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

Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

doi: 10.1359/jbmr.091036

Figure Lengend Snippet: RT-PCR and Q-PCR

Article Snippet: To generate LV-Gsα WT and LV-Gsα R201C vectors, the SalI/HindIII cassette was excised from the rat WT Gsα cDNA (ATCC 63315, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), or the R201C rat Gsα cDNA (ATCC 63317, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), respectively, and inserted into the PmeI/SmaI sites of the pWPXLd-GFP LV transfer vector [provided by D. Trono ( 22 ) ].

Techniques: shRNA

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

Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

doi: 10.1359/jbmr.091036

Figure Lengend Snippet: Analysis of cell proliferation in transduced hBMSCs expressed as (A) population doublings or (B) BrdU incorporation for 2 hrs and 24 hrs. The presence of mutated GsαR201C does not alter BMSC proliferation. Data from 3 experiments are expressed as means ± SD. (C) In vivo transplantation of normal, FD-derived (R201C) and LV- GsαR201C-transduced hBMSCs. Note the formation of abundant bone (b) on hydroxyapatite (ha/tcp, hydroxyapatite/tricalcium phosphate), hematopoietic tissue (hem, meg = megakaryocytes) and adipocytes (ad) in control transplants (upper panels). Transplants of FD-derived BMSCs and transduced BMSCs are recognized by the reduced amount of bone and the fibrous tissue (ft), devoid of hematopoietic cells and adipocytes, filling the marrow space. Polarized light microscopy demonstrates that bone formed by normal BMSCs formed lamellar bone (lb), whereas transduced BMSCs formed woven bone (wb).

Article Snippet: To generate LV-Gsα WT and LV-Gsα R201C vectors, the SalI/HindIII cassette was excised from the rat WT Gsα cDNA (ATCC 63315, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), or the R201C rat Gsα cDNA (ATCC 63317, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), respectively, and inserted into the PmeI/SmaI sites of the pWPXLd-GFP LV transfer vector [provided by D. Trono ( 22 ) ].

Techniques: BrdU Incorporation Assay, In Vivo, Transplantation Assay, Derivative Assay, Control, Light Microscopy

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

Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

doi: 10.1359/jbmr.091036

Figure Lengend Snippet: (A) Schematic representation of the LV vectors expressing the Gsα RNA interfering sequences, under the control of the H1 promoter, and eGFP as a marker. (B) Design of different RNA interfering sequences directed to the human exon1 and human/rat GsαR201C exon 8. (C) Left panel. HeLa cells were first infected with the bidirectional LV-GFP-GsαR201C and subsequently super-infected with the different LV-siGsα vectors as indicated. Specific knock-down of mutated Gsα was obtained only with LV-siGsαR201C-4. Right panel. HeLa GFP-GsαR201C/siCtr or HeLa GFP-GsαR201C/siGsαR201C-4 were superinfected with LV-EF-1α-GsαR201C or LV-EF-1α-GsαWT. A specific down-regulation of the added-in mutated R201C Gsα was observed. In contrast, robust expression of the added-in GsαWT was observed. (D) Left panel. Highly mutated in R201C, FD-derived hBMSCs were mock-infected or treated with LV-siCtr, or LV-siGsαR201C-4 and after 20 days of culture the cells were collected for western blot analysis. A decrease of both long and short endogenous form of Gsα was observed with LV-siGsαR201C-4. Right panel. Normal hBMSCs were untreated or treated with LV-siCtr, LV- siGsαR201C-4 and LV-siGsα human exon 1 (hu). 20 days post-infection cells were processed for western blotting. Both Gsα endogenous WT isoforms (long and short Gsα) were efficiently suppressed with siGsα exon 1 but remained efficiently expressed with specific siGsαR201C-4. (E) cAMP assay on HeLa LV-GFP-GsαR201C/siGsαR201C-4, demonstrates the capacity to restore the cAMP levels similar to that of the basal levels. Data are means ± SD of n=3 experiments in duplicate. b, p < 0.01 versus untransduced HeLa equally treated; c, p < 0.01 versus LV-GsαR201C and LV-GsαR201C/LV-siCtr cells equally treated. (F) cAMP assay on transduced FD-derived BMSCs and normal hBMSCs. A significant reduction of cAMP levels, comparable to that recorded in normal hBMSC, was observed in FD-LV-siGsαR201C-4. a, p < 0.05 of FD-LV-siGsαR201C-4 versus FD mock, FD-LV-siCtr, and hBMSC-GsαR201C.

Article Snippet: To generate LV-Gsα WT and LV-Gsα R201C vectors, the SalI/HindIII cassette was excised from the rat WT Gsα cDNA (ATCC 63315, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), or the R201C rat Gsα cDNA (ATCC 63317, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), respectively, and inserted into the PmeI/SmaI sites of the pWPXLd-GFP LV transfer vector [provided by D. Trono ( 22 ) ].

Techniques: Expressing, Control, Marker, Infection, Knockdown, Derivative Assay, Western Blot, cAMP Assay

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

Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

doi: 10.1359/jbmr.091036

Figure Lengend Snippet: Inhibition of PDE (IBMX) results in marked enhancement of up-regulation of adipogenic markers in BMSCs induced to in vitro adipogenesis. Constitutive activity of Gsα (R201C) results in a less prominent but clear-cut enhanced expression of adipogenic markers. Inhibition of PDE in the presence of constitutively active Gsα, in contrast, results in the block of adipogenic differentiation. In BMSCs induced to in vitro osteogenic differentiation, addition of IBMX enhances the up-regulation of BSP, ALP and RANK-L. Enhanced expression of the same markers is also observed in the absence of IBMX, as an effect of constitutive activity of Gsα. Magnitude of enhancement is schematically represented by red, light red and gray character colors in the notation of the different markers.

Article Snippet: To generate LV-Gsα WT and LV-Gsα R201C vectors, the SalI/HindIII cassette was excised from the rat WT Gsα cDNA (ATCC 63315, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), or the R201C rat Gsα cDNA (ATCC 63317, GenBank {"type":"entrez-nucleotide","attrs":{"text":"M12673","term_id":"204441"}} M12673 ), respectively, and inserted into the PmeI/SmaI sites of the pWPXLd-GFP LV transfer vector [provided by D. Trono ( 22 ) ].

Techniques: Inhibition, In Vitro, Activity Assay, Expressing, Blocking Assay

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

doi: 10.1523/JNEUROSCI.2450-11.2011

Figure Lengend Snippet: SPON neurons express HCN1 and HCN2 channels and have large Ih inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of Ih with 20 μM ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ~1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student’s t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

Article Snippet: Sections were incubated in 2% normal donkey serum in blocking solution overnight at 4°C with one of the following primary antibodies: polyclonal rabbit α -HCN1 (1:250, lot number AN-10; Alomone Labs), which is directed against amino acid residues 6–24 of the intracellular N terminus of rat HCN1 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKB0","term_id":"29840774","term_text":"Q9JKB0"}} Q9JKB0 ); polyclonal rabbit α -HCN2 (1:400, lot number AN-08; Alomone Labs), directed against amino acids 147–161 of the intracellular N terminus of human HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 ); or monoclonal mouse α -HCN2 (1:400, clone N71/37; NeuroMab, UC Davis/NIH NeuroMab Facility) directed against amino acids 761–863 of the C terminus of rat HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKA9","term_id":"83303515","term_text":"Q9JKA9"}} Q9JKA9 ) used in combination with a mouse-on-mouse kit (Vector Laboratories).

Techniques: Immunostaining, Expressing, Activation Assay