Journal: JBMR Plus

Article Title: Cathepsin K inhibitors promote osteoclast-osteoblast communication and engagement of osteogenesis

doi: 10.1093/jbmrpl/ziaf079

Figure Lengend Snippet: Prevalence of ALP + OBs in resorption cavities and level of osteogenic markers after a 72 hr co-culture of OBs and OCs in the presence and absence of CatK inhibitors. (A) Representative images of TRAP-stained OCs (orange) and ALP-stained OBs (purple) under control, T06 and ODN treated conditions. The segmented outline (black dotted) shows the resorption excavation generated by OCs. (B) The quantification of ALP + resorption cavities (±OCs) during 72 hr of OC-OB co-culture indicated the preference of OBs towards pits compared to trenches when normalized with total number of pits and trenches. (C) The quantification of ALP + resorption cavities with OCs (+OCs). Statistics: Kruskal–Wallis’s test, 2 tailed ( * p < .05; *** p < .001); Dunn’s multiple comparisons test ( * p < .057; *** p < .001) compared to the untreated control. (D) ALP activity quantification in the culture supernatant showed significantly enhanced activity under CatK inhibition (Kruskal–Wallis’s test, 2 tailed (ns: not significant; * p < .05; ** p < .01; *** p < .001). Sample size ( n = 4 donors) for control, T06 (300 nM, 500 nM, 1 μM), and ODN (15 nM, 50 nM). For each donor, 3 replicate experiments on individual bone slices were analyzed for all conditions. The median obtained in each experiment are shown as bars. We analyzed between 450 and 800 OC-OB activity per condition per experiment for each of the donors). (E) Western blot analysis of ALP, RANKL, collagen (COL1), CatK and MMP 13 in control, T06 (0.3, 0.5, and 1 μM), ODN (15 and 50 nM) conditions and (F) their quantification ( n = 3 donors). Statistics: Kruskal–Wallis’s test, 2 tailed (ns: not significant; ** p < .01; *** p < .001); Dunn’s multiple comparisons test ( ** p < .02; *** p < .001) compared to untreated control.

Article Snippet: After blocking in 3% (wt/vol) skimmed milk for 3 hr, the membrane was incubated in TBST (Tris-buffered saline with 0.05% Tween-20) overnight at 4 °C for detection of RANKL (MAB6263-SP; R&D systems), ALP (AF2909-SP; R&D systems), COL1 (ab34710; Abcam), MMP 13 (ab39012; Abcam), CatK (MS2), and β-actin (ab8227, Abcam; primary antibody dilution; 1:1000).

Techniques: Co-Culture Assay, Staining, Control, Generated, Activity Assay, Inhibition, Western Blot

Journal: JBMR Plus

Article Title: Cathepsin K inhibitors promote osteoclast-osteoblast communication and engagement of osteogenesis

doi: 10.1093/jbmrpl/ziaf079

Figure Lengend Snippet: Comparative response of OBs to pits and trenches generated during a preculture with OCs subjected or not to CatK inhibition. OB cultures on bone slices which have been pre-treated with OCs subjected or not to CatK inhibitors: prevalence and ALP activity levels in OBs associated with pits and trenches: bone slices were submitted to OC resorption for 72 hr according to the standard protocol under control, T06 (0.3, 0.5, and 1 μM), and ODN (15 and 50 nM), conditions. After stripping of OCs, OBs were seeded on these pre-resorbed bone slices and cultured for another 72 hr (CatK inhibitors −/−). (A-D) Representative images of ALP-stained OBs (purple) after this culture on the bone slices pretreated as indicated. Asterisks and dotted outline highlight the position of resorption cavity. (E) Prevalence of ALP + excavations according to whether they were generated as pits. trenches, and in the presence or absence of CatK inhibitors. Pits and trenches generated in the presence of CatK inhibitors are more likely to attract OBs. (F) Levels of ALP activity in OBs according to whether they are associated with pits and trenches that had been generated in the presence or absence of CatK inhibitors. Horizontal dotted lines represent the average staining intensity of OBs on the non-resorbed bone surface under different conditions. They showed less ALP-activity compared to resorbed excavations. Sample size ( n = 4 donors) for control, T06 (300 nM, 500 nM, 1 μM), and ODN (15 nM, 50 nM). For each donor, 3 replicate experiments on individual bone slices were analyzed for all conditions. The median obtained in each experiment are shown as bars. We analyzed between 500 and 900 OB activities per condition per experiment for each of the donors). Statistics: Kruskal–Wallis’s test, 2 tailed ( ** p < .01; *** p < .001); Dunn’s multiple comparisons test ( ** p < .01; *** p < .001) compared to the untreated control.

Article Snippet: After blocking in 3% (wt/vol) skimmed milk for 3 hr, the membrane was incubated in TBST (Tris-buffered saline with 0.05% Tween-20) overnight at 4 °C for detection of RANKL (MAB6263-SP; R&D systems), ALP (AF2909-SP; R&D systems), COL1 (ab34710; Abcam), MMP 13 (ab39012; Abcam), CatK (MS2), and β-actin (ab8227, Abcam; primary antibody dilution; 1:1000).

Techniques: Generated, Inhibition, Activity Assay, Control, Stripping Membranes, Cell Culture, Staining

Journal: Journal of Orthopaedic Surgery and Research

Article Title: miR-468-3p suppresses osteogenic differentiation of BMSCs by targeting Runx2 and inhibits bone formation

doi: 10.1186/s13018-024-05410-7

Figure Lengend Snippet: miR-468-3p is reduced during osteogenic differentiation. ( A ) BMSC characterization (flow cytometry). ( B ) MiR-468-3p levels in left femurs from ovariectomy (OVX) or sham mice at 6 weeks and 6 months old after surgery (OVX-6w and Sham-6w; 6 months old) (RT-PCR). ( C ) ALP and OC mRNA levels in left femurs after operations (RT-PCR). ( D ) MiR-214 levels (RT-PCR) in Alp + cells by fluorescence-activated cell sorting (FACS) from BMSCs in bilateral femurs from OVX- and sham-operated animals. ( E) OC mRNA levels (RT-PCR) in Alp + cells (FACS) from BMSCs in bilateral femurs from OVX- and sham-operated animals. Six mice/group. ( F ) BMSCs were treated with BMP-2 (200 ng/ml) or not for 4 days. Runx2, OC, and ALP levels by qRT-PCR. GAPDH = internal control. ( G ) BMSCs were treated using BMP-2 and miR-468-3p levels were assayed by qRT-PCR. U6 = internal control. Data are mean values ± SD. Studies were performed three times. * P < 0.05 compared with controls

Article Snippet: The femurs of mice were used to collect stem cells from bone marrow, which were used in FACS analyses with PerCP-labeled antibodies to rat, mouse, and human ALP (R&D Systems, AF2910, 1:50).

Techniques: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Fluorescence, FACS, Quantitative RT-PCR, Control