Journal: Molecular Brain

Article Title: Proteomic profiling of the neurons in mice with depressive-like behavior induced by corticosterone and the regulation of berberine: pivotal sites of oxidative phosphorylation

doi: 10.1186/s13041-019-0518-4

Figure Lengend Snippet: The primary antibodies used for the protein expression in the study

Article Snippet: GR (Ab211) , Rabbit polyclonal antibody , 1:1000 , TA313373, Origene at Shanghai, China.

Techniques: Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk.

doi: 10.1002/jbmr.120

Figure Lengend Snippet: Fig. 1. GR-specific gene silencing impaires osteo/adipogenesis capacity of hBMSCs. (A) Internalization of the glucocorticoid receptor in response to dexamethasone (Dex). hBMSCs were incubated in the presence of 1 mM Dex for 24 hours. (GR green, nuclei blue). (B) Western blot analysis of GR expression after specific knock-down in hBMSCs transduced with pLV-GRi. Control: pLVTHM empty vector (pLV-emp). Sam68 was used as a loading control. Effects of GR silencing on hBMSC adipogenesis (C) and osteogenesis (D). Analysis of GFP expression by flow cytometry. hBMSCs transduced with pLVTHM were harvested, and GFP expression was analyzed by flow cytometry (white). Negative control: noninfected hBMSCs (gray). hBMSCs transduced with either the empty or GRi-containing vector were induced to differentiate in the presence of adipogenic medium (AM) or osteogenic medium (OM). (C) Percentage of mature adipocytes stained with Nile red was estimated by flow cytometry. Percentage of GFPþ and GFP– mature adipocytes after the induction period are represented by bars. Results show the mean SD of triplicate samples. Significance of the difference between GFPþ and GFP– in pLV-GRi cells was determined using Student’s t test; p < .01. (D) Osteogenic induction of transduced hBMSCs. Cells were stained with alizarin red S. Control: absence of OM. Alizarin red calcium quantification is shown in Supplemental Fig. 2A.

Article Snippet: SP600125, RU-486 (Mifepristone), and PDGF-BB were purchased from Sigma and used at the corresponding concentrations. shRNA- and cDNA-transduced cells shRNA and cDNA vectors were constructed by using standard cloning procedures. c-Jun and GR shRNA oligonucleotides were purchased from Sigma-Genosys (www.sigmaaldrich.com): c-Jun (Accession No. NM_002228, sequence 1553–1573)(18) and glucocorticoid receptor (Accession No. NM_000176, sequence 2086–2114; www.origene.com).

Techniques: Incubation, Western Blot, Expressing, Knockdown, Transduction, Control, Plasmid Preparation, Flow Cytometry, Negative Control, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk.

doi: 10.1002/jbmr.120

Figure Lengend Snippet: Fig. 2. Effects of glucocorticoid receptor signaling on hBMSC proliferation. (A) hBMSC proliferation induced by specific GR inhibition in the presence of Dex. Transduced cells were plated at 500 cell/cm2 (day 0) in the presence or absence (control) of dexamethasone (1 mM) and analyzed by flow cytometry on day 10 (left panel). One representative experiment out of three is shown (right pannel). Significance of the difference between Dex-treated and control in pLV-GRi GFPþ cells was determined using Student’s t test (n ¼ 3, p < .01). (B) hBMSC growth arrest induced by increasing concentrations of Dex. Noninfected hBMSCs were plated at 500 cell/cm2, and cell proliferation was estimated using the MTT assay on day 10 of culture. Significance was determined using Student’s t test for paired samples relative to the proliferation levels measured on control cells (n ¼ 3, p < .01). (C) Effects of AM and OM on noninfected hBMSC cell growth. Noninfected hBMSCs were plated at 20000 cell/cm2 in the presence (AM or OM) or absence (control) of differentiation medium. Fold increase was estimated using the trypan blue dye exclusion assay and represents the cell count in relation to the cells plated on day 0. Cell cycle was analyzed by flow cytometry of hBMSCs stained with propidium iodide. The percentages of cells in S/G2/M are indicated. (D) hBMSCs were assayed under the same conditions as in panel C but in the presence of 25 ng/mL of PDGF. AM ¼ adipogenic medium; OM ¼ osteogenic medium. Results show the mean SD of triplicate samples. Significance of the difference between control and PDGF-treated cells from panels C and D is shown in Supplemental Fig. 3.

Article Snippet: SP600125, RU-486 (Mifepristone), and PDGF-BB were purchased from Sigma and used at the corresponding concentrations. shRNA- and cDNA-transduced cells shRNA and cDNA vectors were constructed by using standard cloning procedures. c-Jun and GR shRNA oligonucleotides were purchased from Sigma-Genosys (www.sigmaaldrich.com): c-Jun (Accession No. NM_002228, sequence 1553–1573)(18) and glucocorticoid receptor (Accession No. NM_000176, sequence 2086–2114; www.origene.com).

Techniques: Inhibition, Control, Flow Cytometry, MTT Assay, Exclusion Assay, Cell Counting, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk.

doi: 10.1002/jbmr.120

Figure Lengend Snippet: Fig. 4. Glucocorticoid receptor activation downregulates c-Jun protein expression level and antagonizes the PDGF-induced signaling pathway. (A) Time- and dose-dependent effects of PDGF on c-Jun protein expres- sion level by Western blot analysis. hBMSCs were treated with the indicated concentrations of PDGF for 24 hours (upper panel) or with either 25 ng/mL of PDGF or vehicle control (CTL) for the indicated time (bottom panel). (B) AM- or OM-induced downregulation of c-Jun expres- sion level that antagonizes PDGF-induced upregulation of c-Jun via the JNK signaling pathway. hBMSCs were exposed to AM or OM in the presence of PDGF (25 ng/mL) or vehicle control for 24 hours. c-Jun expression level was measured by Western blot in total cell extracts using the antibody for c-Jun. hBMSCs were pretreated for 6 hours with the inhibitor of JNK SP600125 (4 mM), which significantly inhibited PDGF- induced c-Jun expression in the presence of AM. (C) Downregulation of c- Jun protein expression requires GR activation by dexamethasone. c-Jun protein levels were analyzed in Dex-treated (1 mM, 24 hours) hBMSCs transduced with either the empty or GRi-containing vector, as described in Fig. 1. Sam68 was used as a loading control. Protein quantification is shown is Supplemental Fig. 6B.

Article Snippet: SP600125, RU-486 (Mifepristone), and PDGF-BB were purchased from Sigma and used at the corresponding concentrations. shRNA- and cDNA-transduced cells shRNA and cDNA vectors were constructed by using standard cloning procedures. c-Jun and GR shRNA oligonucleotides were purchased from Sigma-Genosys (www.sigmaaldrich.com): c-Jun (Accession No. NM_002228, sequence 1553–1573)(18) and glucocorticoid receptor (Accession No. NM_000176, sequence 2086–2114; www.origene.com).

Techniques: Activation Assay, Expressing, Western Blot, Control, Transduction, Plasmid Preparation