Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 expression is induced by the DNA damage response in a p53 dependent manner. ( A ) qPCR analysis of Meg3 expression in HUVECs treated with 200 nM, 600 nM and 1 μM of doxorubicin for 12 h. ( B ) qPCR analysis of Meg3 expression in HUVECs treated with 10 μM nutlin-3 for 12, 24 and 48 h. ( C ) qPCR analysis of Meg3 expression in HUVECs treated with 100 μM palmitic acid for 12, 24 and 48 h. ( D ) qPCR analysis of Meg3 expression in HUVECs treated with doxorubicin (40 nM for 12 h) or nutlin-3 (10 μM for 12 h) with or without lentiviral knockdown of p53. ( E ) In situ hybridization of Meg3 in HUVECs treated with or without 0.2 μM doxorubicin for 12 h. The number of red dots per nucleus from 13 cells was counted for each condition. Data show mean ± S.E.M., n = 3 (A–D), n = 13 (E); * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Expressing, In Situ Hybridization

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 knockdown induces DNA damage. HUVECs were transfected with control or Meg3 GapmeRs for neutral comet assay ( A and B ) and western blot analysis ( C ). ( A ) Representative images of DNA tail length and moment under basal condition and in ECs treated with 10 ng/ml TNF-α for 16 h. ( B ) Quantification of DNA tail length and moment under basal conditions and in ECs treated with 10 ng/ml TNF-α for 16 h. ( C ) Western blot analysis of key proteins involved in the DNA damage response under basal conditions. Data show mean ± S.E.M., n ≥ 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Neutral Comet Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Transcriptome analysis identifies p53 signaling as the most significantly upregulated pathway upon Meg3 knockdown. HUVECs were transfected with control or Meg3 GapmeRs. 24 h after transfection, cells were treated with 10 ng/ml TNF-α for 3 h and collected for microarray gene chip analysis. Chromatin immunoprecipitation was performed using transfected cells with or without 10 ng/ml TNF-α treatment for 1 h. ( A ) Volcano plot shows differentially expressed mRNAs in ECs upon Meg3 knockdown. ( B ) KEGG signaling pathways analysis identified significantly regulated pathways among upregulated genes upon Meg3 knockdown. ( C ) Venn diagram shows p53 target genes that are regulated by Meg3. ( D ) qPCR analysis of a group of p53 target genes that were induced upon Meg3 knockdown. (E) Enrichment of p53 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-p53 antibodies followed by qPCR analysis. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Microarray, Chromatin Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Quantitative proteomics analysis identifies p53 signaling as the most significantly regulated pathway upon Meg3 silencing. HUVECs were transfected with control or Meg3 GapmeRs. 24 h after transfection, cells were treated with or without 10 ng/ml TNF-α for 4 h. After treatment, cells were collected for TMT10-plex labeling and mass spectrometry analysis, western blot analysis, or immunoprecipitation. ( A ) Volcano plot shows differentially expressed proteins in ECs upon Meg3 silencing under TNF-α-treated condition. ( B ) KEGG signaling pathways were identified with significant enrichment among upregulated proteins upon Meg3 silencing under TNF-α-treated condition. ( C ) Venn diagram shows proteins encoded by p53 target genes that are regulated by Meg3 under TNF-α-treated condition. ( D ) Western blot analysis of p53, MDM2, and p21. ( E ) Quantifications for (D). ( F ) Co-immunoprecipitation was performed to examine the interaction between p53 and MDM2 in TNF-α-treated HUVECs. ( G ) The level of phosphorylated p53 at serine 15 was examined in immunoprecipitated total p53 in TNF-α-treated HUVECs transfected with control or Meg3 GapmeRs. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, Labeling, Mass Spectrometry, Western Blot, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Meg3 knockdown promotes apoptosis and inhibits proliferation in ECs. HUVECs were transfected with control or Meg3 GapmeRs, treated with or without 10 ng/ml TNF-α for 12 or 16 h. ( A ) Caspase 3/7 activities reflected by luminescence levels were measured, and fold changes were calculated relative to that in ECs transfected with control GapmeRs without TNF-α treatment. ( B ) The number of TUNEL positive cells per high power view were shown. TUNEL positive cells are in red color due to presence of DNA breaks, and DAPI-stained nuclei appear in blue. ( C ) The percentages of EdU positive cells (red) among Hoechst 33342 (blue) stained cells were calculated in transfected cells with or without 12 h TNF-α treatment. ( D ) Cell cycle distribution revealed by flow cytometry. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, TUNEL Assay, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: LncRNA pull-down assay identifies PTBP3 as a binding partner of Meg3. ( A ) LncRNA pull-down assay was used to identify the proteins associated with Meg3 transcript variant 1 (TV1) by incubating the cell lysates with biotinylated sense or antisense Meg3 RNA. The RNA–protein complexes captured by T-1 beads were subjected to silver stain after separation on SDS-PAGE gel. ( B ) The RNA–protein complexes from lncRNA pulldown using antisense Meg3 (negative control RNA), sense Meg3 (TV1), and Meg3 deletion mutants were subjected to western blot analysis of PTBP3 and GAPDH after separation on SDS-PAGE gel. GAPDH was examined as a negative control protein. ( C ) The interaction of endogenous PTBP3 with Meg3 was detected by RNA immunoprecipitation. EC lysates were immunoprecipitated with anti-PTBP3 antibody or Isotype matched control IgG. Meg3 was examined by qPCR in the immuno-precipitates using primer set 2 as shown in (D). LncRNA Neat1 was used as a positive control RNA that interacts with PTBP3. ( D ) Different sets of Meg3 primers were used to detect Meg3 by qPCR following RNA immunoprecipitation using anti-PTBP3 antibodies. Primer sets 1, 2, 5 detect Meg3 transcript variants 1 and 6 (TV1 and TV6); primer sets 3 and 4 detect Meg3 TV1; and primer sets 6 and 7 detect Meg3 TV6. ( E ) Dual-staining of Meg3 and PTBP3 in HUVECs. Linear trajectories (yellow line) crossing the cells with the intensities of Meg3 and PTBP3 signals were presented at the right side of images. White and black arrows indicate partial colocalization of Meg3 and PTBP3 in the nucleus of HUVECs. Data show mean ± S.E.M., n = 3; * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Pull Down Assay, Binding Assay, Variant Assay, Silver Staining, SDS Page, Negative Control, Western Blot, Immunoprecipitation, Positive Control, Staining

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: PTBP3 knockdown phenocopies Meg3′s effects on EC apoptosis and proliferation. HUVECs were transfected with control or PTBP3 siRNAs (A–E), treated with or without TNF-α for 12 or 16 h. HUVECs were transfected with control GapmeRs or Meg3 GapmeRs (F). ( A ) PTBP3 knockdown by siRNA significantly reduces PTBP3 mRNA. ( B ) qPCR analysis of a group of p53 target genes in TNF-α-treated HUVECs. ( C ) Caspase 3/7 activities reflected by luminescence levels were measured, and fold changes were calculated relative to that in ECs transfected with control siRNAs without TNF-α treatment. ( D ) The number of TUNEL positive cells per high power view were shown. TUNEL positive cells are in red color due to presence of DNA breaks, and DAPI-stained nuclei appear in blue. ( E ) The percentages of EdU positive cells (red) among Hoechst 33342 (blue) stained cells were calculated in transfected cells with or without 12 h TNF-α treatment. ( F ) Enrichment of PTBP3 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-PTBP3 antibodies followed by qPCR analysis in TNF-α-treated HUVECs transfected with Ctl GapmeRs or Meg3 GapmeRs. ( G ) Enrichment of PTBP3 at the promoters of indicated genes was identified by chromatin immunoprecipitation using anti-PTBP3 antibodies followed by qPCR analysis in TNF-α-treated HUVECs transduced with lentivirus expressing Ctl shRNA or p53 shRNA. N.S., non-significant. ( H ) qPCR analysis of CDKN1A, MDM2 , and GADD45a promoters after chromatin isolation by RNA purification using control probes or Meg3 probes. Data show mean ± S.E.M., n = 3 or 4 (A–G) or = 2 ( H ); * P < 0.05.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Transfection, TUNEL Assay, Staining, Chromatin Immunoprecipitation, Transduction, Expressing, shRNA, Isolation, Purification

Journal: Nucleic Acids Research

Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response

doi: 10.1093/nar/gky1190

Figure Lengend Snippet: Schematic summary of this study. Under basal conditions, Meg3 protects DNA from damage independent of PTBP3, while it restrains the expression of p53 target genes in cooperation with PTBP3 likely through interactions with the promoters of p53 target genes. DNA damage induces Meg3 expression via a p53 dependent mechanism. Induced Meg3 is likely involved in restoring the homeostasis of DNA damage response through an unknown mechanism, as indicated by a question mark. Meg3 knockdown leads to DNA damage and ATM activation, which in turn stabilizes p53 by disrupting p53 and MDM2 interaction. Activation of p53 signaling results in the expression of p53 target genes, EC apoptosis, and decreased EC proliferation. PTBP3 knockdown phenocopies Meg3′s effects on the activation of p53 signaling, the expression of p53 target genes, EC apoptosis, and decreased EC proliferation.

Article Snippet: In situ RNA detection was performed using RNAscope ® 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics) and RNAscope® Probe - Hs-MEG3 following the manufacturer's instructions.

Techniques: Expressing, Activation Assay

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) RNA in situ hybridization shows robust ADAMTS1 and ADAMTS9 expression in umbilical vein endothelium and tunica media (TM) and in outer arterial TM. Robust ADAMTS4 and ADAMTS5 expression was confined to the venous endothelium, with moderate ADAMTS4 expression and minimal ADAMTS5 expression in SMC (n = 3 umbilical cords for each probe). ( b ) ADAMTS-cleaved aggrecan (anti-NITEGE, red) and versican (anti-DPEAAE, red) both showed strong ADAMTS proteolytic activity throughout the venous wall and the outer artery TM. Unlike aggrecan, extensive versican proteolysis is seen in the arterial intima and sub-intima (n = 4 umbilical cords for each antibody). Wj, Wharton’s jelly. The brackets mark TM boundaries. Scale bars in a-b = 100 μm.

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNA In Situ Hybridization, Expressing, Activity Assay

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) H and E staining of E18.5 wild-type cords showing thicker umbilical arterial (A) and thinner venous (V) wall (n = 6 umbilical cords). ( b ) Aggrecan and versican localization (red, DAPI counterstain blue) in E18.5 wild-type cords showing staining in the arterial inner tunica media (TM) and adventitia but not the vein (n = 3 umbilical cords). ( c ) β-gal (blue) and eosin (red) staining of E18.5 Adamts1 LacZ /+ ( Adamts1 +/- ) cord showing strong Adamts1 expression in venous endothelium and TM and outer artery TM (n = 3 umbilical cords). ( d ) Short umbilical cords in E18.5 Adamts1 -/- and Acan -/- embryos compared to wild type. Red arrowhead indicates an omphalocele in Adamts1 -/- embryos. ( e ) H & E staining of E18.5 wild type, Acan -/- and Adamts1 -/- cord cross-sections showing thinner walls in Acan -/- umbilical vessels and thicker walls in Adamts1 -/- umbilical vessels. ( f–g ) Cord length, TM thickness and vessel luminal area quantifications for Adamts1 -/- ( f ) and Acan -/- mice ( g ) at E18.5 compared to wild-type littermates. Acan -/- umbilical cords show larger lumens and Adamts1 -/- vessels show smaller lumens in (n = 7–11 umbilical cords each, whiskers indicate minimum and maximum values, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). ( h ) Phospho-histone H3 (pHH3) staining shows significantly fewer proliferating cells (white arrowheads) in Acan -/- umbilical vessels. Dotted white lines mark the boundaries of vessel lumens (n = 4 cords each, whiskers indicate minimum and maximum values, **, p<0.001; *, p<0.05). Scale bars = 100 μm in ( a ), 25 μm in ( c ), 100 μm and 50 μm in ( e ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining, Expressing

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) RNA in situ hybridization analysis of E12.5 and E18.5 mouse umbilical cord sections Acan, Vcan and relevant ADAMTS genes (n = 3 umbilical cords for each probe). ( b ) E12.5 and E14.5 wildtype and Acan -/- embryos have a comparable umbilical cord length (n = 2 Acan -/- at E12.5 and n = 3 at E14.5). ( c ) Observed and expected genotype ratios at different embryonic stages from Acan +/- and Adamts1 +/- intercrosses. ( d ) Hematoxylin and eosin staining of E14.5 wild type and Acan -/- umbilical cords shows completion of longitudinal to circumferential reorientation of smooth muscle cells in the mutant arterial tunica media (TM). Upper panels show tangential longitudinal sections whereas the lower panels are taken through the approximate center of each vessel (n = 3 umbilical cords of each genotype). Adv, adventitia; Tm, tunica media. Scale bars in a = 100 μm, 3 mm and 7 mm in ( b ) and 50 μm in ( d ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNA In Situ Hybridization, Staining, Mutagenesis

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) Aggrecan (green) and α-SMA staining (red) in E18.5 umbilical cords show loss of aggrecan and weak α-SMA staining in Acan -/- vessels (n = 3 umbilical cords each genotype). ( b ) Smooth muscle myosin heavy chain (SMMHC, red) and phosphorylated myosin light chain (pMLC, green) staining in E18.5 umbilical cords showing dramatic signal attenuation in the Acan -/- vessels (n = 3 umbilical cords each genotype) ( c ) pMLC (green), endomucin (red), α-SMA (red, center panels) and SMMHC (red, right-hand panels) staining shows blunted dimorphism of Adamts1 -/- umbilical artery and vein with stronger expression of differentiated SMC markers in Adamts1 -/- umbilical vessels and acquisition of endomucin, a venous endothelium marker, by arterial endothelium (n = 3 umbilical cords each genotype) Scale bars = 100 μm in ( a–c ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining, Expressing, Marker

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a,b ) E17.5 Adamts1 -/- umbilical vessels show increased aggrecan staining and reduced anti-NITEGE staining in ( a ), quantified in ( b ) (n = 3 cords each genotype, error bars indicate mean ±S.D.*, p<0.05; **, p<0.01; ***, p<0.001). ( c,d ) Adamts1 -/- umbilical vessels show increased versican ( c ) and reduced anti-DPEAAE staining quantified in ( d ) (n = 3 cords each genotype, error bars indicate mean ±S.D. *, p<0.05; **, p<0.01). Scale bars = 50 μm in ( a–c ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNAscope, In Situ, Software, Gene Expression, Microarray

Journal: Neuron

Article Title: Loss of adaptive myelination contributes to methotrexate chemotherapy-related cognitive impairment

doi: 10.1016/j.neuron.2019.04.032

Figure Lengend Snippet: A) Representative image demonstrating RNAscope visualization of frontal cortex deep layer neurons (NeuN, white), astrocytes (Glast, green), and Bdnf mRNA (red). DAPI, blue. Scale bar = 20 μm

Article Snippet: RNAscope was performed according to Fluorescent Multiplex Reagent Kit protocol using RNAscope probes against BDNF (Advanced Cell Diagnostics, 424821), RbFox3 (NeuN; Advanced Cell Diagnostics 313311-C2) and Slc1a3 (Glast; Advanced Cell Diagnostics, 320851-C3).

Techniques:

Journal: eLife

Article Title: Stereotyped transcriptomic transformation of somatosensory neurons in response to injury

doi: 10.7554/eLife.49679

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RNAscope probe-Mm-S100b , Advanced Cell Diagnostics , ACD: 431731 , .

Techniques: Knock-In, Virus, Sequencing, Modification, Recombinant, RNAscope, Multiplex Assay, Software

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Behavioral screening of LS and HS subpopulations of mice exposed to 3-week CMS. ( B ) Quantitative PCR to examine mRNA expression of cytokines, chemokines, growth factors, and trophic factors in the hippocampus and prefrontal cortex of HS and LS mice. Data are standardized to control group ( n = 4 to 6, each sample in triplicate). ( C ) Enzyme-linked immunosorbent assay to assay the protein level of IL4 in prefrontal cortex, cerebellum, hippocampus, amygdala, and olfactory bulb of control, HS, and LS mice. Each circle represents one mouse ( n = 6, each sample in triplicate). ( D ) Western blotting shows the levels of IL4, IL4 receptor α chain (IL4Rα), STAT6, and pSTAT6 in the hippocampus of control, HS, and LS mice. IL4 and IL4Rα are normalized to β-actin, and the pSTAT6 is normalized to STAT6. ( n = 4 to 6, each sample in triplicate). ( E ) Correlation between sucrose preference or immobility duration in TST, hippocampal Arg1 levels, and hippocampal IL4 levels in CMS-exposed mice. Each circle represents one mouse ( n = 6). ( F ) Morphological characters of hippocampal microglia in control, HS, and LS mice. Scale bars, 50 μm (top) and 10 μm (bottom). The histogram represents the quantification of number and branch length of Iba1 + cells in hippocampus. Each dot in the bar graph represents the average of all micrograph (five to six micrographs for each mouse) in each mouse ( n = 5). ( G ) Immunofluorescence micrographs and quantification of Arg1 + microglia in hippocampus of control, LS, and HS mice ( n = 5). Scale bars, 50 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (B, C, D, F, and G). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Experimental timeline of IL4 overexpression in the hippocampus and CMS protocol. ( B ) Volcano maps indicate differentially expressed genes (DEGs) in the brains of CMS-exposed and/or AAV-IL4–treated mice. ( C ) Quantitative PCR examination of the expression of several DEGs in the hippocampus of mice ( n = 4 to 6). ( D ) Western blotting shows the levels of IL4, Arg1, and iNOS in the hippocampus of CMS-exposed and/or AAV-IL4–treated mice ( n = 5 to 6). ( E ) Morphological characters of Arg1 + microglia in hippocampus. Scale bar, 100 μm. The histogram represents the quantification of number and branch length of Arg1 + -Iba1 + cells in hippocampus. Each dot in the bar graph represents the average of five to six micrographs in each mouse ( n = 6). ( F ) Flow cytometry of single-cell suspension of the whole hippocampus for microglia (CD45 int -CD11b + cells), activated microglia (CD86 + cells), and anti-inflammatory microglia (CD86 + -CD206 + cells). The histogram represents the quantification of CD45 int -CD11b + cells, CD86 + cells, and CD86 + -CD206 + cells ( n = 5 to 6). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 versus AAV + Ctrl group, # P < 0.05, ## P < 0.01, ### P < 0.005 versus AAV + CMS group, two-way ANOVA with Tukey test (C); * P < 0.05, ** P < 0.01, *** P < 0.005, two-way ANOVA with Tukey test (D, E, and F). FITC A, fluorescein isothiocyanate area (FITC-A).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry, Suspension

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Immunofluorescence micrographs and quantification show the hippocampal neurogenesis of LS and HS mice ( n = 5). ( B ) GO enrichment analysis for DEGs in the hippocampus of CMS-exposed mice. ER, endoplasmic reticulum; FDR, false discovery rate. ( C ) Effects of IL4 overexpression and/or microglial IL4Rα down-regulation on NSPC proliferation and differentiation in the hippocampus of CX 3 CR1 Cre/ERT2 mice. Representative fluorescence micrographs showing DCX expression and BrdU incorporation in the subgranular zone (SGZ). Scale bar, 100 μm. Quantification of total number of BrdU + cells, total number of DCX + -BrdU + cells, and percentage of DCX + -BrdU + cells out of all BrdU + cells in the neurogenic zones ( n = 5). ( D ) Effects of IL4 overexpression and/or microglial IL4Rα down-regulation in the hippocampus (before IL4 overexpression) on NSPC survival and maturation. Representative fluorescence micrographs illustrating NeuN expression and BrdU incorporation in the SGZ. Scale bar, 100 μm. Quantification of total number of BrdU + cells, BrdU + -NeuN + cells, and DCX + -NeuN + cells in DG ( n = 5). ( E ) Effects of conditioned medium from microglia isolated from hippocampus on NSPC proliferation. Proliferation was monitored by quantifying the percentage of BrdU + cells ( n = 6). Scale bar, 50 μm. Representative micrographs of NSPCs cultured for 7 days in conditioned culture medium from microglia isolated from hippocampus. Cells were immunolabeled with glial fibrillary acidic protein (GFAP) to identify astrocytes and MAP2 to identify neurons. Percentages of GFAP + cells and MAP2 + cells are quantified ( n = 6). Scale bar, 30 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (A) and two-way ANOVA with Tukey test (C, D, and E).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Immunofluorescence, Over Expression, Fluorescence, Expressing, BrdU Incorporation Assay, Isolation, Cell Culture, Immunolabeling

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Western blotting shows the levels of BDNF and TrkB in the hippocampus of LS and HS mice. BDNF and TrkB are normalized to β-actin ( n = 3). ( B ) Western blotting shows the levels of BDNF and TrkB in the hippocampus of CMS and/or AAV-IL4–treated mice. BDNF and TrkB are normalized to β-actin ( n = 4). ( C ) BDNF was detected in hippocampal microglia (Iba1 + cells) using Bdnf -RNAscope. ( D ) The mRNA expression of BDNF was observed in hippocampal microglia of AAV-IL4–treated mice ( n = 4). ( E ) Double immunohistochemical staining of BDNF and Arg1 in the hippocampus of AAV-IL4 mice. Scale bar, 20 μm. ( F ) Effects of CMS, knockdown of microglial IL4Rα, and overexpression of IL4 on BDNF protein levels in the hippocampus ( n = 5). ( G ) Effects of k252a treatment to block the BDNF/TrkB pathway on hippocampal neurogenesis in AAV-IL4 mice under CMS exposure. Representative fluorescence micrographs showing DCX expression and BrdU incorporation in the SGZ. Scale bar, 100 μm. Quantification of the number of DCX + cells and total number of DCX + -BrdU + cells in the neurogenic zones ( n = 5). DMSO, dimethyl sulfoxide. ( H ) The expression and secretion of BDNF in primary microglia after IL4 treatment in vitro ( n = 6). ( I ) Effects of anti-BDNF antibody or K252a on NSPC proliferation (BrdU + cells) in the presence of the conditioned culture medium from IL4-treated microglia ( n = 5). Scale bar, 20 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (A, G, and I), two-way ANOVA with Tukey test (B, D, and F), and two-tailed t test (H).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Western Blot, RNAscope, Expressing, Immunohistochemical staining, Staining, Knockdown, Over Expression, Blocking Assay, Fluorescence, BrdU Incorporation Assay, In Vitro, Two Tailed Test

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Effects of overexpression of IL4 in the hippocampus on stress-induced depressive-like behaviors. Mice were injected stereotactically with AAV-IL4 or AAV, allowed to recover for 2 weeks, and then subjected to a CMS protocol consisting of exposure to three random stressors daily for 4 weeks. The mice were assessed by SPT and FST ( n = 8). ( B ) Correlations between sucrose preference and percentage of Arg1 + microglia in hippocampus, immobility time in FST and percentage of Arg1 + microglia in hippocampus, sucrose preference and concentration of BDNF in hippocampus, immobility time in FST and concentration of BDNF in hippocampus, sucrose preference and number of BrdU + -DCX + cells in DG, and immobility time in FST and number of BrdU + -DCX + cells in DG. Each circle represents one mouse ( n = 6). ( C ) Effects of microglial IL4Rα down-regulation (before IL4 overexpression) on stress-induced depressive-like behaviors, as assessed by SPT and FST ( n = 8 to 10). ( D ) Assessment of stress-induced depressive-like behaviors in WT mice when treated with K252a or TMZ with sCMS exposure ( n = 8). ( E ) Assessment of stress-induced depressive-like behaviors in AAV-IL4 mice when treated with k252a or TMZ ( n = 8 to 10). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, two-way ANOVA with Bonferroni test (A and C) and one-way ANOVA with Bonferroni test (D and E).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Over Expression, Injection, Concentration Assay

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A–C ) Representative images of wholemount in situ hybridization showing dact1 , dact2 , and wnt11f2 gene expression patterns. ( A ) At 8 hpf, dact2 and wnt11f2 are highly expressed in the dorsal margin and presumptive Nieuwkoop center of the gastrulating embryo, with dact1 being weakly detected (arrowhead). In contrast to wnt11f2 , dact1 , and dact2 are expressed in the presumptive dorsal mesoderm (asterisk). ( B ) Lateral (anterior to the left of page) and anterior (dorsal side toward top of page) views of bud-stage embryos. dact2 and wnt11f2 transcripts are both detected in the tailbud (asterisk) while dact2 is additionally expressed in the axial mesoderm (arrow). dact1 gene expression is concentrated to the paraxial mesoderm and the neuroectoderm (open arrowheads). ( C ) Lateral and flat-mount views of 4 ss embryos. dact2 is expressed in the anterior neural plate and polster (P), notochord (N), paraxial and presomitic mesoderm (PM) and tailbud (TB). In contrast, dact1 is expressed in the midbrain (MB) and the paraxial and presomitic mesoderm. ( D, E ) Representative lateral (anterior to left of page) images of wholemount in situ hybridization showing dact1 and dact2 expression patterns. ( D ) At 24 hpf expression is detected in the developing head. ( E ) At 48 hpf expression is detected in the developing craniofacial structures (arrow). ( F ) Representative images of RNAscope in situ hybridization analysis of dact1 (white) and dact2 (yellow) and irf6 (green) expression in transverse section of 72 hpf embryos. dact1 is expressed in the ethmoid plate (ep) and palatoquadrate (pq) orofacial cartilage, while dact2 is expressed in the oral epithelium (oe). The epithelial marker irf6 is expressed in the oe and surface epithelium (se). Dapi (blue). Scale bar: 100 μm.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: In Situ Hybridization, Gene Expression, Expressing, RNAscope, Marker

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Schematic representations of dact1 and dact2 exons, positions of guide RNA target site, introduced premature stop codon (arrow), and sequences of mutations. ( B ) DNA fragment analysis of dact1+/- and dact2+/- animals showing wildtype (250 and 387 bp, respectively) and mutant (228 and 380 bp, respectively). ( C ) Expression levels of dact1 and dact2 mRNA by RT-qPCR in 12 hpf dact1−/− mutants, dact2−/− mutants, and dact1/2−/− compound mutants. Eight embryos were pooled for mRNA isolation per sample. ( D ) Injection of dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA rescues the rod-shaped ethmoid plate phenotype in dact1/2−/− compound mutants. Representative images of Alcian blue stained dact1/2−/− double mutant treated with 300 pg dact1 mRNA and 300 pg dact2 mRNA. Arrow highlights normal ethmoid plate (EP). Visceral cartilage (VC) also appeared normal. ( E ) Quantification of the mutant craniofacial phenotype observed in a dact1−/−,dact2+/- breeding in-cross. Without mRNA injection, the mutant phenotype was observed at approximately (35%) the expected Mendelian ratio of 25%. Injection with dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA decreased the frequency that the mutant craniofacial phenotype was observed.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Isolation, Injection, Staining

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Inter-cross of compound heterozygotes yield embryos with different degrees of axis extension that correspond to the dact1 and dact2 genotypes. Representative lateral images of embryos at 12 hpf. The yellow line indicates body axis angle measured from the anterior point of the head, the center of the yolk, to the end of the tail. ( B ) Quantification of body axis angle. Numbers represent the difference in angle relative to the average wildtype embryo. Asterisk indicates genotypes with angles significantly different from wildtype. ANOVA p < 0.5 n = 3–21 embryos. Error bars: ± SEM. ( C ) Representative bud stage wildtype and dact1/2−/− mutant embryos stained for gsc (prechordal plate), pax2a (midbrain/hindbrain boundary), and krox20 (rhombomere 3). Asterisk indicates lack of krox20 expression in dact1/2−/− mutant. Scale bar = 200 μm ( D ) Representative flat mounts of 1–2 ss wildtype and dact1/2 mutant embryos stained for zic1 (telencephalon), pax2a and tbx6 (ventrolateral mesoderm). ( E ) Representative flat mounts of 10 ss wildtype and dact1/2−/− mutant embryos stained for ctsl1b (hatching gland), zic1 , pax2a , krox20 , and myo1d (somites).

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Mutagenesis, Staining, Expressing

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Representative brightfield images of wildtype and dact1/2−/− compound mutants at 4 dpf. 100 individuals were analyzed from a dact1/2 +/- double het cross. Lateral and ventral views show d act1/2−/− compound mutants have a hypoplastic midface, medially displaced eyes, and a displaced lower jaw. ( B ) Representative flat-mount images of Alcian blue stained ethmoid plate (EP) and visceral cartilage (VC) elements from 4 dpf wildtype and d act1/2−/− compound mutants. d act1/2−/− mutants have a rod-shaped EP with no distinct lateral and medial elements. No obvious differences were found in dact1/2 mutant VC. ( C ) Representative brightfield image of 4 dpf wildtype and dact1/2−/− mutant. Bar indicates vertebral spine length. Scale bar: 100 μm. ( D ) Representative images of Alcian blue stained dact1/2−/− , wnt11f2−/− , and wnt11f2−/− , dact1 / 2 −/− compound mutants. Embryos resulted from a dact1+/-,dact2+/-,wnt11f2+/- in-cross. Lateral and ventral views show similar craniofacial phenotypes in each mutant. ( E ) Representative flat-mount images of Alcian blue stained EP show a similar phenotype between dact1/2−/− , wnt11f2−/− , and wnt11f2−/−,dact1/2 −/− compound mutants. Scale bar: 200 μm.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Staining, Mutagenesis

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Representative brightfield images of 4 dpf larvae of compound dact1 and dact2 mutant genotypes. Bar represents dact1/2+/+ body length measurement. Scale bar: 100 μm. ( B ) Scatter plot of body length measurement of compound dact1 and dact2 mutant genotypes at 4 dpf. ANOVA p = 0.06. n = 3–4. Error bars: ± SEM.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Mutagenesis

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Representative Alcian blue stained wholemount images of wildtype, dact1/2−/− double mutant, gpc4−/− mutant, and gpc4/dact1/2−/− triple mutants at 4 dpf. Low magnification lateral images of embryos showing tail truncation in dact1/2−/− mutants, shortened and kinked tail in gpc4−/− mutants, and a combinatorial effect in gpc4/dact1/2−/− triple mutants. Higher magnification lateral images show a shortened midface and displaced lower jaw in dact1/2−/− mutants, a shortened midface in gpc4−/− mutant, and a combinatorial effect in gpc4/dact1/2−/− triple mutants. ( B ) Representative flat-mount images of dissected Alcian blue-stained cartilage elements. dact1/2−/− mutants have a narrow rod-shaped ethmoid plate (EP) while gpc4−/− mutants have a broad and shortened EP. dact1/2/gpc4 triple mutants have a combinatorial effect with a short, broad rod-shaped EP. In ventral cartilages (VC), dact1/2−/− mutants have a relatively normal morphology while Meckel’s cartilage in gpc4−/− mutants and gpc4/dact1/2−/− triple mutants is truncated. ( C, D ) Same as above except wls−/− mutant and wls/dact1/2−/− triple mutant, with similar findings. ( E ) Combinatorial genotypes of dact1 , dact2 , and gpc4. dact2 −/− contributed the dact/gpc4 compound phenotype while dact1−/− did not. Scale bar: 200 μm.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Staining, Mutagenesis

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Summary schematic showing similar phenotypes in dact1/2−/− and gpc4−/− mutants at 12 hpf and divergent phenotypes at 4 dpf. Single-cell RNAseq was performed during axis extension to compare and contrast dact1/2−/− and gpc4−/− transcriptional programs. Uniform manifold approximation and projection (UMAP) showing cluster identification. ( B ) UMAP of cell clusters identified by single-cell RNAseq. ( C ) Dot plot showing the most differentially expressed genes between clusters. ( D ) UMAP showing dact1 , dact2 , and gpc4 expression in wildtype embryos.

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Expressing

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A ) Single-cell RNAseq gene expression analysis of capn8 in wildtype and dact1/2−/− mutants. In wildtype embryos, capn8 expression is restricted predominantly to the epidermis whereas capn8 is widely expressed throughout the embryo in dact1/2−/− mutants, especially in the mesoderm. ( B ) Wholemount in situ hybridization of capn8 expression in wildtype and dact1/2−/− mutant embryos at 2 ss. Staining corroborates the single-cell RNAseq data, with expanded ectopic expression of capn8 throughout the embryo. Flat mounts are oriented anterior to the left. Scale bar: 100 μm. ( C ) Brightfield images and Alcian blue staining of the ethmoid plate show ectopic expression of capn8 mRNA (200 pg) at the 1 cell stage in dact1+/- , dact2+/- embryos recapitulates the dact1/2−/− compound mutant craniofacial phenotype. The mutant craniofacial phenotype did not manifest in gfp mRNA (200 pg) injected 1 cell-stage dact1+/- , dact2+/- embryos. Scale bar: 100 μm ( D ) Quantification of mutant and normal craniofacial phenotype in 4 dpf larvae after mRNA injection at the 1 cell stage. Larvae were derived from dact1/2+/- interbreeding. Larvae were uninjected or injected with 200 pg gfp control or capn8 mRNA. A Fisher exact test showed a significant effect of capn8 mRNA injecting in the dact1/2 double heterozygotes. Asterisk indicates a significant difference between conditions (p = 0.013).

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: Gene Expression, Expressing, In Situ Hybridization, Mutagenesis, Staining, Injection, Derivative Assay, Control

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RNAscope probe dact2 , ACDbio , ID_source:identifier 857201-C3 , .

Techniques: CRISPR, Mutagenesis, Reverse Transcription, Gene Expression, Recombinant, Plasmid Preparation, RNAscope, Software

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet: ( A–C ) Representative images of wholemount in situ hybridization showing dact1 , dact2 , and wnt11f2 gene expression patterns. ( A ) At 8 hpf, dact2 and wnt11f2 are highly expressed in the dorsal margin and presumptive Nieuwkoop center of the gastrulating embryo, with dact1 being weakly detected (arrowhead). In contrast to wnt11f2 , dact1 , and dact2 are expressed in the presumptive dorsal mesoderm (asterisk). ( B ) Lateral (anterior to the left of page) and anterior (dorsal side toward top of page) views of bud-stage embryos. dact2 and wnt11f2 transcripts are both detected in the tailbud (asterisk) while dact2 is additionally expressed in the axial mesoderm (arrow). dact1 gene expression is concentrated to the paraxial mesoderm and the neuroectoderm (open arrowheads). ( C ) Lateral and flat-mount views of 4 ss embryos. dact2 is expressed in the anterior neural plate and polster (P), notochord (N), paraxial and presomitic mesoderm (PM) and tailbud (TB). In contrast, dact1 is expressed in the midbrain (MB) and the paraxial and presomitic mesoderm. ( D, E ) Representative lateral (anterior to left of page) images of wholemount in situ hybridization showing dact1 and dact2 expression patterns. ( D ) At 24 hpf expression is detected in the developing head. ( E ) At 48 hpf expression is detected in the developing craniofacial structures (arrow). ( F ) Representative images of RNAscope in situ hybridization analysis of dact1 (white) and dact2 (yellow) and irf6 (green) expression in transverse section of 72 hpf embryos. dact1 is expressed in the ethmoid plate (ep) and palatoquadrate (pq) orofacial cartilage, while dact2 is expressed in the oral epithelium (oe). The epithelial marker irf6 is expressed in the oe and surface epithelium (se). Dapi (blue). Scale bar: 100 μm.

Article Snippet: Commercial assay or kit , RNAscope probe irf6 , ACDbio , ID_source:identifier 555101 , .

Techniques: In Situ Hybridization, Gene Expression, Expressing, RNAscope, Marker

Journal: eLife

Article Title: Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis

doi: 10.7554/eLife.91648

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RNAscope probe irf6 , ACDbio , ID_source:identifier 555101 , .

Techniques: CRISPR, Mutagenesis, Reverse Transcription, Gene Expression, Recombinant, Plasmid Preparation, RNAscope, Software