Journal: Cell Death & Disease

Article Title: Mmu-miR-185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the Bgn-mediated BMP/Smad pathway

doi: 10.1038/s41419-019-1428-1

Figure Lengend Snippet: a Cell Counting Kit-8 (CCK-8) assay reflected cell proliferation of osteoblasts derived from wild-type (WT) or miR-185 -knockout (KO) mice calvaria. b Alkaline phosphatase (ALP) activity determination in WT or miR-185- KO osteoblasts after cultured with osteoblast induction medium (OIM) for 7 days ( n = 3). c Matrix mineralization was quantified in primary osteoblasts after induction in OIM for 14 days ( n = 3). d Representative images of ALP staining of WT or miR-185 KO cells after osteoblast induction for 7 or 14 days. Scale bar = 500 μm. e Representative images of Alizarin Red S staining in cells after osteoblast induction for 14 or 21 days. Scale bars = 500 μm. f The primary osteoblasts were cultured in OIM for indicated times. RNA in cells was extracted with TRIzol reagent, and the expression levels of osteoblast marker genes were quantified by real-time PCR ( n = 3). g The protein levels of osteoblast marker genes in primary osteoblasts cultured with OIM for 0, 3, and 7 days were analyzed by western blot, and expressed as densitometry normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were representative of at least three independent experiments, and were shown as the mean ± S.D (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Cells were washed twice with 1× phosphate-buffered saline, and fixed by ice-cold ethanol for 10 min. Alkaline phosphatase (ALP) staining buffer were prepared according to the manufacturer's instructions (CWBIO, cw0051), and added to the cells.

Techniques: Cell Counting, CCK-8 Assay, Derivative Assay, Knock-Out, Activity Assay, Cell Culture, Staining, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Death & Disease

Article Title: Mmu-miR-185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the Bgn-mediated BMP/Smad pathway

doi: 10.1038/s41419-019-1428-1

Figure Lengend Snippet: MSCs were derived from the bone marrow of wild-type (WT) or knockout (KO) mice, and induced with osteoblast induction medium (OIM) for osteogenic differentiation. a Alkaline phosphatase (ALP) staining in WT or KO MSCs after osteoblast induction for 7 days. Scale bar = 500 μm. b ALP activity determination in MSCs after osteogenic induction for 7 days. c Alizarin Red S Staining in MSCs after osteoblast induction for 21 days. Scale bar = 500 μm. d Matrix mineralization was quantified in MSCs after induction for 21 days. e Real-time PCR showed the messenger RNA (mRNA) expression levels of osterix (Osx), collagen type 1α 1 (Col1a1), and osteocalcin (OC) in MSCs after induction with OIM for 7 days. f The expression of osteoblast marker genes in MSCs cultured for 3 days in OIM was indicated by western blot. Data were shown as mean ± S.D (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Cells were washed twice with 1× phosphate-buffered saline, and fixed by ice-cold ethanol for 10 min. Alkaline phosphatase (ALP) staining buffer were prepared according to the manufacturer's instructions (CWBIO, cw0051), and added to the cells.

Techniques: Derivative Assay, Knock-Out, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Marker, Cell Culture, Western Blot

Journal: Cell Death & Disease

Article Title: Mmu-miR-185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the Bgn-mediated BMP/Smad pathway

doi: 10.1038/s41419-019-1428-1

Figure Lengend Snippet: a Six weeks after ovariectomized (OVX) operation, the mice femurs were dissected and total RNA was extracted. The expression level of Biglycan (Bgn) and Bmp2 were determined by real-time PCR ( n = 3). b The expression of Bgn, Bmp2, t-Smad1, p-Smad1/5/8 in wild-type (WT) or knockout (KO) bones after OVX was detected by western blot. c , d Mesenchymal stem cells (MSCs) were derived from WT or miR-185 KO mice 6 weeks after OVX operation, and transfected with Bgn small interfering (siRNA) or negative control (NC). Cells were cultured with osteoblast induction medium (OIM) for 7 days and alkaline phosphatase (ALP) staining ( c ) and quantification ( d ) were carried out. e , f MSCs were derived from OVX mice, and cultured with OIM. Bgn siRNA or NC transfection was carried out at day 0 and day 7. Alizarin Red S staining ( e ) and mineralization quantification ( f ) were performed at day 14. g The expressions of Alp, Bgn, Bmp2, t-Smad1, p-Smad1/5/8 in MSCs were detected by western blot after OIM induction for 4 days, and expressed as densitometry normalized to GAPDH. Data were shown as mean ± SD (ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Cells were washed twice with 1× phosphate-buffered saline, and fixed by ice-cold ethanol for 10 min. Alkaline phosphatase (ALP) staining buffer were prepared according to the manufacturer's instructions (CWBIO, cw0051), and added to the cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Knock-Out, Western Blot, Derivative Assay, Transfection, Negative Control, Cell Culture, Staining