Journal:

Article Title: Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis

doi: 10.1172/JCI200317778

Figure Lengend Snippet: M5 Ab prevented osteoclast-mediated bone resorption and osteoclast formation in vitro. (a and b) PTH or IL-1α induced calcium releases. PTH and IL-1 induced calcium release from bone in murine neonatal calvaria culture in the presence of M5 Ab (a and b) or anti-β3 integrin Ab (c). Results are expressed as means ± SEM. **P < 0.01 for comparison by Dunnet’s multiple-comparison test with isotype control IgG plus PTH or IL-1α. (d) Mouse bone marrow cells were cultured for 7 days in the presence of M-CSF and RANKL in the presence of control antibody or M5 Ab (200 μg/ml), and TRAP-positive mature osteoclasts numbers were counted and expressed as mean numbers ± SEM of cells. (e) Arthritic mice prophylactically treated with M5 Ab on day 0 and 3 in Figure ​Figure33 were used. Expressions of proinflammatory cytokines, OPN, and integrin mRNA in joint extracts were analyzed using RT-PCR and GAPDH as a housekeeping gene. These are representative data from three separate experiments.

Article Snippet: The cells were incubated with 100 ng/ml murine M-CSF (Immuno-Biological Lab) and 30 ng/ml murine RANKL (Immuno-Biological Lab) in the presence or absence of M5 Ab for 7 days; the culture media was replaced every 3 days.

Techniques: In Vitro, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Combinatorial targeting of cancer bone metastasis using mRNA engineered stem cells

doi: 10.1016/j.ebiom.2019.06.047

Figure Lengend Snippet: MSC engineering using mRNA and in vitro functional validation. (a) PSGL-1/SLEX/CD/OPG MSC display functional rolling on an endothelial layer under physiological shear flow. Native MSC, PSGL-1/SLEX MSC and PSGL-1/SLEX/CD/OPG MSC were flowed on a layer of endothelial cells at different physiological flow-rates 24 h post-MSC engineering. HL-60 leukocytic cells were used as a positive control for rolling. Plot: mean + SD, statistical analysis: Two-way ANOVA test with Dunnett's multiple comparison test to compare each column to Native MSC, *** p ≤ .001, **** p ≤ .0001. (b) PSGL-1/SLEX/CD/OPG MSC inhibit osteoclastic differentiation in vitro . Murine osteoclast precursors (RAW264.7 cells) were plated for 6 days in media with no additional treatment (CT), 100 ng/mL recombinant murine RANKL to induce osteoclastogenesis, and day 2 supernatant of MSC (Native and PSGL-1/SLEX/CD/OPG). 100 ng/mL of recombinant human OPG was used as a positive control for osteoclastogenesis inhibition. Pictures show the TRAP stained culture at day 6 for each condition. Plot: mean + SD, statistical analysis: Kruskal-Wallis with Dunn's multiple comparison test, *** p ≤ .001 compared to PBS + RANKL condition. (c) PSGL-1/SLEX/CD/OPG MSC convert 5-FC into 5-FU in vitro in a cell concentration-dependent manner. 24 h post-engineering, MSC were plated at different concentrations in presence of 400 μg/mL 5-FC. LC-MS/MS was done on conditioned media collected at different days to measure the 5-FU converted from 5-FC. Plot shows mean + SD. (d) PSGL-1/SLEX/CD/OPG MSC kill MDA-MB231 cancer cells in vitro . Native MSC and PSGL-1/SLEX/CD/OPG MSC were plated at different ratios (1:2 and 1:10) on top of cancer cells in the presence of increasing doses of 5-FC, and the viability of the co-culture was determined at day 6. 5-FU was used as a positive control. Graph shows mean ± SD.

Article Snippet: Minimum Essential Medium α, Roswell Park Memorial Institute (RPMI) 1640 Medium, Dulbecco's Modified Eagle Medium, Leibowitz’ L-15 Medium, EGM-2 Endothelial Cell Growth Medium, M-199 Medium, Endothelial Cell Growth Suspension (ECGS), Penicillin/streptomycin solution, 2% gelatin solution, Opti-MEM Reduced Serum Medium, RNAiMAX Lipofectamine, 10× Tris-buffered saline (TBS), Scott's Bluing Solution, Fisherfinest Histoplast paraffin, MX35 Ultra low-profile cryotome blades, polylysine slides, HPLC grade ethyl acetate, acetonitrile, ACS grade glacial acetic acid, 2-propanol, recombinant human TNF-α, recombinant human OPG, recombinant murine RANKL, VybrantTM DiD lipophilic dye, CellTraceTM Calcein Green dye and 7-AAD viability assay dye were purchased from Fisher Scientific.

Techniques: In Vitro, Functional Assay, Biomarker Discovery, Shear, Positive Control, Comparison, Recombinant, Inhibition, Staining, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Co-Culture Assay

Journal: Brazilian oral research

Article Title: Different engagement of TLR2 and TLR4 in Porphyromonas gingivalis vs. ligature-induced periodontal bone loss

doi: 10.1590/1807-3107BOR-2017.vol31.0063

Figure Lengend Snippet: Gingival RANKL mRNA and protein expression levels of P. gingivalis-induced and ligation-induced experimental periodontitis in WT, TLR2 KO, TLR4 KO, and TLR2&4 KO mice. Gingival tissues on the palatal side were collected under a surgical microscope from maxillae and then homogenized for RNA extraction or protein measurement. Gingival RANKL mRNA levels were determined by real-time PCR in the control group, the P. gingivalis infection group, and the ligation group of WT mice (A), TLR2 KO mice (B), TLR4 KO mice (C), and TLR2&4 KO mice (D) (means ± SE, n = 5 mice per group, *p < 0.05, **p < 0.01). Gingival RANKL protein expression levels were measured by ELISA in the control group, the P. gingivalis infection group, and the ligation group of WT mice (E), TLR2 KO mice (F), TLR4 KO mice (G), and TLR2&4 KO mice (H) (means ± SE, n = 5 mice per group, *p < 0.05, **p < 0.01).

Article Snippet: The secreted RANKL level in the gingival homogenate was detected by means of a murine RANKL ELISA development kit (PeproTech, Rocky Hill, USA) following the manufacturer's instructions.

Techniques: Expressing, Ligation, Microscopy, RNA Extraction, Real-time Polymerase Chain Reaction, Control, Infection, Enzyme-linked Immunosorbent Assay