opg sirna  (Santa Cruz Biotechnology)

Journal: Cell death discovery

Article Title: Endogenous osteoprotegerin (OPG) represses ERα and promotes stemness and chemoresistance in breast cancer cells.

doi: 10.1038/s41420-024-02151-8

Figure Lengend Snippet: Fig. 7 OPG downregulation inhibits the breast carcinogenesis of breast cancer cells. MDA-MB-231 and BT-20 cells were transfected with OPG siRNA (OPG-si), while a scrambled sequence was used as control (Ctrl). A Western blot images with GAPDH used as internal control. The numbers underneath each band represent fold changes relative to the control (Ctrl) after normalization to GAPDH. Phosphorylated protein fractions were determined by quantification and normalization against their relative non-phosphorylated forms (B) Cellular analysis using the real-time xCELLigence RTCA System. C Cells (2 × 105) were harvested and stained with propidium iodide (PI), and were analyzed by flow cytometry. The fractions of cells in each phase of the cell cycle are depicted in the boxes. D Cells (1 × 103) were seeded in ultra-low attachment 96‐well plate containing stem cell‐specific medium. The number of spheroids (>100 μm) were counted. Representative photographs of spheroids (left panel), number of formed spheroids (right panel). Error bars represent mean ± SEM (n = 3). *P ≤0.05; **P ≤0.01.

Article Snippet: Transfection with OPG siRNA and universal scrambled sequence (30 nM) (Santa Cruz Biotechnology) was performed using the RNAiFect reagent (Qiagen) following the manufacturer recommendations.

Techniques: Transfection, Sequencing, Control, Western Blot, Staining, Cytometry

Journal: Stem Cells Translational Medicine

Article Title: Skeletal stem cell‐mediated suppression on inflammatory osteoclastogenesis occurs via concerted action of cell adhesion molecules and osteoprotegerin

doi: 10.1002/sctm.19-0300

Figure Lengend Snippet: Skeletal stem cells (SSCs) suppress inflammatory osteoclast (OC) formation through secreted osteoprotegerin (OPG). SSCs (2 × 10 3 SSCs per well) were cultured in the lower compartment of Transwell chambers with a 0.4‐μm‐pore‐size membrane, with CD11b + monocytes (2 × 10 3 CD11b + per well) in the upper compartment. SSC maintained the inhibitory effects without cell‐cell contact. Bars represent 200 μm. B, The tartrate‐resistant acid phosphatase positive (TRAP+) osteoclast formation partially recovered. ** P < .01, compared with no‐Transwell group, n = 5. In addition, (C) OPG mRNA expression in SSCs and (D) SSC‐secreted OPG were detected by quantitative PCR and ELISA, respectively. Both mRNA expression and protein secretion by SSCs were remarkably elevated in the presence of human osteoarthritis synovial fluid (OASF). * P < .05; ** P < .01, compared with no‐OASF group, n = 6. E, Furthermore, the TRAP + osteoclast formation in the SSC/OC coculture system (2 × 10 3 SSC and 2 × 10 3 CD11b + monocytes per well) was partially rescued when OPG in SSC were blocked with OPG‐targeted RNAi and (F) an anti‐OPG‐neutralization antibody (200 ng/mL). * P < .05, compared with NC groups, n = 6

Article Snippet: For the RNAi assay, the murine OPG targeting siRNAs for OPG and a negative control (NC) were obtained from Ribobio ( http://www.ribobio.com ).

Techniques: Cell Culture, Pore Size, Membrane, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Oncotarget

Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

doi: 10.18632/oncotarget.21379

Figure Lengend Snippet: A. The expression of surface antigens on BM-MSCs, AT-MSCs, and T-MSCs were detected by flow cytometry. Cells were negative for hematopoietic cell markers (CD14, CD34, CD45) and positive for CD73, CD90 and CD105. The data show a representative histogram from three experiments. B. BM-MSCs, AT-MSCs, and T-MSCs were harvested and the mRNA expression of TNFRSF11B (OPG encoding gene) was analyzed by real time-quantitative PCR. Data are presented as means ± SEM (* P < 0.05). C. Cell culture supernatants were collected and subjected to western blotting to detect secreted OPG from BM-MSCs, AT-MSCs, and T-MSCs. The pixel densities of the OPG bands were divided by those of the corresponding β-actin bands for normalization. Data are presented as means ± SEM (* P < 0.05). D. The OPG levels in cell supernatants from BM-MSCs, AT-MSCs, and T-MSCs were measured by ELISA. Data are presented as means ± SEM (* P < 0.05). E. Opg -specific siRNA effectively knocked down endogenous OPG expression in T-MSCs. The pixel densities of the OPG bands were divided by those of the corresponding β-actin bands for normalization. Data are presented as means ± SEM (* P < 0.05).

Article Snippet: To reduce endogenous OPG expression, T-MSCs were transfected with OPG-specific siRNA oligonucleotides (Santa Cruz Biotechnology) using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

doi: 10.18632/oncotarget.21379

Figure Lengend Snippet: A. RAW 264.7 cells were cultured on calcium phosphate-coated plates. Naïve CD4+ T cells were added to the RAW 264.7 cell cultures with anti-CD3 Ab, anti-CD28 Ab, IL-6, IL-23, and TGF-β. To test the effect of T-MSC-derived OPG, RAW 264.7 cell cultures were treated with naïve CD4+ T cells and T-CM, OPG-knockdown T-CM, control siRNA knockdown T-CM, or OPG-knockdown T-CM supplemented with rhOPG. After 6 days, resorption activity assays were performed. B. Intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P < 0.05).

Article Snippet: To reduce endogenous OPG expression, T-MSCs were transfected with OPG-specific siRNA oligonucleotides (Santa Cruz Biotechnology) using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions.

Techniques: Cell Culture, Derivative Assay, Knockdown, Control, Activity Assay, Software

Journal: Oncotarget

Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

doi: 10.18632/oncotarget.21379

Figure Lengend Snippet: A. RAW 264.7 cells were cultured on calcium phosphate-coated plates and stimulated with RANKL (50 ng/ml) and SDF-1 (20 ng/ml). Recombinant rhOPG (100 ng/ml), naïve CD4+ T cells (with anti-CD3 and anti-CD28 Abs), or Th17-skewing cytokines alone (IL-6, IL-23, and TGF-β) were added. B. RAW 264.7 cells were cultured with Th17 cells in the presence of RANKL (50 ng/ml) and SDF-1 (20 ng/ml). OPG-knockdown T-CM or control siRNA-treated T-CM were added to RAW 264.7 cell cultures to observe the effect of T-MSC-derived OPG. Exogenous rhOPG was used as a positive control and was also added to OPG-knockdown T-CM. C. - D. After 6 days, resorption activity assays were performed and the intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P < 0.05).

Article Snippet: To reduce endogenous OPG expression, T-MSCs were transfected with OPG-specific siRNA oligonucleotides (Santa Cruz Biotechnology) using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions.

Techniques: Cell Culture, Recombinant, Knockdown, Control, Derivative Assay, Positive Control, Activity Assay, Software

Journal: Oncotarget

Article Title: Androgen deprivation therapy sensitizes triple negative breast cancer cells to immune-mediated lysis through androgen receptor independent modulation of osteoprotegerin

doi: 10.18632/oncotarget.8274

Figure Lengend Snippet: MDA MB 231 (AR-, TNBC) cells were transfected with control or OPG siRNA. A. Amount of OPG in the supernatant of MDA MB 231 cells 48 hours after siRNA transfection. Sensitivity of MDA MB 231 cells to B. CEA-specific CD8+ T cell-mediated lysis or C. TRAIL-mediated lysis cells 48 hours after siRNA transfection. Error bars indicate mean ± S.E.M. for quadruplicate measurements. Statistical analyses were done by Student's t -test, * = P < 0.05 vs . vehicle control. Data are representative of 2 independent experiments.

Article Snippet: OPG expression was inhibited in MDA MB 231 cells using siRNA duplexes targeting OPG sequences and control siRNA duplexes (Origene, Rockville, MD).

Techniques: Transfection, Control, Lysis