Journal: PLoS ONE

Article Title: Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins

doi: 10.1371/journal.pone.0029100

Figure Lengend Snippet: A. RAW 264.7 cells were treated with BSA control or palmitate (250 µM) for 4 hours and conditioned media were collected 20 hours later. BMP 2 and 4 secreted in the media were pulled down on heparin-Sepharose columns and detected by immunoblotting. B. RAW 264.7 cells were treated with BSA control or palmitate for 4 hours, and then incubated with fresh media for 20 hours. BMP2 and BMP4 mRNA levels were analyzed by real-time PCR. Data are illustrated in arbitrary unit relative control and represent the means ± SE from three independent experiments. *p<0.05 compared to control.

Article Snippet: BMP2 siRNA, BMP4 siRNA and scrambled control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins

doi: 10.1371/journal.pone.0029100

Figure Lengend Snippet: A. THP-1 cells differentiated with PMA were stimulated with BSA control or palmitate for 4 hours and conditioned media were collected 20 hours later. Palmitate-conditioned media were treated without or with 2 µg/mL of anti-BMP2, anti-BMP4, or both for 1 hour. Each conditioned media was added to serum starved SMCs for 72 hours. Cell proliferation was analyzed by BrdU incorporation assay. Promotion of SMC proliferation by palmitate-conditioned media was abrogated by combined treatment of anti-BMP2/anti-BMP4. B. Each conditioned media described above was added to SMCs for 24 hours. Cell migration assay was conducted using Boyden chamber assay. Promotion of SMC migration by palmitate-conditioned media was inhibited by treatment of anti-BMP2 or anti-BMP4 or combination of both. C. Each conditioned media described above was added to SMCs for 24 hours. Immunoblots showed that anti-BMP4 or anti-BMP2/anti-BMP4 combination inhibited phenotypic change of SMCs. D. The band intensities were determined by quantitative densitometry. E. THP-1 cell-derived macrophages were transfected with BMP2 or BMP4 or both siRNA for 24 hours. Immunoblots demonstrated knockdown of BMP2 or BMP4 or both BMPs in the macrophages. F. SMCs were treated with palmitate-conditioned media from macrophages without or with knockdown of BMPs for 24 hours. Equal amount of protein were separated by SDS-PAGE, and immunoblots showed that knockdown of BMP4 or both BMPs abrogated phenotypic change of SMCs. Data are illustrated in arbitrary integrator unit relative to β-actin and represent the means ± SE from three independent experiments. *p<0.05 compared to control. **p<0.05 compared to palmitate-conditioned media treatment.

Article Snippet: BMP2 siRNA, BMP4 siRNA and scrambled control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, BrdU Incorporation Assay, Cell Migration Assay, Boyden Chamber Assay, Migration, Western Blot, Derivative Assay, Transfection, Knockdown, SDS Page

Journal: PLoS ONE

Article Title: Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins

doi: 10.1371/journal.pone.0029100

Figure Lengend Snippet: A. Quiescent SMCs were treated with various concentration of BMP2 or BMP4 for 24 hours. Cell proliferation was analyzed by BrdU incorporation assay. Recombinant BMP2 and BMP4 did not have obvious effects on SMC proliferation. B. SMC migration assessment was conducted using Boyden chamber assay and BMP2 and BMP4 significantly promoted SMC migration. C. SMCs were treated with various concentration of recombinant BMP2 or BMP4 for 24 hours. Equal amount of protein was separated by SDS-PAGE and immunoblots showed both recombinant BMPs induced phenotypic change of SMCs with lowering expression of SM α-actin and SM22α.

Article Snippet: BMP2 siRNA, BMP4 siRNA and scrambled control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Concentration Assay, BrdU Incorporation Assay, Recombinant, Migration, Boyden Chamber Assay, SDS Page, Western Blot, Expressing

Journal: Bioactive Materials

Article Title: Graphene oxide/gallium nanoderivative as a multifunctional modulator of osteoblastogenesis and osteoclastogenesis for the synergistic therapy of implant-related bone infection

doi: 10.1016/j.bioactmat.2022.07.015

Figure Lengend Snippet: Investigation of the potential molecular mechanisms of the regulatory effects of GO/Ga nanocomposites on osteoblastogenesis and osteoclastogenesis in MC3T3-E1 and RAW 264.7 cells. (a) Expression features of BMP/SMAD signaling molecules during the osteoblast differentiation of MC3T3-E1 cells after coculture with different nanomaterials for 7 d. (b) Expression features of RANKL-stimulated MAPK signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. (c) Expression features of RANKL-stimulated NF-κB signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. The expression levels of NFATc1 and c-Jun were also confirmed after coculture for 3 d with the stimulation of RANKL (50 ng/mL). Overall, the relative expression levels of p-Smad 1/5, p-JNK, p-P38, p-ERK, p-IkBα, p-P65, NFATc1 and c-Jun were calculated and normalized by β-actin in terms of the gray band intensities as confirmed by ImageJ software. (d) Luciferase reporter gene assessment of BMP-2 and NFATc1 in MC3T3-E1 and RAW264.7 cells, respectively. Stably transfected cells were treated with different nanomaterials for 6 h with specific stimulation, and the luciferase activities were confirmed by a Pierce™ Gaussia Luminescence Assay kit. (e) Molecular docking of Ga3+ with JNK/P38 kinases as demonstrated in binding mode figures using PyMOL visualization software. (f) Schematic diagram of the regulatory mechanisms of GO/Ga nanocomposites involved in osteoblastogenesis and osteoclastogenesis (Created with BioRender.com). *p < 0.01 compared with CTRL and GaNPs, **p < 0.05 and ***p < 0.01 compared with CTRL, #p < 0.01 compared with GO, ##p < 0.05 compared with GO.

Article Snippet: To further confirm the crucial role of corresponding signaling axis involved in the osteogenic and osteoclast differentiation, a small interfering RNA (siRNA) mediated BMP-2 gene knockdown (Ribobio, Guangzhou, China), and specific activators of JNK (Anisomycin, 50 μM, S7409), P38 (Asiatic acid, 10 μM, S2266) and NF-κB (Betulinic acid, 15 μM, S3603) purchased from Selleck Chemicals (Houston, USA) with indicated concentration were used to perform reverse validation experiments aimed at verifying the stimulative effects of GO/Ga nanocomposites on BMP/Smad signaling pathways and the inhibitory effects on MAPK and NF-κB signaling pathways, as previously reported [ [53] , [54] , [55] , [56] ].

Techniques: Expressing, Software, Luciferase, Stable Transfection, Transfection, Luminescence Assay, Binding Assay