Journal: Molecular Cancer
Article Title: Lung endothelial PEAR1 induces tumor cell dormancy
doi: 10.1186/s12943-025-02488-3
Figure Lengend Snippet: PEAR1 sequesters CTSD and LOXL2 to promote tumor cell dormancy. A , MDA-MB-231 cells were treated with increasing concentrations of the extracellular domain of recombinant human PEAR1 containing a His-tag and immobilized on Ni-NTA beads. Cells incubated with Ni-NTA beads only served as a control group. Cell count was determined by flow cytometry analysis ( n = 3 independent experiments). B , MDA-MB-231 cells were treated daily for 4 days with PEAR1 immobilized on Ni-NTA beads, and cells were analyzed by immunoblotting using anti-GAPDH and anti-p27 antibodies. Shown is a representative immunoblot and the statistical analysis ( n = 3 independently performed experiments). C , D , MDA-MB-231 cells expressing mVenus-p27K − were transfected with control siRNA or siRNA directed against SVEP1 or LOXL2 and were incubated in the absence or presence of PEAR1 (100 nM). After 36 h, the cell count (C) and the percentage of p27-positive cells (D) was determined ( n = 3 independent experiments). E , Procedure to search for interaction partners of PEAR1 within the conditioned medium of tumor cells using a pull-down assay with PEAR1 immobilized on Ni-NTA beads. Control Ni-NTA beads and the recombinant extracellular part of human epidermal growth factor receptor immobilized on Ni-NTA beads served as controls. F , The plot shows the proteins that were specifically co-precipitated with PEAR1 compared to control samples. Proteins only found with PEAR1 are indicated by green dots, and proteins found with both PEAR1 and EGFR are represented by grey dots. G , Recombinant CTSD, SEMA6D or LOXL2 were incubated alone or together with purified His-tagged PEAR1. PEAR1 was precipitated with Ni-NTA beads, and precipitates were analyzed by immunoblotting. Shown are representative of 3 independently performed experiments. H , I , MDA-MB-231 cells co-expressing mCherry-luciferase and mVenus-p27K − were co-cultured with HUVECs for the indicated time periods, and cells were treated daily without or with CTSD (100 nM), LOXL2 (100 nM) or SEMA6D (1000 nM). The cell count (H) was assessed using flow cytometry. At the end of the experiment, the percentage of p27-positive cells (l) was determined by flow cytometry ( n = 3 independent experiments). J , K , Co-cultures of HUVECs and MDA-MD-231 cells were incubated for the indicated time periods with 100 nM PEAR1 alone or in combination with 100 nM CTSD or LOXL2. Assessment of cell count (J) was conducted utilizing flow cytometry, and, after 4 days, the percentage of p27-positive cells (K) was analyzed by flow cytometry ( n = 3 independent experiments). L , M , MDA-MB-231 cells co-expressing mCherry and mVenus-p27K − were transfected with control siRNA or siRNA directed against CTSD, LOXL2 or both, and were cultured in the absence or presence of the recombinant extracellular part of human PEAR1 (rhPEAR1; 100 nM). After 96 h, cells were counted (L), and p27 expression was determined (M) ( n = 3 independently performed experiments). Shown are mean values ± S.E.M.; * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A-D, H, J, L, M); multiple two-tailed t-test, Bayesian moderated, Benjamin, Krieger, and Yekutieli-corrected, false discovery rate = 1% (F); one-way ANOVA with Tukey’s multiple comparison test (I, K))
Article Snippet: 6x-His tagged mouse recombinant CTSD protein (catalog #50127-M08H) was purchased from Sinobiological.
Techniques: Recombinant, Incubation, Control, Cell Counting, Flow Cytometry, Western Blot, Expressing, Transfection, Pull Down Assay, Purification, Luciferase, Cell Culture, Two Tailed Test, Comparison
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