Thermo Fisher Scientific) were added to the plates 1 h. The interaction was detected with the homemade anti-COMP antibody , followed by secondary anti-rabbit and OPD substrate. Secondary 104778-14-5 used are listed in Table S1. Evaluation of COMP binding to MDA-MB-231 cells was performed by detaching control cells with Versene solution (Thermo Fisher Scientific), incubating 3 × 105 cells with different concentrations of recombi-nant COMP in PBS at 37 °C under agitation, and finally detecting the cell-bound COMP, utilizing FACS analy-ses with homemade anti-COMP antibody and a secondary anti-rabbit FITC conjugated antibody (Table S1).
Luciferase reporter expression assay
Immunofluorescent & dual link assay
Coverslips were added to 24 well plates, coated with Poly-D-lysine (Sigma), and afterwards 1.5 × 105 cells were seeded. The following day, cells were fixed with 4% formaldehyde (Merck) and permeabilized with 0.1% Triton X-100 (Merck). Proximity ligation assays was performed following manufacturer protocol for Dual link kit (Sigma) using the appropriate antibodies (Table S1).
The mouse experiments were approved by the local Ethical Committees for Animal Experimentation
Fig. 1. COMP expression in breast cancer cells enhances their tumorigenicity in vivo and in vitro (A) MDA-MB-231 COMP-expressing cells transplanted into the mammary fat pad of female NSG mice resulted in higher number of tumors formed compared to control cells injected mice. (B & C) Frequency of cancer stem cells in MDA-MB-231 COMP-expressing cells and control cells were calculated utilizing the ELDA software. COMP expression enhanced the frequency of cancer stem cells in a statistically significant manner (x2 = 28.9 p = 7.8 × 10−8) (D) Representative pictures from tumorsphere formation assay (E) BT-20 and MDA-MB-231 cell lines expressing COMP form larger tumorspheres compared to control cells. The length of the COMP-expressing tumorspheres is reduced, in the presence of DAPT or anti-Jagged1 neutralizing antibody. Two-way ANOVA Bonferroni's multiple comparisons test was used (*b0.05, **b0.01, ***b0.001, ****b0.0001). 110
COMP leads to activation of Notch3
COMP leads to activation of Notch3 111
in Lund, application M142/13. For the serial dilution experiment 1 × 105, 1 × 104 and 1 × 103 MDA-MB-231 COMP-expressing cells and their corresponding control cells in 50 μl PBS were orthotopically transplanted in the 4th inguinal mammary fat pad of female NSG mice (own breeding, n = 4). Tumor formation was evaluated 8 weeks after injection and cancer stem cell frequency calculated using the ELDA software .
C57BL/6 COMPKO mice  were backcrossed with FVB/N-Tg (MMTVPyVT) 634Mul/J transgenic mice for 5 generations. The presence of the MMTV-PyMT transgene and the heterozygous or homozygous knockout of the Comp allele in mice were verified through genotyping. MMTV-PyMT: F, 5′-GGAAGCAA GTACTTCACAAGGG-3′ and R, 5′-GGAAAGTCAC-TAGGAGCAGGG-3′; WT Comp: F, 5′-CAGAAGCAAA GGTCGTGGAA-3′ and R, 5′-TTGCTCGTTAGG-GACTCCAT-3′; KO Comp: F, 5′-CGCCTTCTTGAC-GAGTTCTT-3′ and WT Comp R. Over a period of one-year, mice aged 14 weeks were analyzed for tumors volumes and tissues were collected for further analy-ses. The size of the tumor in each of the ten mammary glands in transgenic MMTV-PyMT mice was measured using a caliper. The tumor volume was calculated as length × width2 × (π/6). To estimate the metastatic spread, lungs were paraffin-embedded. The collected lung lobe was post-fixed in 4% paraformaldehyde for 12 h at 4 °C. The embedded lungs were sectioned saving one section (5 μm) and discarding the following