human erbb2 expression plasmid  (Biogen Inc)

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Exosomes derived from MDA-MB-231 cells stimulate RANKL-induced osteoclastogenesis in vitro . (A) Representative transmission electron microscopy images of exosomes derived from MDA-MB-231 cells. Scale bar, 500 nm (left panel) and 100 nm (right panel). (B) Nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells. (C) Western blot analysis of whole cell lysates (WC) and exosomes (EXOs) prepared from MDA-MB-231 cells. (D) BMMs were treated with exosomes derived from MDA-MB-231 cells in the presence of RANKL and M-CSF for 4 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (E) BMMs were treated with exosomes derived from MDA-MB-231 cells treated with vehicle (Con) or the pan RAS inhibitor, salirasib (10 µ M), in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. ** P<0.01. (F) BMMs were treated with exosomes derived from MCF-7 cells transfected with control vector (Con) or K-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. *** P<0.001. (G) BMMs were treated with exosomes derived from T47D cells transfected with control vector (Con), K-RASV12, H-RASV12 , or N-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (H) BMMs were treated with exosomes derived from control vector (Con) or HER2 vector-transfected T47D cells in the presence of RANKL and M-CSF for 6 days. * P<0.05 and **** P<0.0001. RANKL, receptor activator of nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage colony-stimulating factor; EXOs, exosomes; ALIX, apoptosis-linked gene 2-interacting protein X; HSP, heat shock protein; TSG101, tumor susceptibility 101; GM130, Golgi matrix protein 130; HER2, human epidermal growth factor receptor 2.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Derivative Assay, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Staining, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Identification of osteoclastogenic miRNAs in exosomes induced by RAS activation. (A) Expression levels of 28 miRNAs in exosomes derived from MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. (B) The expression levels of 11 selected miRNAs in exosomes derived from control or KRASV12 vector-transfected MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (C) Cellular expression levels of 11 selected miRNAs in MCF-7 cells transfected with the control or K-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. (D) The expression levels of eight selected miRNAs in exosomes derived from MDA-MB-231 cells treated with the control or salirasib (10 µ M) were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (E) The expression levels of seven selected miRNAs in exosomes derived from T47D cells transfected with the control vector (Con) or N-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05, ** P<0.01 and **** P<0.0001. (F) Representative images of TRAP-positive osteoclasts in BMMs transfected with the indicated miRNAs. BMMs transfected with the indicated miRNAs (20 nM each) were stimulated with RANKL and M-CSF for 4 days. NC, miRNA mimic negative control. ** P<0.01. (G) The expression levels of miR-494-3p, miR-1915-3p, miR-4508 and miR-6869-5p in sera derived from patients with HER2-positive breast cancer (n=19) and triple-negative breast cancer (n=15). The results were normalized to U6 snRNA. * P<0.05, *** P<0.001 and **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; M-CSF, macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; BMMs, bone marrow-derived macrophages.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Activation Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Negative Control, Real-time Polymerase Chain Reaction