Journal: The Journal of Clinical Investigation

Article Title: Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling

doi: 10.1172/JCI77186

Figure Lengend Snippet: (A and B) Relative mRNA expression of Mafb in the course of osteoclast differentiation in vitro (A) and in osteoclast progenitors (B). **P < 0.01; ***P < 0.001, compared with RXR-KO cells on the same day of differentiation. (C) MAFB protein expression in osteoclast progenitors (representative of 3 mice per genotype). (D) Mafb mRNA expression in bone marrow myeloid populations; cells were isolated as shown in Supplemental Figure 1D. (E–G) Proliferation assays in lentiviral-mediated MAFB-overexpressing RXR-KO osteoclast progenitors: number of CFUs (E) and number of cells per CFU (F) in bone marrow cell cultures infected with control lentivirus (WT-GFP and RXR-KO-GFP) and with MAFB lentivirus (RXR-KO-MAFB). n = 6 per group. (G) Flow cytometry analysis of the proliferative responses of osteoclast progenitors infected with control lentivirus (WT-GFP and RXR-KO-GFP) and with MAFB lentivirus (RXR-KO-MAFB). n = 3 per group. *P < 0.05, compared with WT-GFP; #P < 0.01, compared with RXR-KO-GFP. (H–K) siRNA assay: representative TRAP-positive cells (H), number of nuclei (I), Ctsk expression (J), and resorption pit area (K) in osteoclast cultures after 5 days of differentiation from bone marrow cells transfected with control or Mafb siRNAs. *P < 0.05; **P < 0.01, compared with siControl. Resorption activity was measured after culturing day-5 osteoclasts on bone bovine cortical bone slices for 2 additional days; the experiment shown is representative of 3 independent experiments done in triplicate. Scale bars: 100 μm. Data are presented as mean ± SEM. Statistical comparisons were made by unpaired 2-tailed Student’s t test.

Article Snippet: We used predesigned mouse Mafb siRNA (#ID 156036, Ambion) and Srebp1c siRNA (#NM_011480si.1, Eurofins, MWG Operon).

Techniques: Expressing, In Vitro, Isolation, Infection, Flow Cytometry, Transfection, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling

doi: 10.1172/JCI77186

Figure Lengend Snippet: (A) Srebp1c mRNA expression in WT and RXR-KO osteoclast progenitors treated with vehicle (C) or ligands for RXR (LG268) and LXR (T1317). *P < 0.01; **P < 0.001, compared with WT vehicle-treated cells (C). (B) SREBP-1c protein expression in nuclear extracts from osteoclast progenitors treated with vehicle or ligands for RXR (LG268) and LXR (T1317); SMC3 was used as loading control. (C) Luciferase reporter assay in RAW264.7 cells transfected with SREBP-1c or empty cDNA3.1 vector together with the indicated Mafb promoter or pGL3 control reporters; data are presented relative to values obtained with the vehicle-treated pGL3 reporter in the absence of SREBP-1c (cDNA3.1). Indicated fold inductions represent (SREBP-1c)/(cDNA3.1). **P < 0.01; ***P < 0.001, compared with reporter activity in the absence of SREBP-1c. (D) ChIP analysis of SREBP-1c binding to –1.5 kb (Mafb1), –1.0 kb (Mafb2), –0.4 kb (Mafb3), and transcription starting (Mafb4) regions of the Mafb promoter or to a negative control (Igkappa) in osteoclast progenitors. Lower panel, localization of SREBP-1c–binding sites in the Mafb promoter. Arrows indicate the position of primers used for ChIP; the experiment shown is representative of 3 done in triplicate. mRNA expression of Mafb (E) and Srebp1c (F) in osteoclast progenitors transfected with control siRNA (siControl) or Srebp1c siRNA (siSREBP-1c). Data are presented as mean ± SEM (n = 3 per group). *P < 0.01; **P < 0.001, by paired (A) or unpaired (C, E, and F) 2-tailed Student’s t tests.

Article Snippet: We used predesigned mouse Mafb siRNA (#ID 156036, Ambion) and Srebp1c siRNA (#NM_011480si.1, Eurofins, MWG Operon).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Binding Assay, Negative Control