Journal: Journal of Anatomy

Article Title: Sympathetic innervation of human muscle spindles

doi: 10.1111/joa.12309

Figure Lengend Snippet: Immunofluorescence micrographs of human muscle spindles of the deep neck muscles. (A and B) Two consecutive sections of a muscle spindle in the far A region. The NF-positive (red) spindle nerve contains TH-immunoreactive axons (green, arrow). Around both the MYH7b-positive (green) bag fibers (BF) and A4.74-positive (red) chain fiber (CF), very fine but specific labeling with TH (arrowheads) is present and shown at higher magnification in the inset in B. (C) Cross-section of a muscle spindle in the A region showing TH-positive axon (green, arrow) within a spindle nerve alongside with axons labeled with NF (red). In the inset in C, the spindle nerve is shown at higher magnification. (D–F) Longitudinally running axons, with varicose morphology and strongly labeled with the antibody against TH (green, arrowheads) are shown running in parallel with intrafusal fibers (IF) along different parts of the A region of one single muscle spindle from the deep neck muscles. (G and H) Annulospiral endings, strongly labeled with the antibody against NF (red), in the same muscle spindle as above, are shown for comparison. Nuclei are labeled blue with DAPI. C, capsule. Scale bar: 50 μm (A–C and H); 10 μm (D–F); 25 μm (G).

Article Snippet: Antibodies against myosin heavy chain isoforms were used for the purpose of intrafusal fiber typing: mouse MAb ALD19 (kind gift from Donald A. Fischman, Cornell University, New York, USA) and rabbit PAb MYH7b (kind gift from Stefano Schiaffino, CNR Inst. of Neuroscience, Padova, Italy) against slow tonic myosin were used to detect nuclear bag fibers (Liu et al. , ; Rossi et al. ; Janbaz et al. ), mouse MAb A4.74 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) against fast adult myosin was used to detect nuclear chain fibers (Liu et al. , ; Österlund et al. ).

Techniques: Immunofluorescence, Muscles, Labeling, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Orally Bioavailable and Site-Selective Covalent STING Inhibitor Derived from a Macrocyclic Marine Diterpenoid

doi: 10.1021/acs.jmedchem.4c02665

Figure Lengend Snippet: Covalent engagement of hSTING by GHN105 at Cys91. (A) Covalent docking of GHN105 (blue) at Cys91 of apo-hSTING dimer (6NT5). An X-ray diagram of GHN105 was shown. The membrane bilayer environment was simulated to exhibit the embedding position of the hSTING-Cys91-GHN105 complex. (B) HEK293T cells expressing wild type (wt) mSTING or hSTING and the indicated cysteine-to-serine mutants were labeled with 2 μM GHN105 for 15 min ut, untransfected; 2m, C88S/C91S double mutant. GHN105 engagement of mSTING or hSTING was monitored by in-gel fluorescence. Anti-STING Western blot and Coomassie blue stains serve as loading controls for the accompanying fluorescence gels. (C) Scheme created with BioRender.com showing the cGAS-STING signaling cascade in response to intracellular nucleic acids. (D) Western blot analyses of phosphorylated and total STING, TBK1, and IRF3 in NIH3T3 cells pretreated with DMSO or 2 μM of the indicated analogs for 1 h, followed by dsDNA induction of STING signaling for 2 h. Antitubulin blots served as loading controls for the accompanying blots. (E) THP1-Dual KI-hSTING monocytes were pretreated with GNH105 for 1 h prior to cGAMP addition for 24 h. Luciferase reporter activity was normalized to DMSO-treated controls (100%), and the dotted line showed 50% of activity. Error bars, s.e.m. n = 3.

Article Snippet: THP1-Dual KI hSTING cells harboring either WT (R232) or S154 STING variants (Invivogen, thpd-r232, thpd-s154) were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL) and 100 μg/mL normocin (Invivogen, ant-nr-1).

Techniques: Membrane, Expressing, Labeling, Mutagenesis, Fluorescence, Western Blot, Luciferase, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma 1

doi: 10.1016/j.neo.2018.08.007

Figure Lengend Snippet: Binding of NFIA, NFIB, NFIC, and NFIX to NFI binding sites in the HEY1 promoter. Nuclear extracts were prepared from U251 GBM cells transfected with control (pCH), NFIA (pCH-NFIA), NFIB (pCH-NFIB), NFIC (pCH-NFIC), or NFIX (pCH-NFIX) expression constructs. (A) Western blot analysis of transfected cells. Nuclear extracts (20 μg) were electrophoresed through an 8% polyacrylamide-SDS gel, electroblotted onto PVDF membranes, and immunostained with α-HA antibody or α-DDX1 antibody. (B) Electrophoretic mobility shift assays were performed with the indicated radiolabeled probes: −32 bp, −332 bp, −411 bp, and −794 bp. Probes were incubated with the indicated nuclear extracts (2 μg pCH, 3 μg NFIA, 4 μg NFIB, 1 μg NFIC, and 2 μg NFIX). Amounts of protein were adjusted to compensate for differences in expression of transfected HA-NFIs. DNA-protein complexes were electrophoresed through a 6% polyacrylamide gel buffered in 0.5× TBE.

Article Snippet: Total RNA was isolated from GBM cells using the RNeasy Plus Kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies). qPCR was carried out using an ABI 7900HT Fast Real-Time PCR System, with gene-specific oligonucleotides labeled at the 5′ end with the fluorescent reporter dye FAM (NFIA, Hs00325656_m1; NFIB, Hs00232149_m1; NFIC, Hs00907819_m1; NFIX, Hs00958849_m1; GFAP, Hs00157674_m1; B-FABP, Hs00361426_m1; NES, Hs04187831_g1: HEY1, Hs01114113_m1; GAPDH, Hs99999905_m1) and Taqman Fast Master Mix (Life Technologies).

Techniques: Binding Assay, Transfection, Control, Expressing, Construct, Western Blot, SDS-Gel, Electrophoretic Mobility Shift Assay, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma 1

doi: 10.1016/j.neo.2018.08.007

Figure Lengend Snippet: Regulation of HEY1 promoter activity by NFI. U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs. Where indicated (2×), cells underwent two rounds of siRNA transfection. (A) NFIA , NFIB , NFIC , NFIX , and (B) HEY1 mRNA expression was analyzed by qPCR. GAPDH was used as an endogenous control. Similar data were obtained in two separate experiments. (C) U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs, followed 24 hours later by transfection with pGL3/HEY1. Cells were harvested 60 hours later, and luciferase activity was quantified. Changes in relative light units (RLU) are relative to RLU obtained in U251 GBM cells transfected with control (scrambled) siRNA and pGL3/HEY1. The data are from three experiments. SEM is indicated by error bars. Statistical significance, determined using the unpaired t test, is indicated by * ( P < .05) and ** ( P < .01).

Article Snippet: Total RNA was isolated from GBM cells using the RNeasy Plus Kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies). qPCR was carried out using an ABI 7900HT Fast Real-Time PCR System, with gene-specific oligonucleotides labeled at the 5′ end with the fluorescent reporter dye FAM (NFIA, Hs00325656_m1; NFIB, Hs00232149_m1; NFIC, Hs00907819_m1; NFIX, Hs00958849_m1; GFAP, Hs00157674_m1; B-FABP, Hs00361426_m1; NES, Hs04187831_g1: HEY1, Hs01114113_m1; GAPDH, Hs99999905_m1) and Taqman Fast Master Mix (Life Technologies).

Techniques: Activity Assay, Transfection, Control, Expressing, Luciferase

Journal: The Journal of Neuroscience

Article Title: NFIA Controls Telencephalic Progenitor Cell Differentiation through Repression of the Notch Effector Hes1

doi: 10.1523/JNEUROSCI.6167-09.2010

Figure Lengend Snippet: NFIA binds the basal promoter region of Hes1 in vivo and represses Hes1 promoter-driven reporter gene activity. A, Identification of putative conserved NFI binding sites using in silico bioinformatics. The consensus NFI binding site is TTGGC(N5)nnnnnGCCAA (Gronostajski, 2000). Base pairs identified in silico that were identical to the NFI consensus sequence are colored red. The positions of the putative NFI binding sites are given relative to the transcription start site (position 0). The Hes1 promoter has three putative NFI binding sites within 200 bp of the transcription start site. B, EMSA. E18 mouse brain nuclear extracts were incubated with radiolabeled probes for NFI control (lanes 1–3), Hes1-A (lanes 4–6), Hes1-B (lanes 7–9), or Hes1-C (lanes 10–12) consensus sites. Binding reactions included 2× cold (unlabeled) NFI control competitor or specific NFIΑ antibody where indicated. The specific NFIA complex (*) was depleted and produced a supershifted complex (ss) in the presence of the NFIA antibody. FP, Free probe. C, Chromatin immunoprecipitation assays were conducted on E18 wild-type cortices. DNA fragments immunoprecipitated with IgG or anti-NFIA antibodies were analyzed by PCR with primers specifically designed to encompass the predicted NFI binding sites. The NFI binding sites in the Gfap promoter were used as a positive control for NFIA binding (Miura et al., 1990). For both Gfap and Hes1, control primers, designed to an adjacent region of the respective promoters and lacking putative NFI binding sites, were used as negative controls. D, Reporter gene transcriptional assay in HEK 293 cells. Transfection of an Nfia expression vector (Nfia pCAGIG) elicited no luciferase activity, whereas transfection of a luciferase reporter construct under the control of the Hes1 promoter elicited robust induction of the reporter gene. Cotransfection of Nfia with the Hes1 promoter reporter construct resulted in a significantly reduced level of luciferase activity. *p < 0.001, Student's t test. E, Proposed model for the role of Nfia in the regulation of progenitor cell differentiation. Activation of Notch signaling elicits both Nfia transcription (point 1) (Namihira et al., 2009) and Hes1 transcription. NFIA plays a binary role in driving the differentiation of progenitor cells by actively repressing Hes1 expression (point 2) (this study) and activating the transcription of differentiation-specific genes such as GFAP (point 3) (Namihira et al., 2009).

Article Snippet: The primary antibodies used for immunofluorescent labeling were anti-NFIA (1/1000; rabbit polyclonal, Active Motif), anti β-galactosidase (1/1000; mouse monoclonal, Promega), anti-βIII tubulin (Tuj1 clone; mouse monoclonal, 1/1000; R&D Systems), and anti-GFAP (mouse monoclonal, 1/1000; Millipore Bioscience Research Reagents).

Techniques: In Vivo, Activity Assay, Binding Assay, In Silico, Sequencing, Incubation, Produced, Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Transcription Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Construct, Cotransfection, Cell Differentiation, Activation Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 1. Nfic disruption impairs bone formation during postnatal osteogenesis. (A): Nfic expression was evaluated using real-time PCR analyses in bone marrow stromal cells derived from aged mice. n 5 3, *, p < .05. (B): MC3T3-E1 cells were cultured in differentiation media for up to 3 weeks. Nfic, Runx2, and Osx were evaluated using Western blot analyses. (C): Representative micro-CT image of the mandible and (D) the distal femur. (E): 3D micro-CT images of trabecular bone and cortical bone in the distal femur. (F): Micro-CT quan- tification of the distal femur in WT and Nfic2/2 mice aged 6 weeks. n 5 5, *, p < .05. (G): H&E staining (a–f) and von Kossa staining (a0– f0) from distal femurs in WT and Nfic2/2 mice aged 6 weeks. c–f and c0–f0 are higher magnifications of a–b and a0–b0, respectively. c–d and c0–d0, trabecular bone. e–f and e0–f0, cortical bone. Scale bars, a–b and a0–b0 5 500 mm; c–d and c0–d0 5 200 mm; e–f and e0–f0 5 100 mm. (H): Histomorphometric analyses of distal femoral metaphysis in WT and Nfic2/2 mice aged 6 weeks. n 5 5, *, p < .05. (I): Repre- sentative calcein double labeling sections in femurs from WT and Nfic2/2 mice aged 6 weeks. The white arrow represents the MAR. n 5 5, *, p < .05. Data are presented as the mean 6 SD. Abbreviations: BFR/BS, bone formation rate per bone surface; BMD, bone min- eral density; BV/TV, trabecular bone volume; Ct.BV/TV, cortical bone volume; Ct.Th, cortical bone thickness; MAR, mineral apposition rates; MS/BS, mineralizing surface per bone surface; N.Ob/B.Pm, osteoblast number per bone parameter; N.Ob/T.Ar, osteoblast number per tissue area; Ob.S/BS, osteoblast surface per bone surface; Tb.Th, trabecular bone thickness; Tb.N, trabecular bone number; Tb.SP, trabecular separation; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Disruption, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture, Western Blot, Micro-CT, Staining, Labeling

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 2. Nfic-deficiency increases bone marrow fat as seen in osteoporotic patients. (A): H&E staining of distal femurs from WT and Nfic2/2 mice aged 6 and 28 weeks. c–d and g–h are higher magnifications of boxed a–b and e–f, respectively. a–d, 6 weeks. e–h, 28 weeks. Scale bars 5 a–b and e–f 5 500 mm; c–d and g–h 5 50 mm. (B): Representative oil red O staining images (upper left panel) and quantification of oil red O staining (upper right panel). WT and Nfic2/2 BMSCs were cultured in adipogenic induction media for 7 days. PPARc expression was analyzed using real-time PCR in WT and Nfic2/2 BMSCs (lower panel). n 5 3, *, p < .05. (C): H&E and (D) IHC staining from bone specimens of an osteoporotic patient. Left panel, NFI-C; middle panel, CD68; right panel, negative control. Scale bars 5 50 mm. n 5 3. (E): Expression of NFI-C mRNA was analyzed from gene expression dataset GSE35959 deposited in gene expression omnibus. n 5 5, *, p < .05. (F): Effect of H2O2 on NFI-C expression in hBMSCs. Real-time PCR and Western blot analyses were used to determine the level of NFI-C expression in hBMSCs treated with or without H2O2 (200 mM/ml) for 5 days. n 5 3, *, p < .05. Data are presented as the mean 6 SD. Abbreviations: hBMSCs, human bone marrow stromal cells; NFI-C, nuclear factor I-C; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Staining, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Negative Control, Gene Expression, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 3. Nfic accelerates osteoblast differentiation and suppresses adipocyte differentiation. (A): Nfic2/2 bone marrow stromal cells (BMSCs) were transfected with the Nfic expression vector or control empty expression vector and then cultured in osteogenic induction media for 7 days (ALP staining) and 14 days (ARS). (B): Alp (left panel) and Nfic (right panel) expression were analyzed using real-time PCR. n 5 3, *, p < .05. (C): Representative oil red O staining images (left panel) and quantification of oil red O staining (right panel). Nfic2/2 BMSCs were transfected with the Nfic expression vector or control empty expression vector and then cultured in adipogenic induction media for 7 days. (D): Pparc expression was analyzed using real-time PCR. n 5 3, *, p < .05. (E): Pparc promoter activity was assessed in C2C12 cells transfected with pGL3-Luc-Pparc and Nfic expression vectors or control empty expression vector for 48 hours. n 5 3, *, p < .05. (F): Representative micro-CT images and micro-CT quantification of the distal femurs in WT and Nfic2/2 mice trans- planted with Nfic-overexpressing BMSCs or mock-infected BMSCs at 10 weeks of age. n 5 3, *, p < .05. (G): Histological analyses of distal femurs in Nfic2/2 mice transplanted with Nfic-overexpressing BMSCs. Immunohistochemistry (IHC) analysis of the femur transplanted with Nfic-overexpressed or mock-infected BMSCs using Nfic and GFP antibodies. Scale bars 5 50 mm. (H): H&E staining (left panel). Scale bars 5 200 mm. The number of adipocytes in Nfic2/2 mice transplanted with Nfic-overexpressing BMSCs compared to Nfic2/2 mice transplanted with mock-infected BMSCs at 10 weeks of age (right panel). n 5 3, *, p < .05. Data are presented as the mean 6 SD. Abbre- viations: ALP, alkaline phosphatase; ARS, alizarin red S staining; BV/TV, trabecular bone volume; GFP, green fluorescent protein; IHC, immunohistochemistry; Tb.N, trabecular bone number; Tb.SP, trabecular separation; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Activity Assay, Micro-CT, Infection, Immunohistochemistry

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 4. Nfic disruption impairs osteoblast differentiation and reduces osteoclast activity. (A): ALP staining and activity. (B): ARS and quantification of alizarin red S stained mineralized nodules. WT and Nfic2/2 bone marrow stromal cells (BMSCs) were cultured in osteo- genic induction media for 7 days (ALP) and 14 days (ARS). n 5 3, *, p < .05. (C): Expression of Runx2, Osx, Alp, Oc, and Bsp was analyzed using real-time PCR in WT and Nfic2/2 BMSCs after differentiation for 7 days. n 5 3, *, p < .05. (D): TRAP staining of femurs from WT and Nfic2/2 mice aged 6 weeks. Right panels are higher magnifications of boxed left panels. Scale bars, right panels 5 200 mm; left pan- els 5 100 mm. (E): Histomorphometric analyses of the distal femoral metaphysis from WT and Nfic2/2 mice aged 6 weeks. n 5 5, *, p < .05. (F): WT bone marrow-derived macrophages were cocultured with WT and Nfic2/2 primary osteoblasts for 6 days in the absence or presence of VitD3 and PGE2, fixed, and stained for TRAP. (G): TRAP-positive MNCs were counted in (D). n 5 3, *, p < .05. (H): TRAP activity was quantified in cell lysates. n 5 3, *, p < .05. (I): sRankl levels were measured in cell culture media using ELISA kits. n 5 3, *, p < .05. (J): Expression of Rankl and Opg was analyzed in primary osteoblasts using real-time PCR. n 5 3, *, p < .05. Data are presented as the mean 6 SD. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S staining; MNCs, multinucleated cells; N.Oc/T.Ar, osteo- clast number per tissue area; N.Oc/B.Pm, osteoclast number per bone parameter; Oc.S/BS, osteoclast surface per bone surface; PGE2, prostaglandin E2; TRAP, tartrate-resistant acid phosphatase; VitD3, 1,25-dihydroxyvitamin D3; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Disruption, Activity Assay, Staining, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 5. Nfic mediates BMP-2-Runx2-induced Osx expression. (A): Immunofluorescence staining of Osx (red) in trabecular bone of dis- tal femoral metaphysis from WT and Nfic2/2 mice aged 6 weeks. Scale bar 5 100 mm. Western blotting analyses of Runx2, Osx, and Nfic in WT and Nfic2/2 bone marrow stromal cells (BMSCs). (B): Real-time PCR and Western blot analyses to evaluate Osx expression in MC3T3-E1 cells transfected with Nfic and Runx2 expression vectors or control empty expression vector and treated with BMP-2 (300 ng/ml) for 48 hours. n 5 3, *, p < .05. (C): Osx promoter activity assessed in MC3T3-E1 cells transfected with pGL3-Luc-Osx (21,269 to 191) and Nfic expression vectors or control empty expression vector, and treated BMP-2 (300 ng/ml) for 48 hours. n 5 3, *, p < .05. (D): Scheme of putative Nfic-binding motif in the Osx promoter. The primer pairs used for the ChIP assay are shown as arrowheads (upper panel). Detection of Nfic binding to the Osx promoter using ChIP analyses in MC3T3-E1 cells treated with BMP-2 (300 ng/ml) for 48 hours. The putative Nfic-binding motif was PCR-amplified with P1 primers, and a PCR was also performed with negative control locus primers P2 (lower panel). (E): Nfic2/2 osteoblasts were treated with BMP-2 (300 ng/ml) and/or transfected with the Nfic expression vec- tor. Osx expression was analyzed using real-time PCR. n 5 3, *, p < .05. (F): Representative micro-CT images and micro-CT quantification of the distal femurs in WT and Nfic2/2 mice transplanted with Osx-overexpressing BMSCs or mock-infected BMSCs at 10 weeks of age. n 5 3, *, p < .05. (G): Histological analyses of distal femurs in Nfic2/2 mice transplanted with Osx-overexpressing BMSCs. Immunohisto- chemistry (IHC) analysis of the femur transplanted with Osx-overexpressed or mock-infected BMSCs using Osx and GFP antibodies. Scale bars 5 50 mm. (H): H&E staining (left panel). Scale bars 5 200 mm. The number of adipocytes in Nfic2/2 mice transplanted with Osx- overexpressing BMSCs compared to Nfic2/2 mice transplanted with mock-infected BMSCs at 10 weeks of age (right panel). n 5 3, *, p < .05. Data are presented as the mean 6 SD. Abbreviations: BMP-2, bone morphogenetic protein 2; BV/TV, trabecular bone volume; GFP, green fluorescent protein; IHC, immunohistochemistry; Tb.N, trabecular bone number; Tb.SP, trabecular separation; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Expressing, Staining, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Activity Assay, Binding Assay, Negative Control, Micro-CT, Infection, Immunohistochemistry

Journal: Stem cells (Dayton, Ohio)

Article Title: NFI-C regulates osteoblast differentiation via control of osterix expression.

doi: 10.1002/stem.1733

Figure Lengend Snippet: Figure 6. Runx2 mediates BMP-2-induced Nfic expression. (A, B): Nfic promoter activity and mRNA expression were determined using promoter assays and real-time PCR in MC3T3-E1 cells transfected with pGL3-Luc-Nfic (22,520 to 188) and Runx2 expression vectors or control empty expression vector, and treated with BMP-2 (300 ng/ml) for 48 hours. n 5 3, *, p < .05. (C): Western blot analyses to eval- uate Nfic expression in MC3T3-E1 cells transfected with the Runx2 expression vector or control empty expression vector and treated with BMP-2 (300 ng/ml) for 48 hours. (D): Nfic promoter activity was assessed in MC3T3-E1 cells transfected with pGL3-Luc-Nfic (22,520 to 188) and Runx2 siRNA or control siRNA in the presence or absence of BMP-2 (300 ng/ ml) for 48 hours. n 5 3, *, p < .05. Nfic and Runx2 expression were analyzed using Western blot analyses in control and Runx2-inactivated MC3T3-E1 cells. (E): Scheme of putative Runx2-binding motif in the Nfic promoter. The primer pairs used for the ChIP assay were shown as arrowheads (upper panel). Detection of Runx2 binding to the Nfic promoter using ChIP analyses in MC3T3-E1 cells treated with BMP-2 (300 ng/ml) for 48 hours. The putative Runx2-binding motif was PCR-amplified with P1 primers, and a PCR was also performed with negative control locus primers P2 (lower panel). (F): Immunofluorescence staining of Nfic (Red) in femurs from E18.5 WT and Runx22/2 mice. Scale bar 5 100 mm. Total RNA and protein isolated in calvarial bone from WT and Runx22/2 mice. Runx2 and Nfic expression were assessed using real-time PCR and Western blot analyses. n 5 3, *, p < .05. Data are presented as the mean 6 SD. (G): A model of role of Nfic during osteoblast and adipocyte differentiation. Abbreviations: BMP-2, bone morphogenetic protein 2; WT, wild type.

Article Snippet: The samples were sonicated, followed by chromatin immunoprecipitation with rabbit anti-mouse Nfic (30 ml) and rabbit anti-mouse Runx2 (10 ml, Santa Cruz) antibodies.

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot, Binding Assay, Negative Control, Staining, Isolation

Journal: Journal of molecular endocrinology

Article Title: Molecular cloning and characterization of alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells.

doi: 10.1677/jme.1.02180

Figure Lengend Snippet: Figure 5 Identification of transcription factors binding with CCAAT/Sp1 oligonucleotides in (A) GH3 and (B) normal pituitary cells. Eight micrograms nuclear extracts were incubated with DIG- labeled putative CCAAT/Sp1 oligonucleotides in the absence of antibody(laneControl)orinthepresenceofdifferentantibodies(lanes 2–5 in panel A and lanes 2–6 in panel B). Shifted bands are indicated with arrows. To confirm that binding sites for CBF/A and Sp1 are overlapping, 8 mg nuclear extracts were also incubated with DIG- labeled mutated CCAAT/Sp1 oligonucleotides (lane 6 in panel A). C/EBP, CCAAT/enhancer-binding protein; NF-1, nuclear factor-1.

Article Snippet: Antibodies against CCAATbinding factor (CBF)-A (SC-13045), CCAAT/enhancerbinding protein b (SC-746), nuclear factor-1 (NF-1) (SC-870), and Sp1 (SC-14027) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Incubation, Labeling

Journal: Journal of Virology

Article Title: XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity

doi: 10.1128/jvi.00774-22

Figure Lengend Snippet: Upregulation of XAF1 expression in host infected with RNA viruses. (A) Heatmap of ISGs in the PBMCs from rhesus monkeys ( n = 4) subcutaneously injected with ZIKV (10 5 PFU) for the indicated time points. (B) The volcano plot of differentially expressed ISGs in PBMCs of (A), fold change ≥2 or ≤0.5 (1 d postinfection versus Uninfected control). (C) The PBMCs were from the healthy controls ( n = 52), moderate influenza patients ( n = 63), and severe influenza patients ( n = 44). XAF1 mRNA expression of these samples was analyzed by microarray. The microarray raw data were downloaded from GEO (accession no. GSE101702 ). Data are shown as mean ± SD. (D) The PBMCs were from healthy controls ( n = 30) and SARS-CoV-2-positive COVID-19 patients ( n = 65). XAF1 gene expression of these samples was analyzed by RNA-seq. The RNA-seq raw data were downloaded from GEO (accession no. GSE167000 ). Data are shown as mean ± SD. (E and F) qRT-PCR analysis of XAF1 mRNA expression in the PBMCs (E) or A549 cells (F) infected with WSN (MOI 0.1) or PR8 (MOI 0.1) for 8 h. (G and H) qRT-PCR analysis of XAF1 mRNA expression in the PBMCs (G) or A549 cells (H) infected with VSV (MOI 1) or SeV (MOI 0.1) for 8 h. (I) qRT-PCR analysis of Xaf1 mRNA expression in the PMs infected with WSN (MOI 0.1), PR8 (MOI 0.1), VSV (MOI 1) or SeV (MOI 0.1) for the indicated time points. (J) qRT-PCR analysis of Xaf1 mRNA expression in the lung tissues from the mice infected with PR8 (100 PFU) intranasally for 3 d. (K and L) Immunoblot analysis of XAF1 in the PBMCs infected with PR8 for the indicated time points (K) or indicated MOI (L). (M) Immunoblot analysis of XAF1 in the A549 cells infected with PR8 (MOI 0.5) for the indicated time points. (E–J) Data from three independent experiments are presented as mean ± SD; * , P < 0.05; * * , P < 0.01; and ** * , P < 0.001 indicate significant difference by unpaired Student's t test. (K–M) Data are representative results of three independent experiments. The band intensities compared to respective loading control were labeled (L).

Article Snippet: Human Flag-XAF1-expressing plasmids with an FGEH backbone were transfected into the A549 cells, and these cells were selected by 2 μg/mL puromycin (InvivoGen) after 48 h transfection.

Techniques: Expressing, Infection, Injection, Control, Microarray, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Labeling

Journal: Journal of Virology

Article Title: XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity

doi: 10.1128/jvi.00774-22

Figure Lengend Snippet: IRF1 is required for the induction of XAF1 during host antiviral immunity. (A) qRT-PCR analysis of Xaf1 mRNA expression in the PMs transfected with poly(I:C) (1 μg/mL) for 12 h. (B) qRT-PCR analysis of Xaf1 mRNA expression in the PMs transfected with 3p-hpRNA (200 ng/mL) for the indicated time points. (C) qRT-PCR analysis of Xaf1 mRNA expression in the PMs treated with IFN-α (500 U/mL) or IFN-β (500 U/mL) for the indicated time points. (D) qRT-PCR analysis of XAF1 mRNA expression in the A549 cells treated with IFN-β (100 U/mL) for 6 h. (E) qRT-PCR analysis of Xaf1 mRNA expression in the PMs from the WT and Ifnar1 −/− mice infected with IAV (PR8 or WSN, MOI 0.1) for 8 h. (F) qRT-PCR analysis of Xaf1 mRNA expression in the PMs pretreated for 1 h with the indicated inhibitors, followed by IAV infection (PR8 or WSN, MOI 0.1) for 8 h. (G) qRT-PCR analysis of XAF1 mRNA expression in the WT and IRF1 −/− A549 cells infected with WSN (MOI 0.1) for 8 h. (H) qRT-PCR analysis of XAF1 mRNA expression in the WT and IRF1 −/− A549 cells stimulated with IFN-β (100 U/mL) for 8 h. (I) Immunoblot analysis of indicated proteins in the WT and IRF1 −/− A549 cells infected with WSN (MOI 0.1) for 12 h. (J) The IRF1 plasmid (0, 0.5, 1, 2 μg) were transfected into HEK293T cells in a 6-well plate. After 24 h, total cell lysates were subjected to immunoblot analysis of indicated proteins. (K) The model of potential IRF1 binding sites in XAF1 promoter. (L) EV (empty vector), RIG-I(2CARD), MAVS, TBK1, IRF3, or IRF1 vector (100 ng) was cotransfected with the control vector pGL4.17 (100 ng), XAF1 promoter-luciferase reporter constructs (WT-luc, 100 ng), and pRL-TK-Luc vector (10 ng) into HEK293T cells in a 24-well plate. After 24 h, relative luciferase activity was quantified. (M) EV, RIG (2CARD), MAVS, or IRF1 vector (100 ng) was cotransfected with the control vector pGL4.17 (100 ng), XAF1 promoter-luciferase reporter constructs (WT-luc, 100 ng), or mutant XAF1 promoter-luciferase reporter constructs (Mut-luc, 100 ng), and pRL-TK-Luc vector (10 ng) into HEK293T cells in a 24-well plate. After 24 h, relative luciferase activity was quantified. (N) The RIG-I(2CARD) plasmid (5 μg) was transfected into HEK293T cells in a 10-cm dish. After 24 h, total cell lysates were incubated with biotinylated DNA (Mut#1, Mut#2, Mut#1 + 2 [double mutation], respectively), followed by immunoblot detection. (O) The IRF1 plasmid (5 μg) was transfected into HEK293T cells in a 10-cm dish. After 24 h, total cell lysates were incubated with biotinylated DNA (Mut#1, Mut#2, Mut#1 + 2, respectively), followed by immunoblot detection. (A–H, L, and M) Data from three independent experiments are presented as mean ± SD; * , P < 0.05; * * , P < 0.01; and ** * , P < 0.001 indicate significant difference by unpaired Student's t test. (I, J, N, and O) Data are representative results of three independent experiments.

Article Snippet: Human Flag-XAF1-expressing plasmids with an FGEH backbone were transfected into the A549 cells, and these cells were selected by 2 μg/mL puromycin (InvivoGen) after 48 h transfection.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Infection, Western Blot, Plasmid Preparation, Binding Assay, Control, Luciferase, Construct, Activity Assay, Mutagenesis, Incubation

Journal: Journal of Virology

Article Title: XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity

doi: 10.1128/jvi.00774-22

Figure Lengend Snippet: XAF1 protects the host against multiple RNA viruses. (A) The FGEH and FGEH-XAF1 A549 cell clones were infected with PR8-Luc (MOI 1). After 12 h or 24 h, relative renilla luciferase activity was quantified. (B) qRT-PCR analysis of viral RNA (vRNA) in the FGEH and FGEH-XAF1 A549 cell clones infected with WSN (MOI 0.1) for 8 h. (C) Supernatants of (B) were measured for WSN TCID 50 assays. (D) qRT-PCR analysis of viral RNA in the FGEH and FGEH-XAF1 A549 cell clones infected with ZIKV (MOI 0.01) for 24 h. (E and F) qRT-PCR analysis of viral RNA in the FGEH and FGEH-XAF1 A549 cell clones infected with VSV (MOI 1) (E), and SeV (MOI 0.1) (F) for the indicated time points. (G and H) Fluorescence microscopy imaging of GFP in the FGEH and FGEH-XAF1 A549 cell clones infected with VSV-GFP (MOI 1) (G), and SeV-GFP (MOI 0.1) (H) for 8 h; scale bar, 200 μm. (I) qRT-PCR analysis of XAF1 mRNA in the PBMCs transfected with control (Ctrl) or XAF1 siRNA for 48 h, followed by PR8 infection (MOI 0.1) for 12 h. (J) qRT-PCR analysis of viral RNA in the PBMCs transfected with Ctrl or XAF1 siRNA for 48 h, followed by PR8 infection (MOI 0.1) for 12 h. (K) Supernatants of (J) were measured for PR8 TCID 50 assays. (A–F and I–K) Data from three independent experiments are presented as mean ± SD; * , P < 0.05; * * , P < 0.01; and ** * , P < 0.001 indicate significant difference by unpaired Student's t test. Data of (G and H) are representative results of three independent experiments.

Article Snippet: Human Flag-XAF1-expressing plasmids with an FGEH backbone were transfected into the A549 cells, and these cells were selected by 2 μg/mL puromycin (InvivoGen) after 48 h transfection.

Techniques: Clone Assay, Infection, Luciferase, Activity Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Imaging, Transfection, Control

Journal: Journal of Virology

Article Title: XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity

doi: 10.1128/jvi.00774-22

Figure Lengend Snippet: Knockout of XAF1 facilitates RNA virus infection in vitro and in vivo. (A) qRT-PCR analysis of viral RNA in the WT and XAF1 −/− A549 cell clones infected with ZIKV for 24 h. (B) Supernatants of (A) were measured for ZIKV plaque assays. (C and D) qRT-PCR analysis of viral RNA in the WT and Xaf1 −/− fibroblast infected with VSV (MOI 1) (C) or SeV (MOI 0.1) (D) for the indicated time points. (E and F) qRT-PCR analysis of viral RNA in the WT and Xaf1 −/− BMDMs infected with VSV (MOI 1) (E) or SeV (MOI 0.1) (F) for the indicated time points. (G) qRT-PCR analysis of viral RNA in the WT and Xaf1 −/− PMs infected with PR8 (MOI 0.1) for 8 h. (H) TCID 50 assays of supernatants from the WT and Xaf1 −/− PMs infected with PR8 (MOI 0.1) for 12 h. (I) qRT-PCR analysis of viral RNA in the WT and Xaf1 −/− PMs infected with WSN (MOI 0.1) for 8 h. (J) TCID 50 assays of supernatants from the WT and Xaf1 −/− PMs infected with WSN (MOI 0.1) for 12 h. (K) TCID 50 assays of the lung homogenates from the WT ( n = 11) and Xaf1 −/− ( n = 10) mice infected with PR8 (100 PFU) intranasally for 5 d. (L and M) Histological examination of the lung sections from the WT and Xaf1 −/− mice infected with PR8 (100 PFU) intranasally for 5 d was performed by H&E staining (L), and neutrophil infiltrations of the lung sections from the indicated mice were measured by MPO staining (M); scale bar, “—” represents 400 μm; “‐‐‐” represents 200 μm; data are representative results of three independent experiments. (N and O) Survival assays of the WT and Xaf1 −/− mice infected with PR8 (100 PFU) intranasally (WT group: n = 18; Xaf1 −/− group: n = 14). Bodyweight loss curves (N) and Kaplan-Meier survival curves (O) were generated and analyzed from three independent experiments; * , P < 0.05 and ** * , P < 0.001 indicate significant difference. Data of (A–K) from three independent experiments are presented as mean ± SD; * , P < 0.05; * * , P < 0.01; and ** * , P < 0.001 indicate significant difference by unpaired Student's t test.

Article Snippet: Human Flag-XAF1-expressing plasmids with an FGEH backbone were transfected into the A549 cells, and these cells were selected by 2 μg/mL puromycin (InvivoGen) after 48 h transfection.

Techniques: Knock-Out, Virus, Infection, In Vitro, In Vivo, Quantitative RT-PCR, Clone Assay, Staining, Generated

Journal: Journal of Virology

Article Title: XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity

doi: 10.1128/jvi.00774-22

Figure Lengend Snippet: XAF1 maintains IRF1 expression and IRF1-dependent antiviral immunity. (A) Immunoblot analysis of IRF1 protein in the WT and Xaf1 −/− PMs infected with WSN (MOI 0.1) at the indicated time points. (B) Immunoblot analysis of indicated proteins. A549 cells in a 6-well plate were transfected with the vector or XAF1 (1 μg) plasmid, followed by WSN (MOI 0.1) infection for 6 h. (C) Immunoblot analysis of indicated proteins in the WT and Xaf1 −/− PMs infected with WSN (MOI 0.1) for the indicated time points. (D) qRT-PCR analysis of antiviral ISGs ( Ddx58 , Mx1 , Oas2 , and Ddx60 ) mRNA levels in the WT and Xaf1 −/− PMs treated with IFN-β (500 U/mL) for the indicated time points. (E) Immunoblot analysis of indicated proteins in the WT and Xaf1 −/− PMs treated with IFN-β (500 U/mL) at the indicated time points. (F) WT and IRF1 −/− A549 cells in 12-well plates were transfected with the EV or XAF1 (0.5 μg) plasmid, followed by WSN (MOI 0.1) infection for 12 h. The supernatants were measured for TCID 50 assays. (G and H) The WT and IRF1 −/− A549 cells in 12-well plates were transfected with the empty vector (EV) or XAF1 (0.5 μg) plasmid, followed by WSN (MOI 0.1) infection for 6 h. qRT-PCR analysis of OAS2 (G) and DDX60 (H) mRNA levels in cell lysate. (D, F, G, and H) Data from three independent experiments are presented as mean ± SD; * , P < 0.05; * * , P < 0.01; and ** * , P < 0.001 indicate significant difference, and ns, no significant difference by unpaired Student's t test. Data of (A–C and E) are representative results of three independent experiments.

Article Snippet: Human Flag-XAF1-expressing plasmids with an FGEH backbone were transfected into the A549 cells, and these cells were selected by 2 μg/mL puromycin (InvivoGen) after 48 h transfection.

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

Journal: The Journal of comparative neurology

Article Title: DNER and NFIA are Expressed by Developing and Mature AII Amacrine Cells in the Mouse Retina

doi: 10.1002/cne.24345

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: Atlas Antibodies HPA006111 RRID:AB_1854422 1:500 Determined in this study Prospero Homeobox 1 Prox1 Rabbit Polyclonal Mouse Prox1, peptide mapping to amino acids 723–737.

Techniques: Labeling, Purification

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: Nfix is mainly expressed by anti-inflammatory MPs. ( a ) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) of WT mice injected by CTX at D2, D4 and D7, post-injury. Immunostaining for F4/80 (green), Nfix (red) and DAPI (blue) at D4 and D7 after CTX injection; ( b ) Percentage of Ly6C + and Ly6C - sorted MPs positive for Nfix in TA muscles of WT mice injected by CTX at D2, D4 and D7 post-injury; ( c ) Percentage of Nfix + MPs after M1 and M2c polarization (with IFNγ and IL10, respectively). * p < 0.05; *** p < 0.001; for (b) * p < 0.05 Ly6C+ vs. Ly6C + at D4 and D7; # p < 0.05 Ly6C − D7 vs. D2. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: Muscles, Injection, Immunostaining

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: MPs lacking Nfix are unable to adopt an anti-inflammatory phenotype in vivo and in vitro. ( a ) Representative FACS (Fluorescence-Activated Cell Sorting) gate of pro- and anti-inflammatory CD64 + MP populations in TA of Nfix fl/fl and LysM CRE :Nfix fl/fl mice at D2 after CTX injection. (CD64 + /Ly6C + and CD64 + /Ly6C − respectively); ( b ) Ratio of Ly6C +/ Ly6C − MPs sorted from TA of Nfix fl/fl and LysM CRE :Nfix fl/fl mice at D2, D4 and D7, after CTX injection; ( c ) WT BMDM (Bone Marrow Derived Macrophages) were transduced by shScramble and shNfix lentiviral vectors and then polarized into M1 and M2c MPs with IFNγ and IL10 treatment, respectively. MPs were immunolabeled for pro-inflammatory markers (TNFα, iNOS and CCl3) and anti-inflammatory markers (ArgI, TGFβ, CD163 and CD206). The number of positive cells is expressed as percentage out of total cells. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. shScramble M1 MPs. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. shScramble M2c MPs. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: In Vivo, In Vitro, Fluorescence, FACS, Injection, Derivative Assay, Immunolabeling

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: Nfix is expressed after phagocytosis and drive MP phenotypical switch. ( a ) Phagocytosis assay of M1 and M2c Nfix fl/fl and LysM CRE :Nfix fl/fl MPs cocultured 8h with apoptotic mpc. Representative FACS gate of phagocytotic M2c Nfix fl/fl and LysM CRE :Nfix fl/fl MPs (CD64 + CellVue + ) and percentage of phagocytotic M1 and M2c MPs coming from Nfix fl/fl and LysM CRE :Nfix fl/fl BMDM; ( b ) WT MPs were cocultured 16h with apoptotic mpcs. Representative FACS gate of non-phagocytotic (CD64 + CellVue − ) and phagocytotic (CD64 + CellVue + ) WT MPs. Quantification of Nfix expression realized by RT-qPCR on sorted non-phagocytotic and phagocytotic WT MPs and quantification of MPs positive for Nfix (Nfix + /F4/80 + ) realized by IF on non-phagocytotic and phagocytotic WT MPs; ( c ) WT MPs were cocultured for 16 h with apoptotic mpcs, with or without addition of Cytochalasin D. Quantification of F4/80 + MPs were positive for Nfix on a total of F4/80 + MPs; ( d ) Western blot of Nfix expression in WT MPs treated with DMSO (Dimethyl sulfoxide) or Y27632 for 16 h and quantification. Vinculin was used to normalize; ( e ) WT MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells; ( f ) LysM CRE :Nfix fl/fl MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells. * p < 0.05, *** p < 0.001. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: Phagocytosis Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunolabeling