Journal: Oncology Letters

Article Title: microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin

doi: 10.3892/ol.2013.1448

Figure Lengend Snippet: Expression levels of miR-199a and mTOR in OV2008 and C13* cells were detected by qPCR and western blotting. (A) Expression levels of miR-199a were, on average, 83.4-fold higher in the OV2008 cells compared with the C13* cells (P<0.05). (B) Noticeably more mTOR protein was expressed in the C13* cells compared with the OV2008 cells, as shown by western blotting. (C) The mTOR fold change in protein level is shown from three independent experiments. * P<0.05 vs. C13*; ** P<0.05 vs. OV2008. mTOR, mammalian target of rapamycin.

Article Snippet: The Bulge-LoopTM miRNA qPCR primer set for hsa-miR-199a (MQP-0101; RiboBio, Guangzhou, China) and U6 snRNA (MQP-0201; RiboBio) were used according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin

doi: 10.3892/ol.2013.1448

Figure Lengend Snippet: Effect of miR-199a on sensitivity to cisplatin treatment in C13* and OV2008 cells. (A) C13* cells were transfected with miR-199a mimics and miR-mimic negative control (NC), then treated with 40 μM cisplatin for 24 h. Cell apoptosis was measured by flow cytometry. (B) OV2008 cells were transfected with miR-199a inhibitor and inhibitor NC, then treated with 40 μM cisplatin for 24 h. Cell apoptosis was measured by flow cytometry. (C) Cisplatin sensitivity was decreased in miR-199a inhibitor-treated OV2008 cells compared with those treated with NC, and the viability of the cells was evaluated by the CCK-8 assay. (D) C13* cells transfected with mimics of miR-199a exhibited increased sensitivity to cisplatin treatment; the viability of the cells was evaluated by the CCK-8 assay. * P<0.05 vs. NC. mTOR, mammalian target of rapamycin; CCK-8, cell counting kit-8.

Article Snippet: The Bulge-LoopTM miRNA qPCR primer set for hsa-miR-199a (MQP-0101; RiboBio, Guangzhou, China) and U6 snRNA (MQP-0201; RiboBio) were used according to the manufacturer’s instructions.

Techniques: Transfection, Negative Control, Flow Cytometry, CCK-8 Assay, Cell Counting

Journal: Oncology Letters

Article Title: microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin

doi: 10.3892/ol.2013.1448

Figure Lengend Snippet: Regulation of mTOR expression by miR-199a. (A) Expression of mTOR mRNA in OV2008 cells transfected with inhibitor of miR-199a. (B) Expression of mTOR mRNA in C13* cells transfected with mimics of miR-199a. (C) Expression of mTOR protein in OV2008 cells transfected with inhibitor of miR-199a. (D) Expression of mTOR protein in C13* cells transfected with mimics of miR-199a. * P<0.05 vs. NC. mTOR, mammalian target of rapamycin.

Article Snippet: The Bulge-LoopTM miRNA qPCR primer set for hsa-miR-199a (MQP-0101; RiboBio, Guangzhou, China) and U6 snRNA (MQP-0201; RiboBio) were used according to the manufacturer’s instructions.

Techniques: Expressing, Transfection

Journal: Oncology Letters

Article Title: microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin

doi: 10.3892/ol.2013.1448

Figure Lengend Snippet: mTOR may be a target gene of miR-199a. (A) mTOR’s 3′-UTR mRNA includes a highly-conserved binding site for miR-199a. (B) C13* cells were co-transfected with psiCHECK-2™/mTOR 3′-UTR and miR-199a mimics or control oligonucleotides. At 24 h post-transfection, mTOR fluorescence intensity was detected. (C) Luciferase activity was not different between empty vector psiCHECK-2 and controls. * P<0.05 vs. blank. mTOR, mammalian target of rapamycin; UTR, untranslated region.

Article Snippet: The Bulge-LoopTM miRNA qPCR primer set for hsa-miR-199a (MQP-0101; RiboBio, Guangzhou, China) and U6 snRNA (MQP-0201; RiboBio) were used according to the manufacturer’s instructions.

Techniques: Binding Assay, Transfection, Control, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: TLR4 expression of PDLSCs and BMMSCs with or without LPS treatment. (A) Relative gene expression of TLRs (TLR1 to TLR10) of PDLSCs and BMMSCs was analyzed by qRT-PCR. Relative gene expression of TLRs was determined based on the threshold cycle (CT) values. The expression levels of the target genes were normalized to that of the housekeeping gene β-actin. PDLSCs (* P <0.05 versus PDLSCs, n = 3), BMMSCs (n = 3). (B) Western blot analysis showed the protein expression of TLR4, and β-actin was used as the internal control. (C) Relative intensity of the tested protein was quantitatively analyzed using the Adobe Photoshop CS2 software. Data represent the means ± SD (n = 3). BMMSCs, bone marrow mesenchymal stem cells; LPS, lipopolysaccharide; PDLSCs, periodontal ligament stem cells; SD, standard deviation; TLR4, Toll-like receptor 4.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, Control, Software, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: Anti-TLR4 antibody or PDTC effectively blocks the activated NF-κB pathway activated by LPS. (A) The TLR4 antagonist anti-TLR4 antibody (0.5 μg/ml) and the NF-κB inhibitor PDTC (40 ng/ml) were incubated with PDLSCs or BMMSCs, which were stimulated by LPS. After 72 hours, western blot showed that the expression of phospho-NFκBp65 was reduced. β-actin was used as the internal control. (B, C) The relative intensity of the tested protein was quantitatively analyzed using Adobe Photoshop CS2 software. Data represent the means ± SD. * P <0.05 (n = 3). BMMSCs, bone marrow mesenchymal stem cells; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; PDLSCs, periodontal ligament stem cells; PDTC, pyrrolidinedithiocarbamate; SD, standard deviation; TLR4, Toll-like receptor 4.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: Incubation, Western Blot, Expressing, Control, Software, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: TLR4 or NF-κB blockage reverses the impaired osteogenic differentiation of PDLSCs stimulated by LPS. (A, D) Gene expression of Runx2 in PDLSCs and BMMSCs with or without drug treatment was measured by qRT-PCR after osteogenic induction for 14 days. (B, E) The impaired osteogenic differentiation of PDLSCs caused by LPS was reversed by anti-TLR4 antibody or PDTC. Osteogenic differentiation was determined by Alizarin red S staining after osteogenic induction for 28 days and alizarin red was then extracted and measured for light absorbance at 620 nm. (C, F) The osteogenic differentiation of BMMSCs with or without drug treatment was determined by Alizarin red S staining after osteogenic induction for 28 days. Data represent the means ± SD. * P <0.05 (n = 3). BMMSCs, bone marrow mesenchymal stem cells; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; PDLSCs, periodontal ligament stem cells; PDTC, pyrrolidinedithiocarbamate; SD, standard deviation; TLR4, Toll-like receptor-4.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: Gene Expression, Quantitative RT-PCR, Staining, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: Blockage of the TLR4 or NF-κB pathway effectively reduces the bone loss caused by LPS in vivo . (A) Bone loss in the saline group, LPS group, LPS + anti-TLR4 group and LPS + PDTC group. (B) Alveolar bone loss determination of maxillary molars. Seventeen sites per quadrant (three sites for each of five roots and one site for each root furcation of two teeth) were analyzed morphometrically. (C) Mean alveolar bone loss. Data represent the means ± SD. * P <0.05 (n = 3). LPS, lipopolysaccharide; NF-κB, nuclear factor- κB; PDTC, pyrrolidinedithiocarbamate; SD, standard deviation; TLR4, Toll-like receptor 4.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: In Vivo, Saline, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: ALP expression is elevated after treatment with anti-TLR4 antibody or PDTC. (A) ALP positive staining was seen in the periodontal ligament and its expression in the LPS group was significantly reduced compared to that of the control group. After anti-TLR4 antibody or PDTC administration, the ALP expression was greatly increased and was comparable to that of the control group. (B) Quantification of ALP-positive staining. Integrated intensity was measured by Image-Pro Plus 6.0. Scale bar: 125 μm. Data represent the means ± SD. (* P <0.05 versus saline group, n = 3). ALP, alkaline phosphatase; LPS, lipopolysaccharide; PDTC, pyrrolidinedithiocarbamate; SD, standard deviation; TLR4, Toll-like receptor 4.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: Expressing, Staining, Control, Saline, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

doi: 10.1186/scrt456

Figure Lengend Snippet: The number of osteoclasts does not change after anti-TLR4 antibody or PDTC treatment. (A) TRAP staining showed no significant differences in the number of osteoclasts in the alveolar bone among the saline, LPS, LPS + anti-TLR4 antibody and LPS + PDTC groups. Arrowheads, TRAP-positive osteoclasts (purple cells). (B) Quantification of TRAP-positive osteoclasts. Integrated intensity was measured by Image-Pro Plus 6.0. Scale bar: 50 μm. Data represent the means ± SD (n = 3). LPS, lipopolysaccharide; PDTC, pyrrolidinedithiocarbamate; SD, standard deviation; TLR4, Toll-like receptor 4; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: No report so far has described the clinical application of the TLR4 antibody, but there is already a phase I study recruiting for assessing the safety of a human anti-TLR4 monoclonal antibody (NI-0101), which is sponsored by NovImmune SA.

Techniques: Staining, Saline, Standard Deviation