Journal: bioRxiv

Article Title: PTGER2-β-Catenin Axis Links High Salt Environments to Autoimmunity by Balancing IFNγ and IL-10 in FoxP3 + Regulatory T cells

doi: 10.1101/379453

Figure Lengend Snippet: (a) Relative expression level of Active β-catenin (ABC) on ex vivo Treg subpopulations analyzed by flow cytometry (n=11 subjects). Fold change in gMFI over DN were depicted. * P <0.05, ** P <0.01 (one-way ANOVA with Tukey’s multiple comparisons test). gMFI, geometric mean fluorescence intensity. (b) Expression level of ABC between CXCR3 − and CXCR3 + ex vivo Tregs from healthy controls. Representative histogram (left) and summary of results (n=7 subjects) (right). * P <0.05 (two-tailed paired Student’s t -test). (c) Gene expression of Wnt/ β-catenin signaling target genes ( AXIN2 and TCF7 ) assessed by RNA-seq. * P <0.05, ** P <0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (d) Relative expression level of ABC on Tregs stimulated with anti-CD3 and anti-CD28 for 4 days, followed by 4 h PMA/iomomycin stimulation and intracellular cytokine staining for IFNγ and IL-10 (n=12 subjects). Fold change in gMFI over DN were depicted. * P <0.05, ** P <0.01, *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (e) Expression level of β-catenin on Tregs stimulated with anti-CD3 and anti-CD28 in the presence (Th1) or absence (Th0) of IL-12 for 4 days. β-catenin level was determined directly by intracellular staining (left) (n=9 subjects) and β-catenin level on IFNγ positive/negative Treg populations was determined after 4 h PMA/iomomycin stimulation (middle) (n=4 subjects). Representative histogram for β-catenin expression was shown (right). * P <0.05 (two-tailed unpaired Student’s t -test). (f, g) Frequency of IFNγ and IL-10 positive cell number ( f ) and gene expression of IFNG and IL10 by qPCR ( g ). Tregs were stimulated with anti-CD3 and anti-CD28 in the presence of Wnt/β-catenin signaling inhibitor PKF115-584 (PKF), IL-12 (Th1), or IL-12 and PKF115-584 (Th1 + PKF) (n=4 subjects) * P <0.05, ** P <0.01, *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (h, i) Relative frequency of IFNγ and IL-10 positive cell number (fold of scramble shRNA/control condition) ( h ) and gene expression of IFNG and IL10 by qPCR ( i ). Tregs were transduced with a non-targeted shRNA or a CTNNB1 shRNA and cultured in Th0 or Th1 condition for 5 days ( h ; n=7 subjects, i ; n=5 subjects). * P <0.05, ** P <0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Data are representative of two experiments (e (middle), and f) or are from more than three experiments.

Article Snippet: Th1-Tregs induced with human recombinant IL-12 (20 ng/ml) (R&D).

Techniques: Expressing, Ex Vivo, Flow Cytometry, Fluorescence, Two Tailed Test, RNA Sequencing Assay, Staining, shRNA, Transduction, Cell Culture

Journal: bioRxiv

Article Title: PTGER2-β-Catenin Axis Links High Salt Environments to Autoimmunity by Balancing IFNγ and IL-10 in FoxP3 + Regulatory T cells

doi: 10.1101/379453

Figure Lengend Snippet: (a) AXIN2 and TCF7 mRNA expression in IFNγ + and IFNγ − human Treg populations assessed by DNA microarray (n=8 subjects) . * P <0.05 (two-tailed paired Student’s t -test). (b) CTNNB1 gene expression on Tregs transduced with a non-targeted shRNA or a CTNNB1 shRNA and cultured for 5 days (n=10 subjects). *** P <0.001 (two-tailed unpaired Student’s t -test). (c) Flow cytometric analysis of TCF1 expression on ex vivo Treg subpopulations relative to DN (n=8 subjects). * P <0.05, ** P <0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (d) Frequency of IFNγ and IL-10 positive cell number. Tregs were stimulated with anti-CD3 and anti-CD28 in the presence of SGK1 inhibitor GSK650394 (SGK1-i), IL-12 (Th1), or IL-12 and GSK650394 (Th1 + SGK1-i) (n=6 subjects) *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Th1-Tregs induced with human recombinant IL-12 (20 ng/ml) (R&D).

Techniques: Expressing, Microarray, Two Tailed Test, Transduction, shRNA, Cell Culture, Ex Vivo

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Cited2 is expressed broadly by CPN progenitors at E15.5, with expression refining to CPN of the somatosensory cortex by P3. A, Cited2 is highly expressed by CPN (red) relative to CSMN (blue) at critical times during development, as detected by microarray analysis of FACS-purified CPN and CSMN. Error bars denote SEM (Molyneaux et al., 2009). B, Western blot analysis showing that CITED2 protein is highly expressed as early as E15.5 in the neocortex, with expression decreasing postnatally, relative to a β-actin loading control. C–F, Expression of Cited2 is largely restricted to subpallial progenitors at E13.5 (C), but Cited2 is highly expressed in the cortical SVZ at E15.5 (D), the peak of superficial layer CPN birth, with expression maintained in layers II/III and V postnatally (E, F). G, Embryonically, Cited2 is expressed uniformly across the neocortex, detected across the SVZ at E18.5 (arrowheads) and across the cortical plate (CP). H, In the first days postnatally, however, its expression refines and becomes restricted to somatosensory cortex (arrows) by P3. I, At E15.5, Cited2 is highly expressed in the SVZ, extending into the intermediate zone (IZ). J, Cited2 (blue) is largely excluded from PAX6+ (green) radial glial progenitors of the VZ, but is highly expressed by TBR2+ (red) IPCs of the SVZ. K, Cited2 is largely excluded from the highly proliferative Ki67+ (green) VZ and apical mitotic cells, as indicated by pH3 (red), but is expressed by basally proliferating IPCs of the SVZ and IZ. Scale bars: C–F, 500 μm; G–I, 1 mm; C′–H′, J, K, 100 μm; I′, 200 μm. Dotted lines in J and K indicate apical, ventricular surface.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing, Refining, Microarray, Purification, Western Blot

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in specific reduction of TBR2+ IPCs in the E15.5 neocortex. A–A″, At E15.5, the peak of Cited2 expression, there is no change in the overall number of (PAX6+) radial glial progenitors in the Cited2;Emx1-Cre cKO neocortex compared with control littermates (WT). B–B″, There is, however, a significant reduction in the number of TBR2+ IPCs, which largely give rise to superficial layer CPN at this stage of development. C–D″, Reduction in TBR2+ IPCs might result from reduced proliferation of IPCs (C–C″), as indicated by proliferating cell nuclear antigen (PCNA, red) and TBR2 (green) double positivity (N = 11 WT, 6 cKO for A–C); specific reduction in basal cell divisions (D–D″), as indicated by position of pH3-positive mitotic cells (N = 10 WT, 5 cKO); and/or increased cell death (E), as indicated by expression of aC3. There is increased apoptotic cell death in the Cited2 cKO neocortex, both within the progenitor population and postmitotically (N = 11 WT, 6 cKO). F–F″, To directly investigate whether Cited2 cell-autonomously regulates proliferation of IPCs, we electroporated Cre recombinase and GFP into VZ progenitors of Cited2fl/fl and Cited2fl/wt littermates at E14.5 to excise Cited2 in a small subpopulation of neocortical progenitors. We used a BrdU pulse at E15.5, and immunocytochemistry for Ki67 at E16.5 to identify progenitors that continued to proliferate. There is a significant reduction in the number of Cited2-null (Cited2 fl/fl; Cre+) progenitors that incorporate BrdU at E15.5, or express Ki67+ at E16.5, demonstrating that Cited2-null IPCs are less likely to re-enter the cell cycle than their heterozygous counterparts. There is no change in the number of aC3+ cells in the Cited2-null progenitors at E16.5 (N = 4 Cited2fl/wt; 5 Cited2fl/fl). Scale bars, 100 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in reduced superficial layer thickness and total neocortical length at P3. A, At P6, the Cited2 cKO neocortex is smaller than in WT littermate controls, but both CPN (SATB2+) and CSMN (CTIP2+) are present and appropriately positioned. B, C, Anterograde labeling with DiI (B) and retrograde labeling with CTB (C) demonstrate that CPN are present and are targeting the contralateral hemisphere in the Cited2 cKO neocortex. However, both the distribution of retrogradely labeled CPN (C) and CUX1 (red, superficial layers) and CTIP2 (green, deep layers) immunocytochemistry (D) indicate that superficial layers are thinner in the Cited2 cKO neocortex, while the thickness of deep layers is unchanged (N = 4–5 per genotype for A–D). E, Quantitative analysis of neocortical layer thickness at P3 reveals that superficial layers (II–IV; LMO4, red) are significantly thinner in motor, somatosensory, and visual neocortical areas of P3 Cited2 cKO mice compared with those of control littermates. There is no change in deep-layer thickness (V, VI; CTIP2, green) in any region (N = 8 WT, 4 cKO). F, The Cited2 cKO cortex is visibly smaller than control littermates at P3, both in wholemount view and in sagittal sections. G, There is a significant reduction in total neocortical surface rostrocaudal length of ∼10%, as measured on four sagittal sections across the mediolateral axis (N = 8 WT, 4 cKO). Scale bars: A, 1 mm; B, 50 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Labeling, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Neocortical surface length reduction is restricted to layers II/III of somatosensory cortex in the P3 Cited2 cKO neocortex. A–C, Analysis of three broad neocortical areas identified by LMO4 expression at P3 indicates a highly specific and substantial reduction (∼30%) in rostrocaudal surface length of the somatosensory area (blue) in the Cited2 cKO neocortex, entirely accounting for the total cortical surface length reduction (N = 8 WT, 4 cKO). D–G, Reduced somatosensory cortex length (black arrowheads) was confirmed via expression of multiple genes either excluded from superficial layers of the somatosensory cortex (D, E; Cadh8, EphA7), or specifically expressed in the somatosensory cortex (F; ephrinA5). Measurements of the acallosal layer IV somatosensory cortex (black arrows), by contrast (F, G; ephrinA5, Rorβ), reveals that there is no significant difference in non-CPN somatosensory cortex length in Cited2 cKO mice compared with that in WT mice (N = 8 WT, 4 cKO). H–H‴, In the P3 Cited2 WT neocortex, molecular markers of layer IV (green, RORβ; bracket) and the somatosensory cortex in layers II/III (red, Bhlhb5; white line) align at the motor/somatosensory border, shown in sagittal view (rostral to left). I–I‴, In the P3 Cited2 cKO neocortex, by contrast, the boundary of layer II/III expression of Bhlhb5 (white line, with additional low-level expression indicated by dashed line) is located caudal to layer IV RORβ expression (bracket), resulting in a misalignment of molecular areal boundaries between CPN of layer II/III and acallosal layer IV (H‴, I‴; N = 4–5 per genotype). Scale bars: D–G, 500 μm; H–I, 200 μm. Error bars denote SEM. *p < 0.05; **p < 0.001 (Student's t test).

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Additional loss of Lmo4 function does not alter Cited2 cKO neocortical thickness, but does reestablish layer II/III somatosensory neocortical length at the expense of the motor cortex. A–C, Additional loss of Lmo4 function in the Cited2-null neocortex (Cited2fl/fl;Lmo4fl/fl;Emx1Cre/+) does not alter the reduced reduction in superficial layer thickness of the Cited2-null motor or visual cortex, but does increase superficial layer thickness in the somatosensory cortex. Additional loss of Lmo4 function does not alter the overall reduction in total neocortical length (data not shown). D–G, Additional loss of Lmo4 function does, however, re-establish layer II/III somatosensory neocortical length (as measured by Bhlhb5 expression) to normal control length, at the expense of the layer II/III motor cortex. H, Length of motor (rostral to Bhlhb5 layer II/III expression), somatosensory (Bhlhb5 layer II/III positive), and visual (caudal to layer II/III Bhlhb5 expression) cortical areas was measured in control (Emx1-Cre negative), Lmo4 cKO (Cited2+/+;Lmo4fl/fl;Emx1Cre/+), Cited2 cKO (Cited2fl/fl;Lmo4+/+;Emx1Cre/+), and dcKO (Cited2fl/fl;Lmo4fl/fl;Emx1Cre/+) littermates. I–L, In the context of the shortened neocortical surface length in Cited2 cKO mice, additional loss of Lmo4 function re-establishes the length of the somatosensory area boundary, at the expense of the motor cortex length. By contrast, loss of Lmo4 function has no effect on the overall reduced neocortical length or layer II/III thickness of the Cited2 cKO neocortex. Scale bars: C, 100 μm; D–G, 1 mm. For each neocortical area, data were analyzed by a one-way ANOVA with Tukey's post-test. For all experiments, N = 14 controls, 7 Lmo4 cKO, 7 Cited2 cKO, and 8 double cKO mice. Error bars denote SEM. *p < 0.05, **p < 0.001, ***p < 0.0001.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Excision of Cited2 postmitotically via NEX-Cre does not alter neocortical laminar thickness or neocortical area lengths. A, B, NEX-Cre-mediated postmitotic excision of Cited2 does not visibly alter brain morphology or neocortical laminar development at P6 (A; as indicated by DAPI nuclear staining), and there is no significant difference in P3 neocortical length between Cited2; NEX-Cre cKO mice and control littermates (B). C–E, Further, there is no change in the overall neocortical laminar thickness of superficial or deep layers in the P3 Cited2; NEX-Cre cKO neocortex (C–C″), nor is there a change in the length of motor, somatosensory, or visual neocortical area lengths in the Cited2; NEX-Cre cKO neocortex (D, E). For all experiments, N = 4 WT, 4 cKO. Scale bars: A, 1 mm; C, 100 μm. Error bars denote SEM.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Loss of Cited2 function results in aberrant dendritic complexity of superficial layer somatosensory CPN. A, A′, Neuronal soma size (NeuN area) is not affected by loss of Cited2. Increased neuronal density is evident here, consistent with the modest, but significant, increase in cell density in layer II/III of the somatosensory cortex (23% increase, p = 0.01) quantified at P6 (see Results). N = 3 WT, 3 cKO. B–D, Dendritic complexity of layer II/III pyramidal neurons (primarily CPN) was analyzed at P22 by Golgi staining and Sholl analysis in the Cited2;Emx1-Cre cKO neocortex, compared with control littermates. B, B′, D, D′, There is no significant difference in dendritic complexity of layer II/III CPN in the motor cortex (B, B′) or visual cortex (D, D′) of Cited2; Emx1-Cre cKO mice, compared with littermate controls. C, C′, There is, however, a significant increase in dendritic complexity of layer II/III CPN in the somatosensory cortex of Cited2;Emx1-Cre cKO mice (two-way ANOVA p < 0.0001). E, CPN of the motor cortex are more complex than CPN of the somatosensory or visual cortex (two-way ANOVA p < 0.0001). F, In Cited2; Emx1-Cre cKO mice, dendritic complexity of somatosensory CPN is not significantly different from the CPN of the motor cortex (two-way ANOVA p = 0.16), suggesting that somatosensory CPN might be partially “motorized” in the absence of Cited2 function. G–I, Dendritic complexity of layer II/III CPN was analyzed at P22 by Golgi staining and Sholl analysis in a Cited2;NEX-Cre cKO neocortex, compared with control littermates. Following this postmitotic loss of Cited2 function, there is no change in dendritic complexity of superficial layer pyramidal neurons in any of the primary areas examined (motor, somatosensory, or visual cortices). Scale bars, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni's post-test in B–F, G. B–E: motor, N = 17 WT, 12 cKO; somatosensory, N = 27 WT, 14 cKO; visual, N = 18 WT, 9 cKO. G–I: motor, N = 38 WT, 34 cKO; somatosensory, N = 37 WT, 20 cKO; visual, N = 34 WT, 19 cKO.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity

doi: 10.1523/JNEUROSCI.4067-15.2016

Figure Lengend Snippet: Interhemispheric CPN axonal connectivity is disrupted in the adult Cited2 cKO neocortex. A, B, HARDI tractography analysis of interhemispheric connections demonstrates a significant reduction in the size of the CC and in the number of callosal fibers in the juvenile/young adult (9 weeks old) Cited2; Emx1 cre cKO neocortex, compared with littermate controls, particularly within the mid-CC (corresponding to the somatosensory cortex). Reconstructed pathways are superimposed on the mean diffusion-weighted MRI of the brain. Pathways running between right and left are red; dorsal and ventral are green; and anterior and posterior are blue. The images do not include anterior commissure or olfactory bulb fibers; these were removed a priori to focus on the CC. Images in A′ and B′ similarly exclude hippocampal commissure fibers. N = 2 WT, 2 cKO. C, D, Staining for MBP in sagittal sections of Cited2 WT and cKO brains (C) followed by measurement of midsagittal CC area (D) identifies a reduction in CC area in Cited2 cKO brains, but demonstrates the structural integrity of the CC throughout all areas. N = 3 WT, 4 cKO. E–K, To investigate precision of callosal projections in the absence of Cited2 function, a focal injection of the anterograde tracer AAV-GFP was made in the somatosensory (extending into motor) neocortex at P6, and contralateral callosal projections were analyzed at 6 weeks of age. In contrast to the precise homotopic projections observed in Cited2 WTs (G), callosal projections in the Cited2 cKO somatosensory neocortex are diffuse (J). K, Relative GFP fluorescence intensity was measured in matched sagittal sections of the left injected neocortical hemisphere and in the same region in the contralateral projection hemisphere, demonstrating consistent caudal spread of callosal projections in the Cited2 cKO neocortex. N = 3 WT, 3 cKO. ANOVA analysis finds no change in the anterior tail (−1400 to −800 μm) or center (−700 to 700 μm) regions, but the posterior tail (800–1400 μm) of the cKO distribution is significantly different than that of the WT (p < 0.0001). Error bars denote SEM. *p < 0.05, (Student's t test) in C. *p < 0.001 (Bonferroni post-test) in K. Scale bars: A–B′, 1.5 mm; C, D, F, G, I, J, 1 mm; G′, J′, 500 μm.

Article Snippet: Membranes were incubated for 12–20 h at 4°C in rabbit anti-CITED2 (Abcam ab108345) primary antibody diluted in 2% milk/TBS, and developed with goat anti-rabbit HRP IgG (Bio-Rad) diluted in 2% milk/TBS, and signals were detected with chemiluminescence (Pierce).

Techniques: Diffusion-based Assay, Staining, Injection, Fluorescence

Journal: Bioscience Reports

Article Title: Differentially expressed circulating miRNAs in postmenopausal osteoporosis: a meta-analysis

doi: 10.1042/BSR20190667

Figure Lengend Snippet: Characteristics of included miRNA profiling studies

Article Snippet: 12 , Chen C , 2013 , China , Blood , 20 (10/10) , miRNA microarray, LC Sciences , 721 , 7 , 3 , 4 , P <0.05.

Techniques: Clinical Proteomics, Microarray, Sequencing

Journal: Bioscience Reports

Article Title: Differentially expressed circulating miRNAs in postmenopausal osteoporosis: a meta-analysis

doi: 10.1042/BSR20190667

Figure Lengend Snippet: * indicates miRNAs reported in more than one study, # indicates miRNAs reported to be dysregulated in both directions.

Article Snippet: 12 , Chen C , 2013 , China , Blood , 20 (10/10) , miRNA microarray, LC Sciences , 721 , 7 , 3 , 4 , P <0.05.

Techniques: