Journal: BMC Molecular and Cell Biology

Article Title: Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells

doi: 10.1186/s12860-019-0187-2

Figure Lengend Snippet: miR-3198 gain-of-function and loss-of-function experiments. Results of real-time RT-PCR analysis for miR-3198 ( a , b ), OPG and RANKL ( c , d ) expression in HPL cells after transfection of miR-3198 inhibitor ( a , c ) and miR-3198 mimic ( b , d ). n = 3. Biological triplicated. Fold change from the control is shown. Open bar indicates the fold change of OPG expression, and close bar indicates that of RANKL expression ( c , d ). The concentrations of OPG as measured by ELISA after transfection of miR-3198 inhibitor ( e ) and miR-3198 mimic ( f ) are shown ( n = 3). Results of real-time PCR analysis for miR-1207 ( g , h ), OPG and RANKL ( i , j ) expression in HPL cells under the transfection of miR-1207 inhibitor ( g , i ) and miR-1207 mimic ( h , j ). n = 3. Biological triplicated. Fold changes from the control are shown. * indicates P < 0.05 versus control, and NS indicating there was no significant difference between samples

Article Snippet: The concentration of OPG in the culture supernatant was measured using an OPG ELISA kit (Boster Biological Technology, Pleasanton, CA), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: BMC Molecular and Cell Biology

Article Title: Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells

doi: 10.1186/s12860-019-0187-2

Figure Lengend Snippet: miR-3198 regulates the mechanical stress-mediated change of OPG expression. Results of real-time RT-PCR analysis for OPG and RANKL expression in HPL cells in the compression ( a ) and tension ( b ) experiments. n = 3. Biological triplicated. Fold change from the control are shown. Cont, control; Inh, transfection of miR-3198 inhibitor; Mimic, transfection of miR-3198 mimic; press, compression; tens, tension; TF, transfection. Also shown are the OPG concentrations measured by ELISA in the compression ( c ) and tension ( d ) experiments ( n = 3). * indicates P < 0.05 versus control. † indicates P < 0.05 between samples. NS indicates there was no significant difference between samples. e and f Representative image of the western blotting for OPG was shown. g and h Relative band intensity of the western blotting for OPG. *: P < 0.05 versus control. †: P < 0.05 between the groups. NS, no significant difference between the samples

Article Snippet: The concentration of OPG in the culture supernatant was measured using an OPG ELISA kit (Boster Biological Technology, Pleasanton, CA), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: DSCAM Contributes to Dendrite Arborization and Spine Formation in the Developing Cerebral Cortex

doi: 10.1523/JNEUROSCI.2811-12.2012

Figure Lengend Snippet: DSCAM expression during cortical development. A–H, Coronal sections of E16.5 (A, E), E18.5 (B, F), P3 (C, G), and P10 (D, H) cortex hybridized with radioactive RNA probes against DSCAM (A–D) and Fezf2 (E–H), a transcription factor expressed by corticospinal motor neurons that will occupy layer V. DSCAM mRNA is ubiquitously expressed throughout the cortical plate during embryonic and postnatal development (A–D) and is particularly upregulated in developing corticospinal motor neurons. I, Fluorescent image showing Thy1-EYFP-H expression in layer V pyramidal neurons at P42. J, FACS of YFP (FITC)+/PI (APC)− pyramidal neurons from Thy1-EYFP-H adult cortices. Left and right, sorting of PI and YFP, respectively. Cells within box P1 are PI−, and cells within box P2 are PI− /YFP+. K, RT-PCR for DSCAM, Bcl11b, and YFP using samples prepared from YFP−/PI− and YFP+/PI− sorted neurons. L, Assessment of DSCAM protein levels in cortical tissues derived from P1–P28 using Western blot analysis. DSCAM protein levels peak between P7 and P10. Actin immunoreactivity was used as a loading control. M, Subcellular expression of DSCAM, PSD-95, synaptophysin, SynCAM, GDI, and actin in multiple brain fractions including CH, S1, S2, P2′, LS1, LP1, M, and SPM. DSCAM is enriched in SPM fraction. Scale bar: (in A) A–H, 100 μm.

Article Snippet: Sections were washed twice with PBS, permeabilized for 20 min with PBS/0.1% Triton X-100 (PBST), blocked for at least 1 h at room temperature (RT) with heat-inactivated goat serum (5%) and horse serum (5%) in PBS-T, and incubated in anti-Tbr1 1:200 (AbCam) and anti-Bcl11b 1:200 (Santa Cruz Biotechnology) or anti-Cux1 1:1000 (Santa Cruz Biotechnology) and anti-Bcl11b overnight at RT.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: DSCAM Contributes to Dendrite Arborization and Spine Formation in the Developing Cerebral Cortex

doi: 10.1523/JNEUROSCI.2811-12.2012

Figure Lengend Snippet: Lack of DSCAM in DSCAMdel17 mutant causes changes in cortical thickness and lamination in early postnatal animals. A–F, Nissl-stained coronal brain sections (50 μm) from P1 (A, B), P10 (C, D), and P42 (E, F) wt (A, C, E) and DSCAMdel17 mutant (B, D, F) littermates. Sections of the cortex above the hippocampus with the pial surface oriented at the top. G, Quantification of mean cortical thickness at P1, P10, P17, P21, and P42 in wt and DSCAMdel17 mutant littermates. Analysis was performed on the following: P1: wt, n = 4 brains, 69 sections; DSCAMdel17 mutant, n = 5 brains, 94 sections; P10: wt, n = 6 brains, 96 sections; DSCAMdel17, n = 4 brains, 59 sections; P17: wt, n = 3 brains, 57 sections; DSCAMdel17, n = 3 brains, 51 sections; P21: wt, n = 3 brains, 50 sections; DSCAMdel17, n = 3 brains, 52 sections; P42: wt, n = 4 brains, 54 sections; DSCAMdel17, n = 4 brains, 55 sections. H–I, Representative coronal sections derived from E16.5 wt (H) and DSCAMdel17 mutant (I) cortex after a 30 min BrdU pulse. J, Quantitative analysis of BrdU-labeled S-phase cells in the developing neocortex of E16.5 embryos after a 30 min pulse. Each column represents the average number of a single embryo. Error bars show SE. K–L, Representative coronal sections of TUNEL staining in wt (K) and DSCAMdel17 mutant (L) P1 cortices and in ACBD3Myr E12.5 cortex representing as a positive control (M). N, Quantitative analysis of cortical lamination at P1. While the thickness of upper cortical layers II/III (Cux1+) is reduced in DSCAMdel17 mutants, layer V and VI cortical lamination (Bcl11b+ and Tbr1+, respectively) remains intact. Analysis of laminar thickness was performed on the following: Cux1: wt, n = 3 brains, 42 sections; DSCAMdel17 mutant, n = 3 brains, 42 sections; Bcl11b: wt, n = 5 brains, 63 sections; DSCAMdel17 mutant, n = 5 brains, 63 sections; Tbr1: wt, n = 4 brains, 49 sections; DSCAMdel17 mutant, n = 3 brains, 35 sections. O–R, Coronal sections of wt (O, Q) and DSCAMdel17 mutant (P, R) P1 cortex immunostained for Bcl11b (O”, P”, Q”, R”), Tbr1 (O', P'), and Cux1 (Q', R') and counterstained with DAPI (O–R). Cux1+ layers II/III is reduced in thickness (Q”', R”'), whereas Bcl11b+ layer V and Tbr1+ layer VI are normal in DSCAMdel17 mutants compared with wt (O”' and P”'). Mean ± SE, **p < 0.01, ***p < 0.001 compared with wt (Student's t test). Scale bars: (in B, D, L, O) A, B, C–F, K, L, O–R, 200 μm; (in H, M) H, I, M, 100 μm. Arrowheads in K–M mark TUNEL+ cells.

Article Snippet: Sections were washed twice with PBS, permeabilized for 20 min with PBS/0.1% Triton X-100 (PBST), blocked for at least 1 h at room temperature (RT) with heat-inactivated goat serum (5%) and horse serum (5%) in PBS-T, and incubated in anti-Tbr1 1:200 (AbCam) and anti-Bcl11b 1:200 (Santa Cruz Biotechnology) or anti-Cux1 1:1000 (Santa Cruz Biotechnology) and anti-Bcl11b overnight at RT.

Techniques: Mutagenesis, Staining, Derivative Assay, Labeling, TUNEL Assay, Positive Control