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Fig. 4 | Breast Cancer Research

Fig. 4

From: Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts

Fig. 4

miR-218-regulated inhibin βA affects SMAD2/3 signaling in a cell-dependent manner. a and b Western blot analyses of phospho-SMAD2/3 in MDA-231 (a) or MC3T3 (b) that was serum-starved overnight and treated with EV-depleted CM collected from indicated cells for 30 min or 2 h. CM producing cell lines were transfected, PBS washed 48 h after transfection and then incubated with serum-free medium overnight before CM collection and EV depletion by ultracentrifugation. c Western blot analyses of inhibin βA, inhibin βB, and inhibin α in CM from indicated cell lines that was concentrated with medium concentrator columns. Arrows indicate the position of antigen. Control mimic-transfected MDA-231 whole cell lysate (WCL) was used as positive control for inhibin βA and inhibin βB. d Western blot analyses of inhibin βA monomer or dimer (activin A) in CM from indicated cell lines under non-reducing condition and without boiling the samples. Bottom and top arrows indicate inhibin βA monomer and dimer, respectively. e Western blot analyses of inhibin α in MC3T3 CM concentrated from 20 ml CM. WCL of MCF10A was used as a positive control. f and g Western blot analyses of phospho-SMAD2/3 in MDA-231 (f) or MC3T3 (g) that was serum-starved overnight and treated with CM collected from indicated cells for 30 min or 2 h. For MDA-231 cells (f), recombinant inhibin α protein was added to CM as indicated. For MC3T3 cells (g), anti-inhibin α antibody was added to CM as indicated

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