Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: GCs synergize with Runx2 in stimulating a select gene set. (A) ST2/Rx2dox mesenchymal pluripotent cells were treated for 48 h in quadruplicate with vehicle control (C), dox (to induce Runx2; D), the synthetic GC dex (1 μM; G), or dox and dex together (DG). Global gene expression was profiled using the Illumina BeadChip platform and the response of genes to dex in the presence of dox is illustrated as a volcano plot, with red and blue symbols representing genes that were stimulated or repressed, respectively, by dox alone (D/C ≥ 2 or D/C ≤ −2, respectively; FDR P < 0.05). Data representing genes that did not significantly respond to dox alone by these criteria are in gray. NC, no change. Triangles represent genes that were strongly stimulated by dex in the absence of dox (G/C ≥ 5, FDR P < 0.05). Dashed lines highlight a set of 8 genes at the top right that were strongly stimulated (≥ 8 fold; P < 1e–08) by dex in the presence of dox. (B–G) ST2/Rx2dox cells were treated for 24, 48, or 72 h with dox and/or dex at the depicted concentrations, and expression of the indicated genes was measured by RT-qPCR. Data are means and SDs (n = 3) relative to the control values at 24 h, defined as 1. [Color figure can be viewed at wileyonlinelibrary.com].

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: Synergistic stimulation of Wif1 by Runx2 and GCs in primary osteoblast cultures. Pre-osteoblasts isolated from newborn mouse calvariae were transiently transfected with RUNX2 or a control plasmid and then treated for 72 h with 1.0 μM dex or vehicle. Expression of the indicated genes was assessed by either Western blot analysis of whole cell extracts (A) or (B–E). (F, G) Untransfected cells were treated with differentiation medium on day 2 of culture. Dex (1 μM) or vehicle was added for 72 h before lysis, followed by Western blot analysis of Runx2 (F) and RT-qPCR analysis of Wif1 (G) on days 5 and 7. Bars represent mean ± SD (n = 3).

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Western Blot, Lysis, Quantitative RT-PCR

Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: Effect of Wif1 silencing on Wnt target gene expression in ST2 cells expressing Runx2 and treated with GCs. A–B. ST2/Rx2dox cells were transduced with lentiviruses encoding a nonspecific hairpin RNA (shNS) or either of two hairpins (shWif11 or shWif12) targeting distinct regioion in Wif1 mRNA. The derived shRNA-expressing sub-lines were treated for 72 h with dox and/or dex as indicated and Wif1 expression was measured by RT-qPCR (A, Mean ± SD; n = 3) and by Western blotting (B). +/− signs indicate presence or absence of dox and dex in both A and B. The RNA data in A are corrected for 18S RNA and Coomassie blue-stained proteins that remained in the SDS–PAGE gels after transfer are shown as a loading control in B. (C, D) The RNAs from Figure 4A were subjected to RT-qPCR analysis of Axin2 and Ccnd1 (mean ± SD, n = 3).

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Expressing, Transduction, Derivative Assay, shRNA, Quantitative RT-PCR, Western Blot, Staining, SDS Page

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing