Journal: Life Science Alliance

Article Title: Specific N-cadherin–dependent pathways drive human breast cancer dormancy in bone marrow

doi: 10.26508/lsa.202000969

Figure Lengend Snippet: (A) Representative images of tissue sections from biopsies of BC patients (A and S2), hematological malignancy (S3), and benign tumor (S3). The slides were colabeled with anti–CDH2-PE (red), anti–Cx43-AF488 (green), and anti–pan-cytokeratin-AF405 (blue). Images were acquired with EVOS FL Auto 2, 200× magnification. Black arrows show colocalized Cx43 and CDH2 in the white areas (red + green + blue). Inset, zoomed regions of colocalized proteins. (B) Table summarizes the total number of sections positive for colocalized CDH2, Cx43 in pan-cytokeratin + cells. ImageJ software was used to count the colocalized cells in 10 fields/slide. The last column shows the percentages of colocalized CDH2-Cx43. See also Table S1 and and . (C, D) Computational and functional prediction of CDH2–Cx43 interaction using ZDOCK (C) and STRING (D), respectively. CDH2, Cadherin-2 (N-cadherin); TJP1, tight junction protein 1; CTNNB1, β catenin; GJA1, gap junction alpha protein 1(Cx43); JUP, junction plakoglobin; NEDD4, neural precursor cell expressed, developmentally down-regulated 4. (E) Whole cell lysates from MDA-MB-231 and T47D were immunoprecipitated with anti-CDH2 or IgG and then electrophoresed on 12% SDS–PAGE. The membranes were blotted with anti-Cx43 (light band at 43 kD). (F) Whole-cell lysated from MDA-MB-231 cells were subjected to immunoprecipitation (IP) with anti-CDH2 or anti-Cx43. The membrane was blotted with anti-CDH2. (G) MDA-MB-231 was transfected with pCMV2-CDH2 Flag and pcDNA 3.2-Cx43-HA. Protein lysates were immunoprecipitated with anti-Flag or anti-IgG and blotted with anti-Flag or anti-HA. (H) Lysates from cancer stem cells, isolated from MDA-MB-231 were immunoprecipitated with anti-IgG, anti-Cx43, or anti-CDH2. The samples were electrophoresed and then blotted with anti-CDH2. (I) BM biopsy from BC patient (#3) was subjected Proximity Ligation Assay with anti-CDH2 and anti-Cx43. The proximity of the two antibodies was determined using EVOS FL Auto 2, 600× magnification. Control slide was labeled with isotype control. Source data are available for this figure.

Article Snippet: Human CDH2 and Cx43 siRNA and Risc free (control) were purchased from Dharmacon; human cyclin D1 promoter in pGL3-basic (research resource identifiers, RRID:Addgene_32726) and pcDNA 3.2 with Cx43-HA (RRID:Addgene_49851) were obtained from Addgene (which was a donation from Frank McCormick and Anne Brunet laboratory, respectively); pCMV2-CDH2-Flag from Sino Biologicals; and human shRNA pRFP-C-RS with scramble sequence, CDH2-shRNA, or Cx43 shRNA from OriGene Technologies.

Techniques: Software, Functional Assay, Immunoprecipitation, SDS Page, Membrane, Transfection, Isolation, Proximity Ligation Assay, Labeling