Journal: Bone Research

Article Title: Angiogenesis in a 3D model containing adipose tissue stem cells and endothelial cells is mediated by canonical Wnt signaling

doi: 10.1038/boneres.2017.48

Figure Lengend Snippet: Experimental protocol and cell identification. ( a ) Schematic illustration showing total experimental protocol to establish a 3D vascular collagen model in vitro and in vivo . Briefly, GFP-ASCs and RFP-ECs were co-cultured at a 1:1 ratio and suspended in collagen matrices with a Wnt regulator, LiCl, or DKK1, and then gelled at 37 °C and studied in vitro . The gels were implanted into subcutaneous pockets at both dorsal sides of nude mice to set up an in vivo animal model for testing angiogenesis induced by Wnt regulators. Gels were collected and immediately macroscopically imaged by modified CLSM and then sectioned for histological and immunohistochemical analyses. ( b – e ) Identification of ASCs from mouse fat tissue. Oil Red O ( b ) and PPARγ ( c ) show adipogenic differentiation of isolated ASCs, and Alizarin Red ( d ) shows osteogenic differentiation. The stain images shown are representative of four different experiments ( n =4). Flow cytometry ( e ) showing positive staining for CD34, CD146, and Sca-1 in isolated ASCs ( n =3). ( f and g ) Identification of ECs from mouse brain microvascular tissue. Representative image ( f ) showing isolated primary ECs, and factor VIII immunofluorescence ( g ) showing EC marker staining in isolated ECs ( n =3).

Article Snippet: For identification, ASCs were cultured in six-well plates (2×10 5 cells per well) with induced adipogenic media and osteogenic media (Biowit, Shenzhen, China) to show the differentiation abilities toward lipogenesis and osteogenesis.

Techniques: In Vitro, In Vivo, Cell Culture, Animal Model, Modification, Immunohistochemical staining, Isolation, Staining, Flow Cytometry, Immunofluorescence, Marker

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: RUNX2 increases the accumulation of MDA231 cells as micrometastases in bone marrow. (A) Experimental schedule for the bone colonization of MDA231‐derived cells in SCID mice. An osteogenic premetastatic niche (PMN) was established by injecting MDA231 CDH11 high /ITGA5 high extracellular vesicles into SCID mice via the tail vein for 3 weeks (2 doses/week). MDA231 RUNX2‐OE cells and control cells were injected into the mice via the left ventricle. (B) H&E staining images demonstrating osteoblasts (indicated by red triangles) in homeostatic bone (HB) and the PMN. (C) X‐ray images showing the bone mass in the HB and the PMN microenvironments. (D) Bar charts quantifying the osteoblast number as the ratio of osteoblast counts to bone perimeter in /mm (N.Ob/B.Pm) and bone mass as the bone volume fraction (BV/TV). (E) Western blot analysis demonstrating increased RUNX2 protein levels in MDA231 RUNX2‐OE cells compared with those in control cells. (F) Representative X‐ray images, pan‐cytokeratin (pan‐CK) immunohistochemical staining images, and TRAP staining images showing osteolytic lesions, tumor cell distribution, and activated osteoclasts, respectively. (G) Pie charts depicting the incidence of DTCs, micrometastases (micromets), and osteolytic lesions formed by control cells and RUNX2‐OE cells within HB and the PMN. The scale bars in the inset images indicate 20 µm. (H) Bar charts quantifying the tumor surface, erosion surface, and the number of TRAP + osteoclasts normalized to the total bone surface (N.Oc/BS in mm 2 ). (I) Bar charts illustrating the abundance and size of micrometastases in the bone marrow of mice without detectable bone lesions. (J) Representative KI67 fluorescence immunohistochemical staining images. Pan‐CK was used to label the tumor cells, while DAPI was used to stain the nuclei. (K) Bar chart illustrating the reduced numbers of KI67 + tumor cells in micrometastases compared with tumor cells within bone colonization. The data are displayed as the means ± SDs. n.s., not significant; * p <0.05, ** p <0.01, *** p <0.001 compared with the corresponding controls, as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Control, Injection, Staining, Western Blot, Immunohistochemical staining, Fluorescence

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: Abnormal activation of the osteogenic microenvironment reactivates quiescent basal‐like cancer cells. (A) Experimental schedule for PTH‐reactivated bone colonization. MDA231 RUNX2‐OE cells were injected into SCID mice via the left ventricle. The mice were then treated with 100 µg/kg PTH for 10 consecutive days. Mice inoculated with PBS served as controls. (B) Representative BLI, X‐ray, H&E staining, immunohistochemical staining for pan‐CK, and TRAP staining images demonstrating the progression of bone lesions and the activity of osteoclasts. (C) Pie charts displaying the incidence of DTCs/micrometastases and osteolytic lesions in mice treated with PTH and in control mice. (D) Bar charts showing the incidence of osteolytic bone colonization, tumor surface, and erosion surface in PTH‐treated mice and control mice. (E) Experimental schedule involving the injection of MDA231 RUNX2‐OE cells through the left ventricle, followed by treatment with either a single dose of low‐dose estradiol cypionate (E2, 0.3 mg/kg) or weekly high‐dose E2 (2 mg/kg) for 3 weeks via subcutaneous injection, starting 7 days post‐inoculation of cancer cells. Mice injected with an equal volume of the solvent corn oil served as controls. (F) Representative BLI, X‐ray, H&E staining, immunohistochemical staining for pan‐CK, and TRAP staining images illustrating the progression of bone lesions and the activity of osteoclasts. (G) Pie charts depicting the incidence of DTCs/micrometastases and osteolytic lesions in mice treated with various doses of E2. (H) Bar charts illustrating the incidence of osteolytic bone colonization, tumor surface, and erosion surface in mice subjected to low‐dose E2, high‐dose E2, and control conditions. The scale bars in the inset images indicate 20 µm. The data are presented as the means ± SDs. * p <0.05 and ** p <0.01 compared with the control group, as determined by Fisher's exact probability method or Student's t test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Activation Assay, Injection, Staining, Immunohistochemical staining, Activity Assay, Control, Solvent

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: RUNX2 promotes the colonization of luminal‐like MCF7 cells within a highly mineralized osteogenic microenvironment induced by E2. (A) Experimental schedule for bone colonization of MCF7‐derived cells in SCID mice. MCF7 RUNX2‐OE cells and control cells were injected into the mice via the left ventricle. E2 (2 mg/kg) was administered via subcutaneous injection weekly to support MCF7 tumor growth, starting 1 week before tumor cell injection. (B) Representative micro‐CT and H&E staining images showing increased bone mass and a reduced marrow cavity in SCID mice administered E2. Bar graph illustrating the bone volume fraction (BV/TV) in mice treated with E2 compared with in control mice. (C) Western blot analysis of RUNX2 protein levels in MCF7 RUNX2‐OE cells and control cells. (D) Representative BLI images showing the systemic distribution of tumor cells following left ventricular inoculation on day 0. X‐ray and H&E staining images displaying bone lesions in mice after sacrifice. TRAP staining images indicating osteoclast activity. (E) Representative micro‐CT images displaying bone lesions in SCID mice on day 38. The osteolytic lesions are marked by asterisks and arrows. (F) Pie charts illustrating the incidence of osteolytic bone colonization in mice injected with MCF7 RUNX2‐OE cells compared with those in mice injected with control cells. (G) Bar charts showing the tumor surface, erosion surface, and TRAP + surface normalized to the bone surface. The data are displayed as the means ± SDs. ** p <0.01 and *** p <0.001 compared to the control group, as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Control, Injection, Micro-CT, Staining, Western Blot, Activity Assay

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: An unmineralized osteogenic microenvironment exacerbates osteolytic lesions caused by luminal‐like MCF7 cells. (A) Experimental diagram showing the bone colonization of MCF7‐derived cells within different osteogenic microenvironments established by the administration of E2. SCID mice were administered E2 (2 mg/kg) weekly, either alone or in combination with dexamethasone (DEX, 0.5 µg/mL in drinking water) or a low‐calcium diet. (B) Representative BLI images showing the systemic distribution of tumor cells following left ventricular inoculation on day 0. X‐ray and H&E staining images depict bone lesions in mice after sacrifice, whereas the TRAP staining images indicate osteoclast activity. (C) Representative micro‐CT images showing bone lesions in SCID mice on day 38. The osteolytic lesions are marked by asterisks and arrows. (D,E) Bar charts presenting the incidence of osteolytic bone colonization (D), tumor surface, erosion surface, and TRAP + osteoclast surface (E). The scale bars in the inset images indicate 20 µm. The data are displayed as the means ± SDs. * p <0.05, ** p <0.01 and *** p <0.001 compared with the respective E2‐treated mice, as determined by Student's t test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Staining, Activity Assay, Micro-CT

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: Osteoblasts and the bone matrix exhibit distinct effects on RUNX2‐overexpressing breast cancer cells in different osteoblastic microenvironments in vitro. Mouse primary osteoblasts (mOBs) were induced in osteogenic media supplemented with 50 µg/mL L‐ascorbic acid and 10 m m β‐glycerophosphate disodium for 0, 2, 4, 6, 8, 10, or 12 days. mOBs were isolated by digesting the cells with 0.25% trypsin‐EDTA, while the bone matrix (BM) was obtained by removing the cell components by treatment with 20 m m NH 4 OH and 0.5% Triton X‐100 for 5 min. (A) Representative ALP staining and alizarin S staining images after different durations of osteogenic induction. (B) Western blot analysis of osteogenesis‐related protein levels. (C) Staging of the osteoblastic microenvironment in vitro on the basis of various induction durations. (D,E) Effects of mOBs and BM at different stages of differentiation on the chemotactic (D) and proliferative (E) capacities of GFP‐labeled MDA231 RUNX2‐OE cells. Calcium nodules are circled with red dashed lines. (F,G) Effects of mOBs and BM at different stages of differentiation on the chemotactic (F) and proliferative (G) capacities of GFP‐labeled MCF7 RUNX2‐OE cells. Calcium nodules are circled with red dashed lines. The data are presented as the means ± SDs. *** p <0.001 compared with the respective control group (0 days), as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: In Vitro, Isolation, Staining, Western Blot, Labeling, Control

Journal: International Journal of Biological Sciences

Article Title: Abnormal mechanical load aggravates subchondral bone remodeling and uneven tibial plateau settlement in knee osteoarthritis via activation of osteoblast Piezo1-Ca²⁺-JAK2/STAT3 signaling

doi: 10.7150/ijbs.124507

Figure Lengend Snippet: Piezo1 mediates mechanical force induced osteogenic differentiation in vitro . (A) Western blot analysis of Piezo1, RUNX2, and OCN protein expression in MC3T3-E1 and Piezo1-KO MC3T3-E1 cells with or without CTS treatment (n = 3 per group). GAPDH was used as the loading control. (B) Quantification of Piezo1 protein levels normalized to GAPDH (n = 3 per group). (C) Quantification of RUNX2 protein levels normalized to GAPDH (n = 3 per group). (D) Quantification of OCN protein levels normalized to GAPDH (n = 3 per group). (E) Relative mRNA expression of Runx2 detected by qPCR under each condition (n = 3 per group). (F) Relative mRNA expression of Alpl detected by qPCR under each condition (n = 3 per group). (G) Representative immunofluorescence staining of RUNX2 and DAPI in MC3T3-E1 and Piezo1-KO MC3T3-E1 cells under different treatments (scale bar = 50 μm). (H) Quantification of RUNX2-positive cells based on immunofluorescence staining (n = 3 per group). (I) Representative ALP staining to assess early osteogenic differentiation in control and Piezo1-KO MC3T3-E1 cells with or without CTS (scale bar = 500 μm). (J) Representative ARS staining to evaluate matrix mineralization in control and Piezo1-KO MC3T3-E1 cells with or without CTS (scale bar = 500 μm). (K) Quantification of ALP-stained area (%) (n = 3 per group). (L) Quantification of Alizarin Red (OD units) (n = 3 per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After 5 days of osteogenic induction in collagen I-coated BioFlex 6-well culture plates (Flexcell International Corp, Burlington, NC, USA), cells were transferred to a Flexcell FX-5000 Tension System (Flexcell International) and subjected to cyclic sinusoidal tensile strain (10%, 0.5 Hz).

Techniques: In Vitro, Western Blot, Expressing, Control, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Abnormal mechanical load aggravates subchondral bone remodeling and uneven tibial plateau settlement in knee osteoarthritis via activation of osteoblast Piezo1-Ca²⁺-JAK2/STAT3 signaling

doi: 10.7150/ijbs.124507

Figure Lengend Snippet: Inhibition of JAK2/STAT3 signaling attenuates subchondral bone sclerosis and osteogenic marker expression during KOA progression. (A) Representative immunohistochemical staining of p-JAK2 and p-STAT3 in the medial tibial subchondral bone of sham and DMM mice at 2, 6 and 10 weeks post-surgery; red boxes indicate areas shown at higher magnification, and red arrows highlight representative positive cells (scale bar = 50 μm; enlarged views, 5 μm); (B) Quantification of the percentages of p-JAK2⁺ and p-STAT3⁺ cells in the medial tibial subchondral bone (n = 5 mice per group); (C) Schematic of the AG490 treatment regimen. DMM was performed at 12 weeks of age, followed by intraperitoneal AG490 or vehicle for 6 weeks; mice were harvested 6 weeks post-surgery; (D) Representative high resolution coronal micro-CT images (top) and corresponding 3D reconstructions (bottom) of tibial subchondral bone in DMM mice treated with vehicle or AG490 (scale bar = 500 μm); (E) Quantitative analysis of subchondral bone microarchitecture, including BV/TV, BMD, Tb.Th, Tb.N, and Tb.Sp (n = 5 mice per group); (F) Quantification of medial tibial plateau width, medial tibial plateau thickness and TPEA (n = 5 mice per group); (G) Representative SO&FG staining of knee joint sections from DMM mice treated with vehicle or AG490 (scale bar = 200 μm); (H) OARSI histological scores quantifying cartilage degeneration in each group (n = 5 mice per group); (I) Representative immunohistochemical staining of p-JAK2, p-STAT3, OCN and RUNX2 in the medial tibial subchondral bone from DMM mice treated with vehicle or AG490; red boxes indicate areas shown at higher magnification, and red arrows highlight representative positive cells (scale bar = 50 μm; enlarged views, 5 μm); (J) Quantification of p-JAK2⁺, p-STAT3⁺, OCN⁺ and RUNX2⁺ cell percentages on the medial tibial subchondral bone surface (n = 5 mice per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After 5 days of osteogenic induction in collagen I-coated BioFlex 6-well culture plates (Flexcell International Corp, Burlington, NC, USA), cells were transferred to a Flexcell FX-5000 Tension System (Flexcell International) and subjected to cyclic sinusoidal tensile strain (10%, 0.5 Hz).

Techniques: Inhibition, Marker, Expressing, Immunohistochemical staining, Staining, Micro-CT

Journal: International Journal of Biological Sciences

Article Title: Abnormal mechanical load aggravates subchondral bone remodeling and uneven tibial plateau settlement in knee osteoarthritis via activation of osteoblast Piezo1-Ca²⁺-JAK2/STAT3 signaling

doi: 10.7150/ijbs.124507

Figure Lengend Snippet: Conditional deletion of JAK2 in osteoblasts alleviates subchondral bone sclerosis and osteogenic marker expression during KOA progression. (A) Schematic illustrating the TAM induced JAK2 conditional knockout strategy in 12 week old JAK2 f/f and JAK2 ocn mice; (B) Representative locomotor traces from the OFT in sham and DMM groups of JAK2 f/f and JAK2 ocn mice; (C) Quantitative analysis of behavioral parameters including total distance traveled, mean speed, relative activity, and freezing time (n = 5 mice per group); (D) Rotarod performance test showing the time each mouse remained on the rotating rod (n = 5 mice per group); (E) Representative high resolution coronal micro-CT images (top) and corresponding 3D reconstructions (bottom) of tibial subchondral bone in sham and DMM mice with JAK2 f/f or JAK2 ocn genotype (scale bar = 500 μm); (F) Quantification of subchondral bone microarchitectural parameters, including BV/TV, BMD, Tb.Th, Tb.N, and Tb.Sp (n = 5 mice per group); (G) Quantification of medial tibial plateau width, medial tibial plateau thickness, and TPEA (n = 5 mice per group); (H) Representative SO&FG staining of knee joints from sham and DMM groups in JAK2 f/f and JAK2 ocn mice (scale bar = 200 μm); (I) OARSI histological scores quantifying cartilage degeneration (n = 5 mice per group); (J) Representative fluorescent images of calcein and alizarin red S double labeling in the tibial subchondral bone from sham and DMM mice in both genotypes (scale bar = 50 μm); (K) Quantification of mineral apposition rate based on double labeling analysis (n = 5 mice per group); (L) Representative immunohistochemical staining of p-STAT3, RUNX2, and OCN in the medial tibial subchondral bone from each group; red boxes indicate areas shown at higher magnification, and red arrows highlight representative positive cells (scale bar = 50 μm; enlarged views, 5 μm); (M) Quantification of p-STAT3⁺, OCN⁺ and RUNX2⁺ cell percentages on the medial tibial subchondral bone surface (n = 5 mice per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After 5 days of osteogenic induction in collagen I-coated BioFlex 6-well culture plates (Flexcell International Corp, Burlington, NC, USA), cells were transferred to a Flexcell FX-5000 Tension System (Flexcell International) and subjected to cyclic sinusoidal tensile strain (10%, 0.5 Hz).

Techniques: Marker, Expressing, Knock-Out, Activity Assay, Micro-CT, Staining, Labeling, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: Abnormal mechanical load aggravates subchondral bone remodeling and uneven tibial plateau settlement in knee osteoarthritis via activation of osteoblast Piezo1-Ca²⁺-JAK2/STAT3 signaling

doi: 10.7150/ijbs.124507

Figure Lengend Snippet: JAK2/STAT3 signaling mediates mechanical force induced osteogenic differentiation in vitro . (A) Western blot analysis of p-JAK2, total JAK2, p-STAT3, total STAT3, RUNX2, and OCN in MC3T3-E1 cells with or without CTS, in the presence or absence of AG490 (n = 3 per group). GAPDH was used as the loading control. (B) Quantification of p-JAK2 relative to total JAK2, p-STAT3 relative to total STAT3, and RUNX2 and OCN relative to GAPDH under AG490 conditions (n = 3 per group). (C) Relative mRNA expression of Runx2 and Alpl assessed by qPCR under AG490 conditions (n = 3 per group). (D) Western blot analysis of p-STAT3, total STAT3, RUNX2, and OCN in MC3T3-E1 cells with or without CTS in the presence or absence of Stattic (n = 3 per group). (E) Quantification of p-STAT3 relative to total STAT3, and RUNX2 and OCN relative to GAPDH under Stattic conditions (n = 3 per group). (F) Relative mRNA expression of Runx2 and Alpl assessed by qPCR following Stattic treatment (n = 3 per group). (G) Representative immunofluorescence staining of RUNX2 and DAPI with or without CTS under AG490 conditions (scale bar = 50 μm). (H) Representative immunofluorescence staining of RUNX2 and DAPI with or without CTS under Stattic conditions (scale bar = 50 μm). (I) Representative ALP staining with or without CTS under AG490 conditions (scale bar = 500 μm). (J) Representative ALP staining with or without CTS under Stattic conditions (scale bar = 500 μm). (K) Representative ARS staining with or without CTS under AG490 conditions (scale bar = 500 μm). (L) Representative ARS staining with or without CTS under Stattic conditions (scale bar = 500 μm). (M) Quantification of RUNX2-positive cells based on immunofluorescence under AG490 and Stattic conditions (n = 3 per group). (N) Quantification under AG490 conditions of ALP-stained area (%) and Alizarin Red (OD units) with or without CTS (n = 3 per group). (O) Quantification under Stattic conditions of ALP-stained area (%) and Alizarin Red (OD units) with or without CTS (n = 3 per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After 5 days of osteogenic induction in collagen I-coated BioFlex 6-well culture plates (Flexcell International Corp, Burlington, NC, USA), cells were transferred to a Flexcell FX-5000 Tension System (Flexcell International) and subjected to cyclic sinusoidal tensile strain (10%, 0.5 Hz).

Techniques: In Vitro, Western Blot, Control, Expressing, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Abnormal mechanical load aggravates subchondral bone remodeling and uneven tibial plateau settlement in knee osteoarthritis via activation of osteoblast Piezo1-Ca²⁺-JAK2/STAT3 signaling

doi: 10.7150/ijbs.124507

Figure Lengend Snippet: Piezo1 activates the Ca²⁺-JAK2/STAT3 signaling axis to promote osteogenic differentiation. (A) Western blot analysis of RUNX2 and OCN protein expression in control and Piezo1-KO MC3T3-E1 cells with or without Broussonin E (n = 3 per group). GAPDH was used as the loading control. (B) Quantification of RUNX2 and OCN protein levels normalized to GAPDH (n = 3 per group). (C) Relative mRNA expression of Runx2 and Alpl detected by qPCR (n = 3 per group). (D) Representative immunofluorescence staining of RUNX2 and DAPI with or without Broussonin E (scale bar = 50 μm). (E) Representative ALP staining under the indicated conditions (scale bar = 500 μm). (F) Representative ARS staining under the indicated conditions (scale bar = 500 μm). (G) Quantification of RUNX2-positive cells based on immunofluorescence staining (n = 3 per group). (H) Quantification of ALP-stained area (%) and Alizarin Red (OD units) (n = 3 per group). (I) Representative intracellular Ca²⁺ influx traces in cells treated with Con, Yoda1, Yoda1 + BAPTA, or ionomycin. (J) Quantitative analysis of relative intracellular Ca²⁺ fluorescence intensity over time under the indicated treatments (n = 3 per group). (K) Western blot analysis of p-JAK2, total JAK2, p-STAT3, total STAT3, RUNX2, and OCN in cells treated with Con, Yoda1, Yoda1 + BAPTA, or ionomycin (n = 3 per group). (L) Quantification of p-JAK2 relative to total JAK2, p-STAT3 relative to total STAT3, and RUNX2 and OCN relative to GAPDH (n = 3 per group). (M) Representative immunofluorescence staining of p-JAK2 and DAPI under the indicated treatments (scale bar = 50 μm). (N) Representative immunofluorescence staining of p-STAT3 and DAPI under the indicated treatments (scale bar = 50 μm). (O) Representative immunofluorescence staining of RUNX2 and DAPI under the indicated treatments (scale bar = 50 μm). (P) Quantification of p-JAK2-positive, p-STAT3-positive, and RUNX2-positive cells (n = 3 per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After 5 days of osteogenic induction in collagen I-coated BioFlex 6-well culture plates (Flexcell International Corp, Burlington, NC, USA), cells were transferred to a Flexcell FX-5000 Tension System (Flexcell International) and subjected to cyclic sinusoidal tensile strain (10%, 0.5 Hz).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Fluorescence