Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Inducible RNase H1 transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of RNase H1-mediated knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Expressing, In Vitro, Staining, Marker, Knockdown, Mutagenesis, Binding Assay, Construct, Control, Western Blot, Concentration Assay, Virus, Immunocytochemistry, Transduction, Derivative Assay, Immunoprecipitation, In Silico

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Cell specifications and transcriptomic alterations in RH1 transgenic cultures. A, B) Gene set enrichment analysis of scRNA-seq data from RH1ΔMLS and BFP control hiPSC-differentiated cultures. (A) Cnet plot and (B) dot plot showing significant gene ontologies up- (left) and down-regulated (right) following RH1ΔMLS transgene induction. Dot color indicates fold change (cnet plot) or adjusted p- value (dot plot), dot size indicates number of genes in category. n = 2 replicates from 2 cell lines/group. C) Combination of clusters into broad cell type classes, including glia (green), neurons (purple), and NPCs (blue). Gray clusters did not show canonical markers of these major neural cell types. D) RH1ΔMLS cells have significantly (p < 0.0001, Wilcoxin Rank Sum test) higher RNASEH1 expression than BFP controls (mean normalized expression: RH1ΔMLS = 2.45, BFP = 0.94). Cells from each condition were split into two groups according to RNASEH1 normalized expression value (> 1.25 = hi; ≤ 1.25 = lo) for downstream analyses. E) Bar charts showing difference in cell proportions across BFP or RH1ΔMLS transduced cells, stratified by RNASEH1 expression level. There is a significant reduction in the proportion of glia (p = 0.015) and an increased proportion of neurons (p = 0.036) in RH1ΔMLS- RNASEH1 high relative to BFP- RNASEH1 low groups (Two-way ANOVA with Dunnett’s Multiple Comparison test; n = 2 replicates from 2 cell donors per group; error bars denote S.E.M.; n.s. = not significant.) F, G) Volcano plots showing differentially expressed genes in scRNA-seq ( F ) and bulk RNA-seq ( G ) from RH1ΔMLS hiPSC-differentiated cultures and BFP controls. Green dots indicate genes more highly expressed in control cells (FDR < 0.05, log 2 fold change < 0) and purple/magenta dots indicate genes more highly expressed in RH1ΔMLS cells (FDR < 0.05, log 2 fold change > 0). H) Venn diagram showing overlap of upregulated genes in RH1ΔMLS hiPSC-differentiated cultures from scRNA and bulk RNA-seq, and promoter-bound R-loops that are knocked down following RNase H1-transgene expression. RF = Representation factor, which signifies more overlap of the two gene sets than expected based on a background list of genes if > 1. P-values calculated via hypergeometric test. I) Gene ontology of genes with upregulated expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures. Numbers within gene ontology images indicate number of genes in the enriched set that occur within the ontology class, legend denotes -log 10 enrichment FDR. J) Venn diagram showing overlap of genes that have upregulated gene expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures and R-loop “primed” genes in germinal matrix . RF = Representation factor, p-values calculated via hypergeometric test. K) Heatmap indicating overlap of genes that have upregulated gene expression following promoter-bound R- loop loss in RH1ΔMLS hiPSC-differentiated cultures and disease risk genes. p-values calculated via hypergeometric test. L) Optical intensity from immunofluorescence images of the R-loop-specific antibody S9.6, and the synaptic proteins PDS-95 and synapsin, in RH1ΔMLS and RH1ΔMLS D145N hiPSC-differentiated cultures. P-values calculated via two-tailed student’s t test; Error bars denote S.E.M.; n = 25 images per cell line / group. M) Representative immunofluorescence images of the synaptic protein synapsin (green) in RH1ΔMLS hiPSC- differentiated cultures and RH1ΔMLS D145N controls, along with the nuclear marker DAPI (blue) and the neuronal marker MAP2 (magenta). Scale bar: 20 mm.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Transgenic Assay, Control, Expressing, Comparison, RNA Sequencing, Gene Expression, Immunofluorescence, Two Tailed Test, Marker

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1-mediated R-loop loss during neuronal differentiation in vitro results in electrophysiological deficits. A) Top left: Electrophysiological recordings were taken with an Axion multielectrode array (MEA) system to assess population-wide neuronal activity. Schematic of the 48-well MEA plate used for electrophysiological recordings. Bottom Left: Schematic of an individual well within the MEA plate, containing 16 recording electrodes. Right: Screen shot of the MEA recording on week 8, with BFP-expressing hiPSC-neurons plated on the left half of the plate and RNase H1-overexpressing neurons on the right. B) Representative spike raster plots over the 10-minute recording at week 8 for BFP-expressing (left) and RNase H1-overexpressing (right) hiPSC-neurons. Each row denotes a single recording electrode within a single well of the 48-well MEA plate. C-E) The number of spontaneous spikes ( C ), weighted mean firing rate (wMFR) in Hertz ( D ), and number of bursts ( E ) were recorded weekly from BFP-controls and RNase H1 transgene expressing hiPSC-neurons from two donor lines. Data from the two donor lines were combined and a two-way ANOVA was performed with Sidak’s multiple comparisons test to test significant differences in each metric between weeks 2-8. Each condition contained at least 14-20 viable well replicates after removing wells that had less than 10 active recording electrodes. F) Statistics were additionally calculated for each electrophysiological metric at week 8 (unpaired two-tailed student’s t test) between RNase H1-transgene hiPSC-neurons and BFP controls. Individual donors represented with either filled in or hollow shapes.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: In Vitro, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1 overexpression leads to reduced dendritic complexity and spine density. Expression of the RNASEH1 ΔMLS transgene, delivered via in utero electroporation into neural precursor cells of the ventricular zone in E15 mouse embryos, affects dendrite and spine morphology of cortical projection neurons. A) Expression plasmids of GFP combined with tdTomato are used as controls, while GFP combined with RNASEH1 ΔMLS are used to destroy neural R-loops in mice during prenatal development. B) Overview of the in utero electroporation technique. TdTomato or RH1ΔMLS along with GFP reporters are transfected and electroporated unilaterally in E15 mouse ventricular zone. In adult mouse, P28, strong unilateral GFP expression is observed. Scale bar = 1 mm. C) In utero electroporation experimental timeline. D) Expression of exogenous (human) RNASEH1 in RH1ΔMLS mice was confirmed in FAC-sorted GFP+ neurons, whereas no difference was observed for endogenous expression of (mouse) Rnaseh1 between groups via qPCR (Normalized to Gapdh expression). Student’s two-tailed t test; n.s. = not significant. E) Expression of human RH1ΔMLS reduces S9.6 immunoreactivity in the nucleus of mouse neural cells at P0. F) FlSH images showing RNA expression of RNASEH1 (red) in the P28 cortex of tdTomato controls and RH1ΔMLS mice, and colocalization of GFP (green) and RNASEH1 in RH1ΔMLS cells. Scale bar = 100 mm. G) Representative images of (left) control and (right) RH1ΔMLS-expressing frontal pyramidal neuron dendritic trees at P28. H) Sholl analysis, showing reduced dendritic complexity in (left) control and (right) RH1ΔMLS-expressing pyramidal neurons at P28 (tdTomato control n = 3, RH1ΔMLS n = 4). I) Representative images of (left) control and (right) RH1ΔMLS frontal pyramidal neuron dendritic spines at P28. J) Apical and basal spine densities, assessed by Imaris tracing + FOCM (see text), are significantly decreased in RH1ΔMLS-expressing prefrontal pyramidal neurons. N = 3/group. *P < 0.05, **P < 0.01 determined by Two-Tailed Student’s t-test.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Over Expression, Expressing, In Utero, Electroporation, Transfection, Two Tailed Test, RNA Expression, Control

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic showing the rat placentation site. Invaded trophoblast cells are depicted in green. b . Relative expression of Cited2 transcripts in the ectoplacental cone ( EPC ), whole placenta ( P ), junctional zone ( JZ ), and labyrinth zone ( LZ ) of the rat placenta during gestation. Values depicted were normalized to EPC 9.5 samples. c . Relative expression of Cited2 transcript in postnatal day 1 ( PND1 ) rat neonatal tissues and gd 14.5 JZ tissue. d . Relative expression of Cited2 transcripts within the uterine-placental interface during gestation. e . In situ hybridization showing Cited2 transcript distribution (top left) and Cited2 and Prl7b1 (invasive trophoblast marker) transcript co-localization in rat gestation day ( gd ) 18.5 placentation site (bottom left). Higher magnification images of the area outlined by a yellow rectangle (bottom left) are shown to the right. Scale bar=500 μm (left panels), scale bar=100 μm (right panels). Uterine-placental interface ( UPI ), spiral artery ( SpA ). The histograms presented in panels b, c, and d represent means ± SEM, n=5-10, 3-6 pregnancies. One-way ANOVA, Tukey’s post hoc test, * p < 0.05, ** p < 0.01, **** p<0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, In Situ Hybridization, Marker

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic of the Cited2 wild type and null alleles. A 1477 bp deletion was generated using CRISPR /Cas9 genome editing. The deletion included the entire coding sequence of the Cited2 gene. b . Western blot for CITED2 protein in rat junctional zone tissue samples from Cited2 +/- x Cited2 +/- breeding on gd 18.5. c . Fetal and placental weights from Cited2 +/- x Cited2 +/- breeding for the rat. Values represent mean ± SEM, n=17-35, unpaired t-test, **p<0.01, ***p<0.001, **** p<0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Generated, CRISPR, Sequencing, Western Blot

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Immunohistological analysis of vimentin in wild type ( WT, +/+ ) and null (-/-) placentas from Cited2 +/- x Cited2 +/- breeding on gd 18.5. Scale bar=1000 μm. UPI uterine-placental interface, JZ junctional zone, LZ labyrinth zone. b . JZ and LZ weights from Cited2 +/- x Cited2 +/- breeding on gd 14.5 and gd 18.5. c . Simplified schematic depicting the strategy for achieving trophoblast specific CITED2 knockdown in vivo. d . Relative expression of Cited2 transcripts in control ( CTRL ) and CITED2 shRNA-exposed gd 14.5 JZ. e . JZ, LZ and fetal weights from control and gd 14.5 trophoblast specific Cited2 knockdown. CTRL, control knockdown, KD, Cited2 knockdown. Shown are mean values ± SEM, n=12-20, unpaired t-test, **p<0.01, **** p<000.1.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Knockdown, In Vivo, Expressing, Control, shRNA

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Significantly upregulated and downregulated transcripts from RNA-seq analysis of rat 14.5 junctional zone tissue from wild type (+/+) and Cited2 null (-/-) rats, n=4, log 2 -fold change in either direction>1.5, p<0.05. b . Heatmap showing select transcripts from RNA-seq analysis of wild type (+/+) and Cited2 null (-/-) rat 14.5 junctional zone tissue. c . Significantly upregulated and downregulated transcripts from RNA-seq analysis of differentiated wild type (+/+) and Cited2 null (-/-) rat trophoblast stem ( TS ) cells, n=3, log 2 -fold change in either direction>1.5, p<0.05. d . Heatmap showing select transcripts from RNA-seq analysis of differentiated rat TS cells from RNA-seq analysis of wild type (+/+) and Cited2 null (-/-) differentiated TS cells. e . Gene Ontology ( GO ) enriched terms for DEGs from wild type (+/+) versus Cited2 null (-/-) rat 14.5 junctional zone RNA-seq analysis. f . GO enriched terms for DEGs from wild type (+/+) versus Cited2 null (-/-) rat TS cell RNA-seq analysis.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Representative images of wild type (+/+) and Cited2 null (-/-) rat gd 13.5, 15.5 and 18.5 placentation sites immunostained for cytokeratin (green). Scale bars=1000 μm. b . Relative expression of transcripts associated with invasive trophoblast ( Prl7b1 ) from wild type (+/+) and Cited2 null (-/-) gd 13.5 decidual tissue and uterine placental interface tissue at gd 15.5 and 18.5. Graphs depict mean values ± SEM, n=11-24, unpaired t-test, *p<0.05. c . In situ hybridization showing Ceacam9 (red) and Prl7b1 (invasive trophoblast marker, green) transcript localization in gd 15.5 wild type (+/+) and Cited2 null (-/-) rat placentation sites, scale bar=1000 μm. d . Relative expression of Ceacam9 transcripts in gd 15.5 uterine placental interface tissue. Shown are mean values ± SEM, n=12-14, unpaired t-test, **p<0.01. Uterine placental interface ( UPI ), decidua ( DEC ), junctional zone ( JZ ), labyrinth zone ( LZ ).

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, In Situ Hybridization, Marker

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: Single cell-RNA sequencing was performed on wild type (+/+) and Cited2 null (-/-) gd 18.5 uterine-placental interface tissue samples. a . UMAP plot showing cell clustering in wild type (+/+) and Cited2 null (-/-) gd 18.5 uterine-placental interface tissue. UD, unidentified cluster. b . Number of analyzed cells in the invasive trophoblast cell cluster. Graph represents mean values ± SEM, n=3, unpaired t-test, *p<0.05. c . Bar plot showing select DEGs in the invasive cell cluster (upregulated shown in red; downregulated shown in blue). d . Gene Ontology enriched terms for DEGs from the invasive trophoblast cell cluster.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic of the experimental timeline for hypoxia exposure. b . Representative images of wild type (+/+) and Cited2 null (-/-) rat gd 13.5 placentation sites exposed to ambient or hypoxic (10.5% oxygen) conditions. Sections were immunostained for cytokeratin (green), perforin (red), and DAPI (blue). Scale bar=500 μm. Uterine placental interface ( UPI ), Decidua ( DEC ), Junctional Zone ( JZ ), labyrinth zone ( LZ ), and Spiral artery ( SpA ). c . Depth of invasion was quantified, and fold changes calculated for hypoxic relative to ambient conditions, n=5-9. d . Resorption rate assessed on gd 18.5 for individual genotypes from Cited2 +/- females bred to Cited2 +/- males and exposed to ambient of hypoxic conditions, n=20-33. e . Schematic of the experimental timeline for polyinosinic:polycytidylic acid ( polyI:C ) treatment, f . Relative expression of Isg15, Mx2, Ifi27I2b , and Oasl2 in junctional zone tissue from control (saline treated; CTRL ) and polyI:C treated ( IC ) wild type (+/+) and Cited2 null (-/-) animals.; n=8-17. Shown are mean values ± SEM, one-way analysis of variance, Tukey’s post-hoc test. *p<0.05, **p <0.01, ***p <0.001. ****p <0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, Control, Saline

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . In situ hybridization showing CITED2 transcript localization in first trimester human placenta. C ITED2 transcripts (black) were co-localized with E-cadherin ( CDH1 , red; marker of cytotrophoblast progenitor cells) transcripts. High magnification image of an EVT cell column and villus (yellow box) are shown in the right panel. The scale bar in the magnified image is 100 μm. b . Schematic showing human trophoblast stem ( TS ) cell differentiation from stem/progenitor state to EVT cells. c . Relative expression of CITED2 and EVT cell signature transcripts ( MMP2 and HLA-G ) in human TS cells in the stem state and following eight days of EVT cell differentiation, n=5. d . Relative expression of CITED2 transcript levels in EVT cells expressing control ( CTRL ) shRNA or CITED2 shRNA, n=3. Graphs represent mean values ± SEM, unpaired t-test, *p<0.05, ***p <0.001. ****p <0.0001. e . Significantly upregulated and downregulated transcripts from RNA-seq analysis of CTRL shRNA versus CITED2 shRNA samples, n=3, and log 2 -fold change ( FC ) in either direction>1.5, p<0.05. f . Heatmap showing select transcripts from RNA-seq analysis of CTRL shRNA versus CITED2 shRNA treated EVT cells. g . Gene Ontology enriched terms for DEGs from CTRL shRNA versus CITED2 shRNA treated EVT cells.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: In Situ Hybridization, Marker, Cell Differentiation, Expressing, Control, shRNA, RNA Sequencing

Journal: International journal of cancer

Article Title: Serpin B5 is a CEA-interacting biomarker for colorectal cancer.

doi: 10.1002/ijc.28494

Figure Lengend Snippet: Figure 1. Identification of serpin B5 as differently expressed in CEA-suppressed LoVo cells. (a) 2-DE of protein extracts from LoVo and its CEA-suppressed derivative cell line. Typical electrophoretic patterns are shown, with circles showing the different protein pat- terns in CEA-suppressed cell extracts. (b) Analysis of the protein spot differently expressed in CEA-suppressed LoVo cells by MALDI- MS. The protein spot was excised from the gel and digested with trypsin; the peptides were analysed by MALDI-MS. (c) Downregula- tion of serpin B5 expression after transfection with a shRNA against CEA in LoVo cells. LoVo cells were transfected with a CEA shRNA or a control nonspecific shRNA, and Western blot of lysates with antibodies against CEA and serpin B5 proteins. Abbreviations: CEA, carcinoembryonic antigen; 2-DE, two-dimensional gel electro- phoresis; MALDI-MS, matrix-associated laser desorption ionisation mass spectrometry; Ctrl, control.

Article Snippet: After blocking, the membranes were probed with antibodies against serpin B5 (Santa Cruz Biotechnology, Santa Cruz, CA), CEA (AbFrontier, Seoul, Korea), or actin (Sigma).

Techniques: Expressing, Transfection, shRNA, Control, Western Blot, Two-Dimensional Gel Electrophoresis, Mass Spectrometry

Journal: International journal of cancer

Article Title: Serpin B5 is a CEA-interacting biomarker for colorectal cancer.

doi: 10.1002/ijc.28494

Figure Lengend Snippet: Figure 2. Effect of serpin siRNA on CEA expression and interactions between serpin B5 and CEA. (a) Expression of CEA and serpin B5 in human colon cancer cell lines. Western blot analysis was performed in human colon cancer cell lines. Reference levels of CEA expression in each cell line are as follows26–28 (ng/106 cells): SNU-81 (800.0), SNU-407 (85.4), SNU-C4 (<100), SNU-C5 (<10), Caco-2 (27), DLD-1 (<2), HCT-116 (<2), and LoVo (882). CEA-depleted cell lines showed absent or weak serpin B5 positivity. (b) Downregulation of CEA expression in LoVo, SNU-81, and SNU-C4 cells transfected with serpin B5 siRNA. LoVo, SNU-81, and SNU-C4 cells were transfected with plasmids con- taining serpin B5 siRNA and negative control siRNA. The expression of CEA was decreased in the cells transfected with serpin B5 siRNA compared with nonsilencing siRNA. (c) Western blot analysis of proteins from LoVo cells immunoprecipitated by anti-serpin B5 and anti-CEA antibodies. The immunoreactive serpin B5 signal was detected in immunoprecipitates of CEA and vice versa. Normal mouse IgG was used as a negative control. (d) Co-localisation of endogenous serpin B5 and CEA in cultured LoVo, SNU-81, and SNU-C4 cells. Cells were fixed and stained with fluorescently labeled antibodies and DAPI; localisation of the proteins was examined by confocal microscopy. The merged image of serpin B5 (green), CEA (red), and DAPI-stained DNA (blue) clearly shows co-localisation of serpin B5 with CEA. Abbreviations: CEA, carcinoembryonic antigen; siRNA, small interfering RNA; IP, immunoprecipitation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: After blocking, the membranes were probed with antibodies against serpin B5 (Santa Cruz Biotechnology, Santa Cruz, CA), CEA (AbFrontier, Seoul, Korea), or actin (Sigma).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Immunoprecipitation, Cell Culture, Staining, Labeling, Confocal Microscopy, Small Interfering RNA

Journal: International journal of cancer

Article Title: Serpin B5 is a CEA-interacting biomarker for colorectal cancer.

doi: 10.1002/ijc.28494

Figure Lengend Snippet: Figure 3. Serpin B5 levels in sera and tissues from patients with colorectal cancer. (a) ELISA of serpin B5 in sera from normal controls and colorectal cancer patients. Box plots display serpin B5 expression in sera from normal healty controls and colorectal cancer patients with subcategories indicating the CEA expression levels (see Table 1). (Upper panel) Serpin B5 concentrations indicated according to the CEA level in the colorectal cancers. Elevated serpin B5 levels were noted in the normal controls and high-CEA colorectal cancers; *p < 0.001, **p < 0.001, and ***p 5 0.006 between the indicated groups. (Lower panel) Serum serpin B5 levels were significantly decreased in those patients with CRC and a CEA concentration <10 ng/mL compared with the healthy controls (p < 0.001). (b) Western blots of serpin B5 pro- tein levels in 10 colorectal cancers and matched non-tumourous colonic mucosa, and metastatic liver tumours. (c) Relative quantification of serpin B5 xpression indicates strong serpin B5 expression in colorectal tumours compared with non-tumour tissue (p 5 0.029), but there is no significant difference between the T and LM groups (p 5 0.342) or the NT and LM groups (p 5 0.294), or among the three groups (p 5 0.111). (d) Representative cases of immunohistochemical serpin B5 staining in normal colonic mucosa (left) and colon cancer mucosa (middle and right). (left) Moderate cytoplasmic serpin B5 expression, (middle) strong cytoplasmic and nuclear serpin B5 expression, (right) negative-to- minimal serpin B5 staining. Abbreviations: Ca, cancer; CRC, colorectal cancer; NT, non-tumour; T, tumour; LM, liver metastasis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: After blocking, the membranes were probed with antibodies against serpin B5 (Santa Cruz Biotechnology, Santa Cruz, CA), CEA (AbFrontier, Seoul, Korea), or actin (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Western Blot, Immunohistochemical staining, Staining

Journal: International journal of cancer

Article Title: Serpin B5 is a CEA-interacting biomarker for colorectal cancer.

doi: 10.1002/ijc.28494

Figure Lengend Snippet: Figure 4. Prognostic value of serpin B5 in patients with CRC. Kaplan-Meier estimates of disease-free survival (DFS) and overall survival (OS) according to high and low serpin B5 levels indicate a significant association between strong expression of serpin B5 and a shorter (A) DFS (p 5 0.001) and (B) OS (p 5 0.017). High expression was defined as an immunoreactive score 20. Abbreviation: CRC, colorectal cancer.

Article Snippet: After blocking, the membranes were probed with antibodies against serpin B5 (Santa Cruz Biotechnology, Santa Cruz, CA), CEA (AbFrontier, Seoul, Korea), or actin (Sigma).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Expression of SAA1 in the human placenta at term. (A) In situ hybridization showed the presence of SAA1 mRNA (red brown) in the nuclei of the syncytial layer of the villous tissue. Scrambled RNA probe was used as the negative control (nc). (B) Immunohistochemical staining showed the presence of SAA1 (red) in the syncytial layer of the villous tissue. Non-immune serum was used as negative control (nc). (C) Immunofluorescence staining showed the colocalization of SAA1 (red) and 11β-HSD2 (green), a marker of syncytiotrophoblast, in the syncytial layer of the villous tissue. Nuclei (blue) were counterstained blue with DAPI.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Expressing, In Situ Hybridization, Negative Control, Immunohistochemical staining, Staining, Immunofluorescence, Marker

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Effect of syncytialization and LPS, TNF-α and cortisol on SAA1 expression in human placental trophoblasts . (A) Hematoxylin-eosin staining of cultured trophoblasts showed cell fusion in vitro . (B) Changes of SAA1 mRNA and secretion before (3 h after cell plating) and after (48 h after cell plating) syncytialization ( n = 3–4). (C–E) LPS (5 ng/mL, 24 h, C ), TNF-α (10 ng/mL, 24 h; D ), and cortisol (1 μM, 24 h; E ) increased SAA1 mRNA and secretion in syncytiotrophoblasts ( n = 4–5). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Expressing, Staining, Cell Culture, In Vitro

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Increased SAA1 abundance in human placental tissue and maternal blood in labor at term and preterm. (A,B) Abundance of SAA1 mRNA and protein in human placenta tissue collected from elective cesarean section without labor at term (TNL) ( n = 12) and spontaneous labor at term (TL) ( n = 12). (C) Dynamic changes of serum SAA1 in pregnant women before, at the onset of, and 24 h after labor ( n = 5). (D) Changes of SAA1 in maternal blood obtained from iatrogenic preterm birth without histologic chorioamnionitis ( n = 15) and infection-induced preterm birth with histological chorioamnionitis ( n = 12). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Effects of SAA1 on the expression of genes pertinent to parturition in human placental syncytiotrophoblasts. (A,B) Effects of SAA1 (10 ng/mL, 24 h) on IL-8, TNF-α mRNA, and secretion in the presence or absence of a TLR4 antagonist CLI095 (5 μM, 24 h) ( n = 3–4). (C) Effects of SAA1 (10 ng/mL, 24 h) on COX-2 mRNA and protein in the presence or absence of a TLR4 antagonist CLI095 (5 μM, 24 h) ( n = 3). Top panel is a representative immunoblot. (D) Effects of SAA1 (10 ng/mL, 24 h) on PGE2 and PGF2α secretions ( n = 4). Data are mean ± SEM. * P < 0.05 vs. control (0); ** P < 0.01 vs. control (0); # P < 0.05 vs. SAA1.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Expression of SAA1/2 in mouse placenta and fetal membranes. (A–C) Immunohistochemical staining showed the presence of SAA1 (red) in the junctional zone of the placenta (A) and the yolk sac membrane (B) , but not in the amnion (C) at gestational day 18.5. (D) negative control. (E,F) Increased SAA1/A2 abundance in mouse placenta and fetal membranes from gestational days 16.5 to 18.5 ( n = 3). Data are mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Induction of proinflammatory factors in placenta and fetal membranes by intraperitoneal administration of LPS or SAA1 in the mouse. (A,B) Induction of SAA1/2 expression in mouse placenta (A) and fetal membranes (B) by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) at gestational day 16.5 ( n = 3). (C–E) Induction of IL-1β (C) , TNF-α (D) and COX-2 (E) in the mouse placenta by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) and SAA1 (8 μg/kg BW, 6 h) at gestational day 16.5 ( n = 4). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

doi: 10.3389/fimmu.2020.01038

Figure Lengend Snippet: Pregnancy outcome in mice administered with SAA1 and PBS.

Article Snippet: The villous tissue was fixed with 4% paraformaldehyde in 1%0 diethyl pyrocarbonate (DEPC), and the paraffin-embedded tissue was sectioned at 4 μm in thickness for subsequent in situ hybridization using a customized kit containing digoxigenin-labeled antisense RNA probe against SAA1 mRNA (Boster, Wuhan, China).

Techniques:

Journal:

Article Title: Homologue of Macrophage-Activating Lipoprotein in Mycoplasma gallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures

doi: 10.1128/JB.185.8.2538-2547.2003

Figure Lengend Snippet: Expression, purification, and cleavage of recombinant p47 and Northern blot analysis of p47 gene transcript. Gene p47-mut was cloned and expressed with the pGEX4T-1 expression vector. The fusion product was purified over a glutathione Sepharose column and cleaved with thrombin. The fusion product and the cleaved fusion products, together with molecular weight standards (Novex) or prestained molecular weight standards (New England Biolabs), were separated by SDS-12.5% PAGE and either stained with CBB or Western transferred. Total RNA of M. gallisepticum strain ts-11 cells was extracted and, together with RNA standards (Promega), electrophoresed, blotted onto nylon membrane, and probed with the 233-bp region encoding the carboxyl-terminal end of p47. (A) CBB-stained gel of E. coli-expressed p47 protein following purification over a glutathione Sepharose column (lane 1) and after cleavage with thrombin (lane 2). (B) GST-p47 products immunostained with rabbit anti-GST sera (lane 1), rabbit anti-p47 sera (lane 2), or MAb B3 (lane 3). (C) Ethidium bromide-stained total RNA of M. gallisepticum strain ts-11 cells (lane 1). Northern blot hybridized with DNA from the carboxyl region of the p47 gene. The probe bound a 1.98-kb band (arrowed, lane 2).

Article Snippet: Total RNA of M . gallisepticum strain ts-11 cells was extracted and, together with RNA standards (Promega), electrophoresed, blotted onto nylon membrane, and probed with the 233-bp region encoding the carboxyl-terminal end of p47. (A) CBB-stained gel of E . coli -expressed p47 protein following purification over a glutathione Sepharose column (lane 1) and after cleavage with thrombin (lane 2). (B) GST-p47 products immunostained with rabbit anti-GST sera (lane 1), rabbit anti-p47 sera (lane 2), or MAb B3 (lane 3). (C) Ethidium bromide-stained total RNA of M . gallisepticum strain ts-11 cells (lane 1).

Techniques: Expressing, Purification, Recombinant, Northern Blot, Clone Assay, Plasmid Preparation, Molecular Weight, Staining, Western Blot