Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: Species name, accession numbers, host/substrate and geographical origin of the Pythium strains sequenced in this study.

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques:

Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: Assembly and annotation statistics † of thirteen stramenopiles.

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques:

Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: The predicted proteomes of the seven Pythium species were clustered using OrthoMCL to identify orthologs and close paralogs. The number of gene families shared between the species and total number of clustered genes (numbers in parentheses) are indicated. The numbers outside the Venn diagram show the total number of orthologous clusters and number of genes (in parentheses) within those clusters for each species. Pap, Pythium aphanidermatum; Par, Pythium arrhenomanes ; Pir, Pythium irregulare ; Piw, Pythium iwayamai ; Puls, Pythium ultimum var. sporangiiferum ; Pult, Pythium ultimum var. ultimum ; Pve, Pythium vexans.

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques:

Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: The CAZymes coding genes were annotated using the CAZymes Analysis Toolkit- CAT according to the CAZy database in combination with protein family domain analyses. Gene families absent in at least 2 species are underlined. Comparison of total CAZymes from different classes is listed in . CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyl transferase; PL, polysaccharide lyase; Pap, Pythium aphanidermatum ; Par, Pythium arrhenomanes ; Pir, Pythium irregulare ; Piw, Pythium iwayamai ; Pult, Pythium ultimum var. ultimum ; Puls, Pythium ultimum var. sporangiiferum ; Pve, Pythium vexans ; Phin, Phytophthora infestans ; Phrm, Phytophthora ramorum ; Phsj, Phytophthora sojae ; Hpa, Hyaloperonospora arabidopsidis ; Thps, Thalassiosira pseudonana ; Phtr, Phaeodactylum tricornutum .

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques: Comparison

Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: (A) The typical architecture of a YxSL[RK] effector candidate inferred from 141 sequences from seven Pythium species, Phytophthora infestans , and Phytophthora sojae . The consensus sequence pattern of the YxSL[RK] motif was calculated using WebLogo . The bigger the letter, the more conserved the amino acid site. Please note that the numbers in the sequence logo refer to the corresponding positions in the alignment and thus differ from the average position of the motifs. (B) The YxSL[RK] motif distribution in the proteomes of Pythium species, Phytophthora infestans and Phytophthora sojae is shown. The YxSL[RK] sequence is over-represented in the secretome of Pythium and Phytophthora species relative to the non-secreted proteome ( P ≤0.05). The YxSL[RK] motifs were counted only if they were within the first 30 to 150 residues from the signal peptide. The frequency was calculated as percentage of either all secreted proteins or all non-secreted proteins. Pult, Pythium ultimum var. ultimum ; Puls, Pythium ultimum var. sporangiiferum ; Pir, Pythium irregulare ; Piw, Pythium iwayamai ; Par, Pythium arrhenomanes ; Pap, Pythium aphanidermatum ; Pve, Pythium vexans ; Phin, Phytophthora infestans ; Phrm, Phytophthora ramorum ; Phsj, Phytophthora sojae ; Hpa, Hyaloperonospora arabidopsidis . (C) The typical architecture of an LxLYLAR/K effector motif inferred from 129 sequences from 7 Pythium species. The consensus sequence pattern of the LxLYLAR/K motif was calculated using WebLogo . The bigger the letter, the more conserved the amino acid site. Please note that the numbers in the sequence logo are referring to the corresponding positions in the alignment and thus differ from the average position of the motifs. (D) Number of CRN effector proteins in oomycetes. The number of candidate CRN effectors estimated by Hidden Markov Model (HMM) searches in combination with two other computational methods is shown. The number of CRN effectors from Pythium ultimum var. ultimum , Phytophthora species and H. arabidopsidis were taken from published genome datasets , , , . Pult, Pythium ultimum var. ultimum ; Puls, Pythium ultimum var. sporangiiferum ; Pir, Pythium irregulare ; Piw, Pythium iwayamai ; Par, Pythium arrhenomanes ; Pap, Pythium aphanidermatum ; Pve, Pythium vexans ; Phin, Phytophthora infestans ; Phrm, Phytophthora ramorum ; Phsj, Phytophthora sojae ; Hpa, Hyaloperonospora arabidopsidis .

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

doi: 10.1371/journal.pone.0075072

Figure Lengend Snippet: The circle is a graphical representation of the selected regions from Pythium arrhenomanes (contigs 8, 17, 26, 41, 68, 131, 170, 285, 707) Pythium irregulare (contigs 28, 92, 103, 106, 119, 123, 129, 132, 140, 163, 195, 226, 372, 396), Pythium aphanidermatum (scaffolds 4, 6, 23, 80, 88, 96, 115, 150, 327), Pythium iwayamai (contigs 18, 28, 29, 61, 235), Pythium ultimum var. sporangiiferum (contigs 4, 6, 34, 106, 121, 134, 150, 173, 181, 222, 231, 257, 319, 404, 437, 458, 533, 726), Pythium vexans (contigs 9, 31,42, 94, 151, 160, 209, 220, 347), Phytophthora infestans (supercontig 1.2), Hyaloperonospora arabidopsidis (scaffolds 5, 6, 7, 8, 9) and Thalassiosira pseudonana (chromosome 3). Numbers along each ideogram are sequence lengths in kbp. Syntenic regions were identified through reciprocal best matches between gene models and block identification using MCscan . Each line radiating from Py. ultimum var. ultimum (scf1117875581354) links a syntenic gene pair. Each species is represented by a genus-species abbreviation and colored as Pythium ultimum var. ultimum (Pult) in blue, Pythium arrhenomanes (Par) in orange, Pythium irregulare (Pir) in yellow, Pythium aphanidermatum (Pap) in dark brown, Pythium iwayamai (Piw) in green, Pythium ultimum var. sporangiiferum (Puls) in dark red, Pythium vexans (Pve) in purple, Phytophthora infestans (Phin) in brick red, Hyaloperonospora arabidopsidis (Hpa) in olive green, and Thalassiosira pseudonana (Thaps) in light purple.

Article Snippet: Py. arrhenomanes , 324.62 , ATCC 12531 , Zea mays , WI, USA.

Techniques: Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

doi: 10.1101/2020.06.04.133462

Figure Lengend Snippet: Technical considerations for headloop PCR. ( A ) Comparison between results obtained with a proofreading (Phusion Hot Start II) or a non-proofreading (REDTaq) DNA polymerase for three target loci (A, B, C) of slc24a5 amplified with the PCR primers used for sequencing (seq) or when one is replaced by a headloop primer (HL). Samples were uninjected controls. Orange arrowheads mark the 300-bp ladder band. ( B ) Headloop primer designs, using slc24a5 locus G as an example. To perform headloop PCR, the forward or reverse primer from a previously verified primer pair is modified with a 5’ tag sequence and used in conjunction with its unmodified partner. The sequence of the headloop tag is selected so that the predicted Cas9 cleavage site (dashed line) is located towards the 5’-end of the tag. (left) If the modified primer and the gRNA binding site are in the same direction (headloop tag is added to the forward primer and gRNA binding site is on the 5’-3’ genomic strand), the reverse-complement of the gRNA binding site is sufficient (grey underlay). (right) If the modified primer and the gRNA binding site are in opposite directions (headloop tag is added to the reverse primer while gRNA binding site is on the 5’-3’ genomic strand), a sequence which includes the protospacer adjacent motif (PAM) and shifted from the gRNA binding site is sufficient. In both cases, after second strand elongation, the tag is able to bind the target sequence and direct elongation (hatched sequences) to form a hairpin, suppressing exponential amplification of the wild-type haplotype. Framed: headloop tag; grey font: gRNA binding site; grey underlay: headloop tag binding site.

Article Snippet: Each PCR well contained: 9.4 μL PCR mix (as described above), 0.25 μL forward primer (100 μM), 0.25 μL reverse primer (100 μM), 0.1 μL Taq DNA polymerase (ThermoFisher), 1 μL genomic DNA (same lysates as used for Illumina MiSeq); for a total of 11 μL.

Techniques: Comparison, Amplification, Sequencing, Modification, Binding Assay

Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: B cells were harvested from the spleen of an Exosc10COIN/LacZ mouse and fixed and prepared for 3D-STORM after 72 hrs of treatment with (1) stimulation cocktail and (2) 4OHT+stimulation cocktail. Reconstructed two color 3D STORM (super-Aresolution) image from a data set of 50,000 frames with Atto488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus. Three dimensional views of the boxed region show spatial distribution of AID and Mtr4 molecules inside the nuclei of B cells isolated from (A) wild type cells and (D) Exosc10 knockout cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in the B cell nucleus of (B) wild type & (E) Exosc10 knockout cells and in the corresponding cytoplasms (C) & (F), by using Matlab (2014b, MathWorks) software. (G) Comparison of the distribution of paired interaction of AID and Mtr4 in the nucleus versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. Comparison of the distribution of paired interaction of AID and Mtr4 were calculated in the cytoplasm versus nucleus of (H) wild type & (I) Exosc10 knockout B cells using a Student’s t-test in Matlab (2014b, MathWorks) software and P values are noted in the graph. (J) Comparison of the distribution of paired interaction of AID and Mtr4 in the nuclear center versus displaced from center versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. (K) Reconstructed two color 3D STORM (super-resolution) image with Alexa488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus of wild type Exosc10 cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in (L) nucleus center and (M) displaced from center of wild type cells, by using Matlab (2014b, MathWorks) software. All of the 3D STORM imaging was performed in three different B cells (from independent experiments) and repeated three or more times. 3D STORM super resolution image magnification is ×100. Scale bar: 1μm(K). Error bars indicate S.D. (P values: ** <0.01, *** <0.001)

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Labeling, Isolation, Knock-Out, Software, Comparison, Imaging

Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Modification, Electron Microscopy, Shear, Transgenic Assay, Sequencing, Subcloning, Software

Journal: bioRxiv

Article Title: Immunogenomic landscape of hematological malignancies

doi: 10.1101/618918

Figure Lengend Snippet: A . HLA I score (log2 geometric mean of B2M, HLA-A, HLA-B , and HLA-C ) is shown as boxplots comparing main cancer types and normal cell populations in Hemap. B . HLA II score (log2 geometric mean of HLA-DRA, HLA-DRB1, HLA-DPA1 , HLA-DPB1, HLA-DMA , and HLA-DMB) is shown as in A. C . HLA II score colored on Hemap t-SNE map. Specific clusters with low HLA II score are highlighted with circles. D . Comparison of HLA II score and HLA II surface protein expression level in blasts in a validation cohort of AML BM samples (n = 37) profiled using both RNA-seq and flow cytometry for HLA-DR. E . CIITA expression, HLA II score, and methylation of CIITA and HLA II genes in TCGA AML data shown as a heatmap. F . Methylation of CIITA colored on the TCGA AML t-SNE map. NPM1 and IDH1, IDH2 , and TET2 mutation status is labeled for cluster with low HLA II score. G . Differentially methylated cytosines (DMCs) in the CIITA region between samples with low and high HLA II score in GSE86952 ERRBS dataset. Histogram indicates the negative log10 P value of differential methylation at each cytosine, with red and blue colors indicating hypermethylated and hypomethylated cytosines in HLA II low samples, respectively. CpG areas, including CpG islands, CpG shores (< 2 kb flanking CpG islands), and CpG shelves (< 2 kb flanking outwards from CpG shores) are shown above CIITA exons belonging to isoforms pIII (lymphoid) and pIV (IFNγ-inducible). Transcription factor binding sites (TFbs) are shown below. E . Heatmap showing methylation of cytosines at CIITA regions significantly hypermethylated in the HLA II low group compared to high in the AML GSE86952 ERRBs dataset. 0 indicates no methylation and 1 indicates complete methylation. Patients (columns) are grouped by HLA II score and PML-RARA status. Rows correspond to cytosines at the CpG island, shores, and inter-CGI area (> 4 kb outwards from a CpG island) shown on the right. Major AML genetic alterations, HLA scores, and FAB classification are shown. F . Percentages of HLA-DR+ MOLM13 cells measured by flow cytometry after 72 h treatment with indicated concentrations of decitabine (DAC) and/or 10 ng/mL IFNγ. Dots indicate individual technical replicate wells. Data are shown for one of two independent experiments. P values are obtained using two-sided Wilcoxon rank sum test. See also Figure S4 and Table S4.

Article Snippet: Cell were treated with 10, 100 or 1,000 nM decitabine (Selleck) or DMSO as a control, both in the presence or absence of 10 ng/mL recombinant human interferon gamma (Peprotech), all conditions in triplicate wells.

Techniques: Comparison, Expressing, Biomarker Discovery, RNA Sequencing, Flow Cytometry, Methylation, Mutagenesis, Labeling, Binding Assay

Journal: EBioMedicine

Article Title: Cholangiopathies – Towards a molecular understanding

doi: 10.1016/j.ebiom.2018.08.024

Figure Lengend Snippet: Single Cell RNA sequencing experiments of liver, or cells differentiated into liver cells.

Article Snippet: Human In vitro: 2D culture of iPSCs (TkDA3–4, University of Tokyo) undergoing hepatic differentiation and 3D culture of liver bud organoids derived from hepatic cells differentiated from the iPS cell line, cocultured with HUVECS (Lonza) and MSCs (Lonza) In vivo: Adult (three donors: donor 1, female, 55; donor 2, male, 65; donor 3, male, 21) and fetal (two donors, gestation weeks 10.5 and 17.5) Mouse E14.5, E15.5, and E16.5 , Liver bud organoid cells: Liver bud organoids, different constellations of cells: 177 cells dissociated, no selection. Isolation of adult human liver cells: 256 cells from human adult liver. Protocol of hepatocyte or other cell isolation from adult liver published in [ ]; liver is dissociated and cell types separated using centrifugation steps. Isolation of fetal human cells: 238 cells from fetal stages, dissociated and briefly cultured (12h) on laminin-coated plates to remove red blood cells, followed re-dissociation of cells. Isolation of mouse hepatoblasts: 92 cells from mouse liver, dissociated, erythrocytes were lysed, and magnetic bead sorted for Dlk1. , C1 Fluidigm chip 1–5 million reads per cell. Cells were excluded from further analyses if they had < 100,000 reads, < 1,000 expressed genes or failed to express housekeeping genes ACTB or GAPDH , This manuscript does not explicitly identify cholangiocytes, but provides valuable insight into which culture systems better support in vitro differentiation faithful to in vivo hepatoblast growth. , iPSC-derived hepatoblasts undergoing culture in liver bud organoids more closely resemble fetal liver hepatic cells than do 2D cultured iPSC-derived hepatoblasts. Ligand-receptor pair analyses of co-cultured cells in organoids showed a KDR/VEGFA signaling pair in which VEGFA secreted by immature hepatocytes stimulates KDR on endothelial cells, which in turn support hepatoblast growth. , [ ] .

Techniques: RNA Sequencing, Sequencing, Isolation, Expressing, Comparison, Marker, Lysis, Modification, In Vitro, Derivative Assay, In Vivo, Selection, Cell Isolation, Cell Culture, Control

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , General morphology of livers 2 days after PHx. b , H&E staining of liver sections 2 days after PHx. c , d , Ki67 + percentages in HNF4α + hepatocytes ( c ) and HNF4α − NPCs ( d ) 2 days after PHx. Means ± SEM are shown, n=3 per group, **P < 0.01 (two-tailed unpaired t test). n.s., not significant. e , EpCAM was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). Stellate cells (GFAP) were not altered around the colony. f , CK19 was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). CD133 was highly expressed in the colony. g , Sox9 and CD44 were highly expressed in biliary epithelial cells (arrowhead), but was not upregulated in the colony (dashed line) compared to surrounding hepatocytes. h , AFP showed no difference between the colony (dashed line) and the surrounding tissue. i , Liver/body weight ratios after PHx. Means ± SEM from 3 or more mice analyzed for each time point and group are shown. j , k , Immunofluorescent staining of SKO liver sections 3 weeks after PHx. Colonies were at various sizes and locations as shown by arrows in j . Macroscopic colonies were found as shown by arrows and dashed lines in k . Immunofluorescent image in k corresponds to the magenta arrow in the liver image. PV, portal vein; CV, central vein. l , Vasculature shown by PECAM did not distinguish the colony (arrow). m , HGF expressed by NPCs was not concentrated in the colony (dashed line). Scale bars, 1 cm ( a ) 100 μm ( b , e - g , i - l ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence on liver tissue sections 2 days after PHx. HNF4α is a hepatocyte marker and Ki67 is a proliferation marker. Arrow in a points to an area enriched with proliferating hepatocytes. Dashed line in b shows an area with continuous CD133 expression. c , Quantification of proliferating rate in hepatocytes in CD133-positive and -negative areas in SKO livers 2 days after PHx. Each dot indicates one area. Data were collected from 3 mice. Means ± SEM are shown. ****P < 0.0001 (two-tailed unpaired t test). d , While WT hepatocytes proliferated at high frequency everywhere, proliferating hepatocytes in SKO liver were mostly located in patchy areas marked by CD133 expression. e , Immunofluorescence of CD133 on liver tissues at day 0 or 2, and 3 weeks (0d, 2d, 3 wk) after PHx. CD133 + hepatocyte clusters were only found in SKO livers after PHx (light green arrows). In WT livers, CD133 expression was only seen in bile duct epithelial cells (arrowheads). Scale bars, 100 μm ( a , e ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Marker, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Representative image of CD133 + colonies shown by immunofluorescence on SKO liver tissues 3 weeks after PHx. Dashed lines, vasculatures. b , H&E staining of SKO liver sections 3 weeks after PHx. Yellow dashed line: boundary between the colony and surrounding tissue; white dashed lines: vasculatures. c , Immunofluorescence on SKO liver sections at indicated time points after PHx. Arrowheads and dashed lines indicate the boundaries between the colonies and surrounding tissues, which was clear at 3 weeks but disappeared at 5 weeks. d , Unstained thick tissue section of SKO liver 3 weeks after PHx. e , Colonies ( C ) and non-colony control areas ( N ) were dissected out from thick tissue sections of SKO livers 3 weeks after PHx, and analyzed by immunoblotting. Random areas from WT livers 3 weeks after PHx, bile duct ( BD ) and E17.5 liver ( E ) were used for comparison. f , Illustration of epithelial lineages in the liver. Scale bars, 100 μm ( a - d ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Comparison

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Schematic illustration of mosaic rescue experiment of Shp2 in Shp2-deficient liver. b , Immunofluorescence showing Shp2-rescued hepatocytes also labeled with GFP. c , GFP + hepatocytes were proliferating (Ki67 + ; arrows), while other proliferating cells were not hepatocytes (arrowheads). Areas with colonies were not included here. d , Colonies were frequently constituted by GFP-negative hepatocytes (dashed line). Orange dots: non-specific stains of debris caused by HTVi. e , Some GFP + hepatocytes showed expression of CD133 (arrowheads). Dashed lines show CD133-positive areas. f , qRT-PCR analysis of WT and SKO liver lysates 2 days after PHx. n=3, means ± SEM are shown, *P < 0.05, **P < 0.01 (two-tailed unpaired t test). g , Immunofluorescence on SKO liver section 2 days after PHx. White dashed lines mark Porcupine + cells enriched in CD133 + colonies, rarely detected in non-colony area (arrowheads). Pink dashed lines: vasculature. h , Immunofluorescence on the colonies (arrows) in Shp2-deficient hepatocyte culture in vitro. Arrowhead, dying cells with high autofluorescence. i , Immunoblot analysis of WT and SKO liver lysates 2 days after PHx. j , qRT-PCR analysis of SKO liver tissue lysates without PHx (0d, 3 mice) and with PHx (2d, 3 mice). Means ± SEM are shown. **P < 0.01 (two-tailed unpaired t test). k , qRT-PCR analysis of hepatocyte and NPC fractions from SKO livers (3 mice) 2 days after PHx. HNF4α and CD45 were used as positive controls for the fractionation. β-actin was used for normalization, because GAPDH was highly expressed by hepatocytes. Means ± SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). l , qPCR analyses of PLC cells treated with Mek inhibitor and Porcn inhibitor. Means ± SD from 4 wells are shown. *P < 0.05, **P < 0.01, (two-tailed unpaired t test). n.s., not significant. Scale bars, 100 μm ( b - e , g , h ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Labeling, Expressing, Quantitative RT-PCR, Two Tailed Test, In Vitro, Western Blot, Fractionation

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence of liver sections 2 days after CCl 4 injection. Proliferating hepatocytes were scattered in WT livers, whereas they were highly concentrated in CD133 + colonies (white dashed line) in SKO livers as shown in a . CD133 + /EpCAM + /HNF4α - bile duct epithelial cells (arrowheads) were not associated with the colonies. Pink dashed lines, injured areas. PV, portal vein. c , Immunofluorescence of Met hep-/- liver sections 2 days after PHx or CCl4 injection. White dashed lines, CD133 + colonies. Pink dashed line, injured area. d , qRT-PCR analysis of PLC cell lysates treated with Shp2 or MEK inhibitors (Shp2i and MEKi). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. e , Immunoblotting of PLC cell lysates treated with inhibitors or transfected with Shp2 targeting CRISPR vector. Guide RNA targeting the AAVS1 safe harbor site was used as a control (sgCtrl). f , qRT-PCR analysis of MCF10A cell lysates treated with the inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. g , Immunoblotting of MCF10A cell lysates treated with inhibitors. h , qRT-PCR analysis of lysates from various cell lines treated with Shp2 or MEK inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. Scale bars, 100 μm ( a - c ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Injection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transfection, CRISPR, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence on in vitro colonies of primary hepatocytes from SKO liver. CD133 was localized at filament-like structures in E-Cad + colonies as shown by arrowheads in a , which were connected between different hepatocytes as shown by arrows in b . c , 3D-reconstituted confocal image of immunofluorescence on PLC cells. Lower panel shows the Z-plane section of the orange box area. Arrowheads indicate the CD133 signal on continuous filament like structures bridged between neighboring cells. Pink dashed lines indicate the cell surface. d , Immunofluorescence of MCF10A cells treated with Shp2 inhibitor. e , f , Super-resolution STORM images of immunofluorescence on PLC cells without ( e ) or with ( f ) CD133 overexpression. Colocalization of CD133 and β-tubulin was analyzed as Pearson’s coefficient. Mismatched green and magenta channels from shuffled ROIs were measured as controls . Means ± SEM from 6 images are shown. **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). g , Immunofluorescence and Immuno-Gold EM images of cryo-ultramicrotome sections of SKO liver tissue after PHx. Cyan arrowheads and asterisk indicate apical lumens. White arrowheads indicate the CD133 signals aligned between apical lumens of neighboring cells. Light green arrow, CD133 staining (12 nm colloidal gold); Magenta arrows, α-tubulin staining (18 nm colloidal gold). h , Immunoblotting of CD133 + vesicles isolated from MEK inhibitor (MEKi) -treated PLC cells. Markers for different fractions were analyzed. CD133 antibody used for the vesicle isolation was IgG produced in mouse, which was detected by anti-mouse IgG antibody, showing efficient capture by the beads. Despite the efficient capture, the DMSO-treated PLC cells did not have much CD133+ vesicle to be bound with the antibody. Scale bars, 100 μm ( a ), 25 μm ( d ), 1 μm ( e , f , Fluorescence in g ), 100 nm (EM in g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, In Vitro, Over Expression, Two Tailed Test, Staining, Western Blot, Isolation, Produced, Fluorescence

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a - c , Immunofluorescence on PLC cells transfected with CD133-Myc-tag fusion protein expression construct. Cells with different expression levels are shown as representatives. In a and c , exposure times for the CD133 signals were adjusted separately to clearly demonstrate the patterns, rather than intensities. Note that cells with higher CD133 expression displayed bulky pattern of the filaments, with maintained colocalization with tubulin filaments. Arrowheads, CD133 + filaments. As shown in b , the overexpressed CD133 primarily localize to the filaments, without detectable membrane localization (arrowheads). With extreme overexpression, CD133 can also localize to the cellular membrane surface, altering the morphology of the surface (right panels). d , Immunofluorescence on HeLa and MC38 cells. e , Colocalization of CD133 and β-tubulin was analyzed as Pearson’s coefficient. Mismatched green and magenta channels from shuffled ROIs were measured as controls. f , Immunofluorescence on PLC cells transfected with cMet-GFP. cMet, an HGF receptor, shows the cell surface. CD133 did not colocalize with cMet-GFP on the cell surface, but instead localized to the filaments. g , Immuno-Gold EM images of cryo-ultramicrotome sections of SKO liver tissue after PHx. Light green arrowheads, CD133 staining (12nm colloidal gold); Magenta arrowheads, α-tubulin staining (18nm colloidal gold). Scale bars, 5 μm ( a , c ), 50μm ( b , d ), 25μm ( f ) and 50nm ( g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Transfection, Expressing, Construct, Membrane, Over Expression, Staining

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunofluorescence for endosomal markers on PLC cells. b , Immunoblotting of CD133+ vesicles isolated from SKO liver after PHx. CHMP2B is a protein that is generally involved in vesicle formation. c , qRT-PCR analysis of housekeeping genes in RNAs extracted from WT (3 mice) and SKO tissues (3 mice) and from CD133 + vesicles and CD133-negative fractions (4 mice for both). Means ± SEM are shown. d , qRT-PCR analysis of RNAs extracted from CD133 + vesicles from PLC cells treated with MEK inhibitor. e , RNA-seq analysis showing enrichment scores of IEG contents in the two vesicle types compared to the whole cells. f , Immunofluorescence on SKO liver 2 days after PHx. Asterisks indicates vasculature. g , Immunofluorescence on MCF10A cells treated with inhibitors. h , qRT-PCR analysis of PLC cell lysates after treatment with CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. RNase and Triton X-100 were used to digest the RNA content of the vesicles. *P < 0.05, **P < 0.01 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 replicates are shown. Scale bars, 25μm ( a ) and 50μm ( f , g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Western Blot, Isolation, Quantitative RT-PCR, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Agarose gel electrophoresis of total RNAs extracted from WT and SKO livers and CD133-positive vesicle and negative fractions from SKO liver after PHx. Arrowheads show rRNAs and arrow shows microRNAs. b , qRT-PCR analysis of RNAs extracted form WT (3 mice) and SKO tissues (3 mice) and from CD133 + vesicles and CD133 − fractions (4 mice for both). Means ± SEM are shown. *P < 0.05, **P < 0.01 (uncorrected Dunn’s multiple comparison test, performed after Kruscal-Wallis test). n.s., not significant. c , RNA-seq analysis of the different cell types and the whole cells. The bars indicate proportions between numbers of deficient and enriched gene transcripts in each RNA types. d , Comparison of IEG contents between the different vesicle types with RNA-seq. e , RNA-FISH for MYC mRNA and immunostaining for CD133. f , Quantitative colocalization analysis of CD133 and MYC mRNA in PLC cells shown in e . Means ± SEM from 14 images are shown. g , Experimental design to detect the traffic of CD133+ intercellsome between neighbor cells. h , Immunostaining of Myc-tag and CD133 on GFP+ and mCherry+ PLC cells mixed as shown in g . Note that Myc-tag only indicates exogenous CD133, while CD133 indicates both endogenous and exogenous CD133. Myc-tag was primarily detected in the GFP+ cells, but also detected on the bridges (arrowheads) and in the mCherry+ cells (arrows). GFP was not detected at the same locations (arrowheads and arrows), indicating the specific traffic of CD133-Myc-tag. Scale bars, 10 μm (e) and 50 μm ( h )

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Agarose Gel Electrophoresis, Quantitative RT-PCR, Comparison, RNA Sequencing, Immunostaining

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunoblotting of HuR in the CD133 + vesicles from SKO liver after PHx. GAPDH and HNF4α were used as controls for cytoplasmic and nuclear fractions, respectively. b , Immunofluorescence on PLC cells. Arrows, peri-nuclear areas enriched with HuR in the cytoplasm; Arrowheads show colocalization of HuR on the CD133 + filament bridging two cells. c , Immunofluorescence on PLC cells treated with the Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. d , Immunofluorescence on PLC cells transfected with CD133 expression vector treated with a Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. e , Immunoblotting of CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. f , qRT-PCR analysis of PLC cell lysates after treatment with CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. RNase and Triton X-100 were used to digest the RNA content of the vesicles. *P < 0.05 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 replicates are shown. g , A model and predictions for single cell RNA-seq data analysis. h , Total IEG expression levels. Cyclin D1-positive and -negative cells were analyzed separately to evaluate the influence of cell cycle on the IEG analysis. See Methods section for what the bars and dots represent. i , j , Box plots (Tukey’s) of IEG diversity within each cell (calculated as entropy) and IEG variations among cells. Analyses were focused on cyclin D1 + cells in all groups for fair comparison. k , l , Plot of intracellular IEG diversity against total IEG expression levels in SKO hepatocytes 2 days after PHx. Blue color gradient indicates cyclin D1 expression levels. For the simulation in l , the parameters used were: Group of 5 cells, X=1/12, model 3 (see also and Methods). Green and Black arrows show typical profiles of CD133-positive and -negative cells, respectively. m , Box plot (Tukey’s) of intracellular IEG diversity after simulation. The analysis was not limited to cyclin D1-positive or -negative cells. Note the simulation of the IEG exchange attracted the cells from cyclin D1-low profile to cyclin D1-high profile ( k - m ). Statistics was performed by Wilcoxon rank sum test adjusted by FDR in i , j and m . Scale bars, 50 μm ( b , c ), 5 μm ( d ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Western Blot, Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Isolation, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a, Heatmap analysis of single hepatocytes. b , Detection of CD133 expression in the single cell RNA-seq data. c , Expression levels of indicated hepatocyte subtype markers and stem cell-like markers. CD133- and CD133+ hepatocytes in the SKO liver 2 days after PHx were compared. d , Principal component analysis with the IEGs. e , tSNE analysis. Clusters likely reflect spatial locations within the tissue.

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence ( a ) on liver sections of WT and Prom1 KO mice 2 days after PHx with or without Shp2 deletion using AAV-Cre and quantification of Ki67 + ratio in hepatocytes in the indicated genotypes ( b ). Separate analyses of pericentral and periportal hepatocytes showed insignificance of zonal difference. **P < 0.01, (two-tailed unpaired t test). Means ± SEM are shown. n = 3, 3, 4 and 5 mice, respectively. c , d , Immunofluorescence ( c ) on primary hepatocytes isolated from SKO and Shp2/Prom1 double KO (DKO) mouse livers and quantification of Ki67+ ratio ( d ). Images of representative colonies are shown. ***P < 0.001, (two-tailed unpaired t test). Means ± SD from 4 wells are shown. e , Experimental design with primary hepatocytes isolated from GFP labeled SKO liver and unlabeled DKO liver. E-Cad+ colonies were analyzed. f , Immunofluorescence images of E-Cad+ colonies in SKO, DKO and mixed culture as shown in e . The arrows show a GFP+ SKO cell forming part of the colony with the surrounding DKO cells. g , Quantification of the Ki67 ratio in E-Cad+ colony-forming cells shown in f . ***P < 0.001 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 wells are shown. h - j , scRNA-seq analyses of DKO liver compared to SKO liver 2 days after PHx. Porcn was used as a marker to represent the Prom1 promoter active hepatocyte populations in both SKO and DKO ( h ). A proliferation marker CyclinD1 was reduced in Porcn+ DKO hepatocytes compared to Porcn+ SKO hepatocytes ( i ). The IEG variations among cells were analyzed ( j ). See also a model in , regarding the predicted relationship between the intercellsome exchange and IEG variations among cells. Scale bars, 100 μm ( a , c , f ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Two Tailed Test, Isolation, Labeling, Marker

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunofluorescence on WT and Prom1 KO mouse intestinal tissue sections and intestinal organoids. b , c , Immunofluorescence ( b ) on WT and Prom1 KO mouse intestinal organoids treated with MEK inhibitor (MEKi) and quantification of Ki67+ ratio in the crypt cells ( c ). Dashed lines indicate the crypt buds. *P < 0.05, (two-tailed unpaired t test). Means ± SD from 3 wells are shown. Each dot represents each crypt buds, and symbols indicate each well. Scale bars, 20 μm ( a ) and 50 μm ( b ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Two Tailed Test

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Schematic of library generation by OTTR or ligation-based protocols from a single pool of RNase I derived RPFs (green) from a sucrose cushion. The pool of RPFs were split unevenly after T4 PNK treatment with only 1:10th of the RPFs used in OTTR. In OTTR, each step before cDNA size selection occurs in 4 hours in a single tube. First, input RPF RNA was labeled by either ddA or ddG on the 3′ end before unincorporated ddRTPs were inactivated by rSAP. Lastly, two ordered jumps, the first initiated from the +1Y DNA/RNA primer duplex and the second initiated from a non-templated dG addition to the RPF cDNA to jump to the 3’C adapter template, yields a cDNA molecule with a 5’ and 3’ adapter flanking the complement of the RPF input. In ligation-based, the 3’ adapter is first adenylated on its 5’ end. THen, 3’ adapter ligation is carried out, followed by gel-based size selection and overnight elution. THe next day, material is precipitated and primer hybridization for reverse transcription occurs. Following reverse transcription, cDNA is purified by gel-base size-selection. After elution, cDNA is circularized. In these illustrations green/light green denoted the RPF sequence, orange/light orange denoted the R1 adapter sequence, blue/light blue denoted the R2 adapter sequence, gray/dark gray denoted the unique molecular identifier sequence, brown/light brown denoted the barcode sequence, red octagon denoted polymerase blocking groups, magenta triangle denoted a 3’ddR, and a magenta square denoted the dG non-templated addition. b, Comparison of gene-level ribosome occupancy estimates from libraries generated in (a). Read counts are for RPFs aligned to verified CDSs excluding those RPFs that are aligned to the first 15 and last 10 codons. Read counts for each gene were normalized by DESeq2. c, Comparison of mean codon-level occupancy estimates from libraries generated in (a). Aligned RPFs were assigned to an A-site codon and counted. These counts were then rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and averaged across the translatome. d-e, Metagene averages around the start (left) and stop (right) codons for either (d) OTTR or (e) ligation-based libraries. Aligned RPFs for each CDS were first rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and then averaged across the translatome. Footprints were tabulated according to either the 5′ aligned position alone (shown at top as a black line), or both 5′ aligned position and read length (shown at bottom as a matrix of distinct RPF lengths and positions). f, Per-codon contributions to iχnos machine learning models of RPF occupancy profiles. A model based on a widow of 13 codons (−7 to +5) around the A-site was compared with thirteen additional models, each omitting one codon from the model. The contribution of a codon position to RPF occupancy profile was inferred from the change in Pearson’s correlation coefficient between the predicted ribosome occupancy versus actual ribosome occupancy changed when the codon was omitted (Y-axis).

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Ligation, Derivative Assay, Selection, Labeling, Hybridization, Reverse Transcription, Purification, Sequencing, Blocking Assay, Comparison, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Size-selection of P1 nuclease RPF cDNA from OTTR by direct imaging of Cy5, the dye covalently linked to the 5′ end of the +1dY DNA/RNA adapter duplex primer (see Fig. 1a). The 30 nt and 40 nt RNA oligonucleotides used for RPF size selection (not shown) were also used in OTTR reactions parallel to those using input RPFs, to generate cDNA size-selection markers. Bromophenol blue formamide loading dye was used to resuspend OTTR cDNA for size selection to avoid xylene cyanol interference during Cy5 imagining. A 0.6X TBE 8% urea-PAGE was chosen for cDNA size selection since xylene cyanol and the no-insert OTTR adapter-dimer cDNA (~75 nt) co-migrate. For these reasons, xylene cyanol was included only in the peripheral lanes. Horizontal black lines indicate the boundaries for cDNA gel slice excision to remove adapter-dimer from desired cDNA library. All lanes are from the same gel. P1 RPF were either from sucrose cushion purified human 293T or sucrose cushion purified S288C yeast material after nuclease digestion. A 30 nt and 40 nt template control OTTR reaction was used to synthesize OTTR cDNA to enable cDNA size selection equivalent to RNA size selection. b, Read length distribution of yeast RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates. c, Read length distribution of human RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates d, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for yeast libraries generated by P1 nuclease or RNase I digestion. e, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for human libraries generated by P1 nuclease or RNase I digestion. f, Average per-base read coverage of cytosolic 18S and 25S or 28S rRNA from yeast (left) or human (right) ribosome profiles with P1 nuclease (red) or RNase I (blue). Coverage was represented in reads per million total reads, including reads mapping to rRNA, tRNA, ncRNA, mRNA, and other genomic loci) to emphasize relative proportion from the entire library. Material was purified from a sucrose cushion. g, As in (f) but for 5.8S and 5S rRNA coverage. h, As in (f) for mitochondrial rRNA coverage.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Selection, Imaging, cDNA Library Assay, Purification, Control, Sequencing, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Fraction of RNase I RPF cDNA library sequencing reads mapped to each transcript class. Library generation artifacts included sequences that were adapter-only, shorter than 15 bases, or unmapped. b, Read length distribution of OTTR (blue) and ligation-based (red) RNase I RPFs from the CDS, excluding those RPFs that are aligned to the first 15 and last 10 codons. Counts were represented in RPM and averaged across replicates c, For each read length from 26 to 29 nt, the fraction of RPF alignments with mismatches at the 5′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. d, For each read length from 26 to 29 nt, the fraction of RPF alignments with an adenosine (A) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. e, For each read length from 26 to 29 nt, the fraction of RPF alignments with a thymine (T) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Comparison, Ligation, cDNA Library Assay, Sequencing

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Metagene average profiles around the start (left) and stop (right) codons from sucrose cushion purified yeast RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. The 5′ ends of aligned reads were counted, and counts for each gene were rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, prior to averaging. b, As in (a), for sucrose cushion purified human 293T cell RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. c, Gene-level ribosome occupancy estimates from sucrose cushion purified yeast RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. d, Gene-level estimates from sucrose cushion purified human 293T cell RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. e, Average profile of yeast footprints at start codons for P1 nuclease (red, 30 – 40 nt) and RNase I (blue, 25 – 29 nt) libraries. Footprint alignments were counted separately for each gene monitoring read length as well as 5′ end position (left) and 3′ end position (right), then averaged as in (a). A heatmap shows footprint abundance according to length and end position (below), and the end position average summed across all lengths is shown (above each heatmap matrix. The 5′ and 3′ end averages are shown to the left and to the right, respectively, of a black vertical bar. A diagram of a translating ribosome footprint (top) indicates mRNA cleavage positions of P1 nuclease (red triangle) and RNase I (blue triangle). f, Average profile of human cell footprints at start codons for P1 nuclease (red, 33 – 40 nt) and RNase I (blue, 27 – 32 nt) libraries, as in (e). g-h, Comparison codon-level ribosome occupancy estimates from (g) yeast or (h) human cell RPFs generated by P1 nuclease and RNase I digestion, as in Fig. 1c. In (h), arginine codons are shown in red. i, Schematic of proposed P1 nuclease and RNase I cleavage sites around an mRNA-engaged ribosome. Increased frequency of an RPF terminal position is indicated by increasing color saturation.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Purification, Generated, Comparison

Journal: Nature Communications

Article Title: RNA-mediated double-strand break repair by end-joining mechanisms

doi: 10.1038/s41467-024-51457-9

Figure Lengend Snippet: a Schemes of sense and antisense-genetic constructs expressing RNA transcripts that differ by sequence and transcription level. (left) Sense constructs with sgRNA A and B: Sense, BranchΔ, and pCMVΔ (right) Sense constructs with sgRNA E and J: Sense/ BranchΔ/pCMVΔ, Antisense constructs with sgRNA C(C’) and D: Antisense and 5′-SplicingΔ. All these constructs contain the DsRed gene (blue-framed box) with an intron (green-framed box) in the sense or antisense orientation, respectively. The sense and antisense transcript RNAs (in red) are depicted after intron splicing (thin green mark) or carrying the intron (thick green line) for the splicing-mutant constructs. The single-guide RNA (sgRNA) A (orange line), sgRNA B (purple line), sgRNA E (brown line) and sgRNA J (blue line) with Cas9 endonuclease (light gray ovals) bind to the complementary DNA of the sense constructs to generate a DSB. The sgRNA C (orange line) or C’ (dark orange line) and D (purple line) with Cas9 endonuclease bind to the complementary DNA of the antisense constructs to generate a DSB. Black arrows: CMV , cytomegalovirus promoter, or EF1α , human eukaryotic translation elongation factor 1 alpha promoter. Transcription activity by a cryptic promoter is indicated with a yellow arrow. Dark green box: branch site of the intron or 5′-splice site for the sense and antisense constructs, respectively. b Scheme of DNA products for the Sense construct obtained following DSB repair by different DSB repair mechanisms. NHEJ non-homologous end joining, MMEJ microhomology-mediated end joining, DSB (black parallel lines); an example of a microhomology pair (light blue lines); DNA repaired by NHEJ (blue zigzag line).

Article Snippet: Both the PCR fragment containing the EF1α promoter and the antisense plasmid were digested by AvrII restriction enzyme (New England BioLabs) and ligated by the T4 DNA ligase to merge them.

Techniques: Construct, Expressing, Sequencing, Mutagenesis, Activity Assay, Non-Homologous End Joining

Journal: Nature Communications

Article Title: RNA-mediated double-strand break repair by end-joining mechanisms

doi: 10.1038/s41467-024-51457-9

Figure Lengend Snippet: a Pie charts showing frequencies of sequencing reads displaying intron retention or pop-out following a double-strand gap by the sgRNAs A and B or sgRNAs E and J in the Sense, BranchΔ, and pCMVΔ constructs of wild-type and RNase H2A KO cells, or a double-strand gap by the sgRNAs C/C’ and D in the Antisense and 5′-SplicingΔ constructs of wild-type cells. Percentages represent an average of 4 repeats with standard deviation in parenthesis; N = 4. The percentages of sequences with and without intron are bolded. * p = 0.029 comparing frequencies of the BranchΔ or the pCMVΔ with those of the Sense construct, or comparing frequencies of the Antisense with those of the 5′-SplicingΔ construct via the two-tailed Mann–Whitney U test. b Individual (left) and comparison (right) variation-distance graphs illustrating sequence variations for the indicated samples. Refer to Fig. and Supplementary Fig. for details. The sequences are reverse-complemented prior to computing xy -coordinates so that they correspond to the forward-strand sequence coordinates. c Sum of MMEJ frequencies from all microhomology pairs of exon-exon MMEJ (sense constructs, see Supplementary Fig. , antisense constructs, see Supplementary Fig. ), following a double-strand gap by the sgRNAs A and B or sgRNAs E and J in the Sense (red), BranchΔ (green), and pCMVΔ (yellow) constructs of wild-type and RNase H2A KO cells, and the sgRNAs C/C’ and D in the Antisense (red) and the 5′-SplicingΔ (green) constructs of wild-type cells. Plotted data are the mean ± s.d. of the 4 biological replicates with the individual values shown as dots; N = 4. * p = 0.029 (two-tailed Mann–Whitney U test). d Boxplot showing specific ratios of MMEJ frequencies. Twelve Exon1-Exon2 microhomologies (black dots) are shown for MMEJ following the double-strand gap in the sense constructs generated by the sgRNAs A and B. Twenty-two Exon1-Exon1 microhomologies (black dots) are shown for MMEJ following the double-strand gap in the sense constructs generated by the sgRNAs E and J. Ten Exon1-Exon2 microhomologies (black dots) are shown for MMEJ following the double-strand gap in the antisense constructs generated by the sgRNA C/C’ and D. For details see Fig. legend. Source data are provided as a Source Data file.

Article Snippet: Both the PCR fragment containing the EF1α promoter and the antisense plasmid were digested by AvrII restriction enzyme (New England BioLabs) and ligated by the T4 DNA ligase to merge them.

Techniques: Sequencing, Construct, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Comparison, Generated

Journal: Nature Communications

Article Title: RNA-mediated double-strand break repair by end-joining mechanisms

doi: 10.1038/s41467-024-51457-9

Figure Lengend Snippet: Frequency of intron flipping caused by re-capture of the intron (or exon segment for sgRNAs E&J) via NHEJ following a double-strand gap by the sgRNAs A and B or sgRNAs E and J in the Sense (red), BranchΔ (green), and pCMVΔ (yellow) constructs of wild-type and RNase H2A KO cells, and the sgRNAs C/C’ and D in the Antisense (red) and the 5′-SplicingΔ (green) constructs of wild-type cells. Plotted data are the mean ± s.d. of the 4 biological replicates with the individual values shown as dots; N = 4. * p = 0.029 (two-tailed Mann–Whitney U test). Source data are provided as a Source Data file.

Article Snippet: Both the PCR fragment containing the EF1α promoter and the antisense plasmid were digested by AvrII restriction enzyme (New England BioLabs) and ligated by the T4 DNA ligase to merge them.

Techniques: Construct, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: RNA-mediated double-strand break repair by end-joining mechanisms

doi: 10.1038/s41467-024-51457-9

Figure Lengend Snippet: a Schemes of yeast constructs expressing RNA transcripts that differ by sequence. All these constructs contain the his3 gene (blue-framed box) with an intron (green-framed box) in the antisense orientation. The antisense transcript RNAs (in red) are depicted after intron splicing (thin green mark) or carrying the intron (thick green line) for the BranchΔ construct. The single-guide RNA (sgRNA) C (orange line) and sgRNA D (purple line) with Cas9 endonuclease (light gray ovals) bind to the complementary DNA of the yeast constructs to generate a DSB. Black arrows: pHIS3 , his3 promoter, or pTEF1 , TEF1 promoter. Dark green box: branch site of the intron. b Scheme of DNA products for the yeast constructs obtained following different DSB repair mechanisms. NHEJ, non-homologous end joining; MMEJ, microhomology-mediated end joining; DSB (black parallel lines); an example of a microhomology pair (light blue lines); DNA repaired by NHEJ (blue zigzag line). c Pie charts showing frequencies of sequencing reads displaying intron retention or pop-out following a double-strand gap by the sgRNAs C and D in the Antisense and BranchΔ constructs of wild-type cells following 48 h DSB induction. The NHEJ blue sector contains a DNA product having an identical sequence with the spliced RNA which could be a result of RNA-templated DNA repair (R-TDR), cDNA-templated DNA repair (c-TDR), or NHEJ. The frequencies of this DNA product are 0.03% (Antisense, ±0.03%) and 0.03% (BranchΔ, ±0.01%). Percentages represent an average of 4 repeats with standard deviation in parenthesis; N = 4. The percentages of sequences with and without intron are bolded. * p = 0.029 comparing frequencies of the BranchΔ with those of the Antisense construct via the two-tailed Mann–Whitney U test. d Pie charts showing frequencies of sequencing reads displaying intron retention or pop-out following a double-strand gap in the Antisense and BranchΔ constructs of wild-type and ku70 mutant cells following 18 h DSB induction. Survival of each construct on YPD and YPGal medium is shown under each pie chart. Percentages represent an average of 4 repeats with standard deviation in parenthesis; N = 4. Source data are provided as a Source Data file.

Article Snippet: Both the PCR fragment containing the EF1α promoter and the antisense plasmid were digested by AvrII restriction enzyme (New England BioLabs) and ligated by the T4 DNA ligase to merge them.

Techniques: Construct, Expressing, Sequencing, Non-Homologous End Joining, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Mutagenesis

Journal: Experimental & Molecular Medicine

Article Title: Origin of congenital coronary arterio-ventricular fistulae from anomalous epicardial and myocardial development

doi: 10.1038/s12276-022-00913-x

Figure Lengend Snippet: a Comparative RNA-seq analysis shows that 376 genes are differentially expressed (DEG) between mutant ( G2-Gata4 Cre/+ ; Itga4 flox/flox ) ( n = 4) and control ( G2-Gata4 +/+ ; Itga4 flox/+ ) ( n = 5) E11.5 ventricles ( p .value < 1e −3 ); 134 genes are increased and 242 are decreased. Each replicate represents a pool of three ventricles. b Gene Ontology (GO) enrichment analysis (an Over Representation Analysis with a Hypergeometric distribution with a Benjamini–Hochberg adjustment for multiple comparison) reveals important differences between the two genotypes. Pathway enrichment is expressed as the –log [P] adjusted for multiple comparison. The direction of the bars indicates which category was over/underrepresented. All categories in red are overrepresented in mutant animal, whereas all categories in blue are overrepresented in control animals. RT-qPCR analysis of E11.5 embryonic ventricles ( n = 5 in control and n = 3 in mutant; each replicate represents a pool of three ventricles) does not reveal significant variations in Vcam1 gene expression ( c ), but Vcam1 protein mislocalisation is evident in ventricular cardiomyocytes (compare d with e ; arrowheads mark the reduced Vcam1 distribution in the lateral plasma membrane of cardiomyocytes). Statistical significance was obtained by a non-parametric one-way analysis of variance with a Kruskal–Wallis post-hoc test ( p < 0.05). BrdU uptake ( f , g , arrowheads) is significantly reduced in E11.5 mutant ventricular compact myocardium (dashed lines) ( n = 5) as compared with stage-matched control ( n = 5) ( p < 0.01, 3 asterisks, h ). The statistical significance was assessed by a t-test and a post hoc Lubischew coefficient analysis. For the quantification of BrdU-positive cells, each replicate represents the mean value from quantifications in three ventricular sections. Abbreviations: BrdU bromodeoxyuridine; CVM compact ventricular myocardium; DAPI diamidino-2-phenylindole; TnI troponin I; TVM trabeculated ventricular myocardium. Scale bars: d , e : 25 µm; f , g : 50 µm.

Article Snippet: Proliferating cardiomyocytes were estimated with the IMARIS® software (see supplementary information).

Techniques: RNA Sequencing, Mutagenesis, Control, Comparison, Quantitative RT-PCR, Gene Expression, Membrane

Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Constitutive expression of rpfG restores motility and EPS production of the mutant Δ tfmR . And Xoc TfmR binds directly to the promoter of RpfG and activates its transcription. ( A ) Constitutive expression of rpfG restores motility of the mutant Δ tfmR . ( i ) Example photo of a bacterial strain. ( ii ) Mean measurements of colony diameter for each strain on “swarming” plates. ( iii ) Mean measurements of colony diameter for each strain on “swimming” plates. Data shown are the mean ± SD ( n = 10). Significance was determined by ANOVA and Dunnett’s post hoc test for comparison with to the wild type. ** p < 0.01; n.s., not significant. ( B ) Constitutive expression of rpfG restores the yield of the mutant Δ tfmR EPS. ( i ) Xoc strains were grown on NA plates supplemented with 2% sucrose for 3 days. ( ii ) Xoc strains were cultured in NB medium supplemented with 2% sucrose for 3 days and EPS was precipitated from the culture supernatant. Values given are the means ± SD of triplicate measurements from a representative experiment, and significance was determined by analysis of variance (ANOVA) and Dunnett’s post hoc test for comparison with the wild type. * p < 0.05; n.s., not significant. Similar results were obtained in two other independent experiments. ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of rpfG ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) ß–Glucuronidase (GUS) activity of the gusA reporter of the rpfG gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) Detection of Δ tfmR mutant and wild–type expression of rpf genes in NB medium ( i ), or XOM2 medium ( ii ), revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( F ) Fold enrichment of the promoter region of rpfG in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t -test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( G ) In vitro transcription experiments showing TfmR activates the transcription of rpfG . RNA was produced from a 323 bp template DNA fragment containing the rpfG promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 150 bp template DNA fragment of the rpfG coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Expressing, Mutagenesis, Comparison, Cell Culture, Electrophoretic Mobility Shift Assay, Negative Control, Activity Assay, Standard Deviation, Quantitative RT-PCR, In Vitro, Produced, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Xoc TfmR binds directly to the promoter of HrpX and activates its transcription. ( A ) ß–Glucuronidase (GUS) activity of the gusA reporter of the hrpG and hrpX gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( B ) Detection of Δ tfmR –mutant and wild-type expression of T3SS genes in NB medium ( i ), or XOM2 medium ( ii ) revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of hrpX ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) Fold enrichment of the promoter region of hrpX in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t –test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) In vitro transcription experiments showing TfmR activates the transcription of hrpX . RNA was produced from a 371 bp template DNA fragment containing the hrpX promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 161 bp template DNA fragment of the hrpX coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Activity Assay, Mutagenesis, Standard Deviation, Expressing, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Negative Control, In Vitro, Produced, Sequencing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were exposed to increasing LPS concentrations, thereafter washed and co‐cultured with PMA‐activated neutrophils. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Scale bars: 50 μm. Quantification of NET‐bound elastase indicative of NET formation. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA‐histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS‐primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non‐primed MSC (middle panels) or PBS‐injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10 μm (upper row) and 50 μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Incubation, Positive Control, Negative Control, Injection, Expressing, Activation Assay, Control, Comparison

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graph shows quantification of ROS by measuring fluorescence intensity of DHR123 dye. The MSCs were primed with increasing concentrations of 10 ng/ml, 100 ng/ml, and 1,000 ng/ml LPS followed by co‐culture with neutrophils. The MSCs were then incubated with ROS‐specific dye DHR123, and the fluorescence was read after 60 min of incubation at 488/520 nm with spectrophotometer. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Fluorescence, Co-Culture Assay, Incubation, Spectrophotometry, Positive Control, Negative Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: A Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat‐map shows the gene expression profile of non‐primed MSCs and MSCs which were LPS primed for 6 and 24 h. Red color represents up‐regulation, while blue color depicts down‐regulation in gene expression; each data point represents FPKM in log 2 value. B, C Pathway analysis depicts that cytokine–cytokine receptor interactions, genes encoding secreted soluble factors, an ensemble of genes encoding extracellular matrix, chemokine receptors which bind distinct chemokines, GPCR ligand binding, cytokine and inflammatory response, chemokine signaling pathway and IL‐5 signaling are the most dominant pathways as assessed by transcriptional changes when non‐primed and LPS‐primed MSCs were compared at 6 and 24 h after LPS priming. D Venn diagram analysis displays up‐regulated and down‐regulated gene numbers indicated in blue and pink colors, respectively. Purple color indicates the number of shared genes of the up‐regulated and down‐regulated genes at 6 h and 24 h after LPS stimulation. This implies rapid changes of expression of the same genes. E–G Validation of the selected genes uncovered from global RNAseq analyses by qRT–PCR analyses displays up‐regulation of CXCL‐6, IL‐8, and IL‐1β after LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. H, I ELISA results depict an up‐regulated expression of CXCL‐6 after 24 h of LPS treatment, while a significantly increased IL‐8 expression after 6 and 24 h of LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. J Western blot analysis showed up‐regulation of IL‐1β expression upon LPS priming of LPS‐primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6 h of LPS stimulation compared to non‐primed MSCs. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Gene Expression, Ligand Binding Assay, Biomarker Discovery, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Human MSCs were treated with LPS (100 ng/ml) for 24 h. The mRNA expression of MyD88 and IL‐6 was assessed by qRT–PCR analysis at 24 h. MyD88 (upper left panel) and IL‐6 gene expression (upper right panel) revealed an increase in MyD88 and IL‐6 in LPS‐primed MSCs. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. The TLR4 signaling pathway is depicted. Upon LPS binding, conformational changes in the TLR4 receptor led to recruitment of intracellular TLR domains harboring adaptor proteins. A bifurcation in MyD88‐dependent signaling converges to NF‐κB and pro‐inflammatory cytokine release, among them IL‐6, and the TRIF signaling pathway relaying its signals to downstream effectors like the transcription factor IRF which transactivates type 1 interferons. The right panel depicts representative Western blot analyses with a time‐dependent regulation of TLR4 and components of the TLR4 signaling pathway after exposure of MSCs with LPS. p65, a component of the heterodimeric NF‐κB transcription factor, and the IRF‐1 transcription factor are swiftly induced at 0.5 h, but down‐regulated to basal levels at 6 h after LPS challenge of MSCs. The target genes of NF‐κB are induced at 0.5 h, and this induction is differentially maintained. The expression of indicated proteins was analyzed in the cell lysates from LPS (100 ng/ml)‐ or PBS‐treated MSCs, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Binding Assay, Western Blot

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS‐primed MSCs into wounds increased the expression of CXCL6, IL‐8, and IL‐1β. Graphs display numbers of double‐positive cells for h‐β2M + CXCL6, h‐β2M + IL‐8, and h‐β2M + IL‐1β, respectively. MSC‐injected wounds served as controls. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50 μm. Results show that LPS‐primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL‐8 in mice and is known as neutrophil chemoattractant. Similar results were found for IL‐1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP‐2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double‐positive cells for Ly6G MIP/KC, IL‐1β, and LIX, respectively. PBS and non‐primed MSC‐injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Scale bars: 50 μm.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Expressing, Comparison, Functional Assay, Sequencing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were either treated with a scrambled control siRNA (Scr. siRNA) or with four different siRNAs targeting the TLR4 receptor (siRNA smart pool). Forty‐eight hours after transfection, TLR4‐silenced and non‐silenced MSCs were analyzed for RNA expression by qPCR. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Seventy‐two hours after transfection, TLR4‐silenced and non‐silenced MSCs were treated with 100 ng/ml LPS for 16 h and subjected to Western blot analysis for protein expression. Densitometric assessment shows the percentage of the change in TLR4 expression in TLR4‐silenced and non‐silenced MSC. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control, Transfection, RNA Expression, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative microphotographs of TLR4‐silenced and non‐silenced MSCs primed with LPS and co‐cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA‐histone, red). Of note, TLR4‐silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non‐silenced, LPS‐primed MSCs co‐cultured with PMA‐activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co‐culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50 μm. Quantitative assessment of NET‐bound elastase employing a specific ELISA in identical experimental setting as described in Fig . Similarly, to immunostaining, a significant reduction of NET‐bound elastase was observed in TLR4‐silenced LPS‐primed MSCs when co‐cultured with activated neutrophils as opposed to high NET‐bound elastase in non‐silenced LPS‐primed MSCs co‐cultured with activated neutrophils. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. High‐resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS‐primed MSC co‐cultured with PMA‐activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non‐primed MSCs co‐cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4‐silenced LPS‐primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4‐silenced and non‐primed MSCs display reduced NETs in co‐cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA‐activated neutrophils alone served as a positive control with highly enhanced NETs (upper row, outer right panel) and DMSO‐treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil‐derived granules. Scale bars: 0.1 μm

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Immunostaining, Negative Control, Positive Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Activation Assay, Derivative Assay

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative clinical pictures of murine wounds at 0, 3, 5, 7, and 10 days after wounding. Enhanced wound healing in the LPS‐primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non‐primed MSCs, LPS‐primed MSCs, non‐primed scrambled siRNA‐treated (Scr) MSCs, LPS‐primed Scr MSCs, and LPS‐primed TLR4‐silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one‐way ANOVA. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G + neutrophils (green) and F4/80 + macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bar: 100 μm. Quantification of Ly6G and F4/80 double‐positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantification of Ly6G + neutrophils and F4/80 + macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Single‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double‐stained for TGFβ‐1 (red) and for F4/80 + macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Scale bar: 50 μm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α‐SMA (indicative of myofibroblasts differentiation) after injection of LPS‐primed MSCs, non‐primed MSCs, LPS‐primed TLR4‐silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Double‐positive macrophages stained for TGFβ‐1 and F4/80 were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantitative analysis of CD31‐positive endothelial cells in sections of wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced LPS. Cell counting was performed on immunostained wound sections; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depict enhanced α‐SMA protein expression in wounds injected with LPS‐primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Confocal Microscopy, Injection, Staining, Double Staining, Control, Comparison, Cell Counting, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of sections from differently injected day 3 wounds stained for Ly6G + neutrophils (green) and caspase 3 (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 3 wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate apoptotic neutrophils. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Quantification of Ly6G and caspase double‐positive cells on sections of differently injected day 3 wounds. A significant increase in double‐positive cells (Ly6G and caspase 3) in sections of wounds injected with LPS‐primed MSCs as opposed to wounds injected with LPS‐primed TLR4‐silenced MSCs and PBS control. Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Staining, Double Staining, Comparison, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graphical summary, depicting the molecules involved in sensing, signaling, and raising an adaptive response in MSCs. MSCs sense bacterial intruders as modeled by the key molecule LPS, a widely distributed PAMP and wall component of Gram‐negative bacteria. LPS is sensed via the TLR4 receptor, and the signal is relayed via MyD88 to downstream effectors which, in consequence, shape the adaptive MSC response. This response is enforced by fundamental transcriptomic reprogramming which is responsible for the release of factors critical for neutrophil and macrophage recruitment to the wound site. The adaptive response of LPS‐primed MSCs depicts a bifurcation with the activation of neutrophils with enhanced microbicidal NET formation and ROS release to directly counteract invading bacteria. In addition, in a second line of tissue protection and repair, apoptotic neutrophils are phagocytosed by macrophages. Neutrophil engulfment constitutes a strong signal for macrophages to release TGFβ‐1 which subsequently enhances differentiation of myofibroblasts and accelerates wound contraction and thus wound closure. Acceleration of wound closure together with enhanced NET formation and ROS release effectively counteracts microbial invasion. These data may stimulate new avenues to refine MSC‐based therapies for difficult‐to‐heal wounds and/or infected wounds.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Bacteria, Activation Assay, Infection

Journal: Cell Death & Disease

Article Title: Tubule-derived CCN1 drives renal repair via α v β 5 -STAT6-ARG1-dependent reprogramming of macrophages

doi: 10.1038/s41419-025-08340-2

Figure Lengend Snippet: A BayesPrism workflow of scRNA-seq ( GSE174324 ) and bulk RNA-seq transcriptomic data integration and deconvolution based on BayesPrism to infer joint. B Cell-type fraction of deconvolution analysis across all samples. C Comparison of cell-type fractions following CCN1 treatment under normal and LPS-stimulated conditions. n = 4 per group. * P < 0.05. D , E The protein and mRNA expression of ARG1 in BMDMs. F , G Contour plot and histogram of flow cytometry of the proportion of ARG1 + cells in BMDMs. H The concentration of urea and Arginase activity in BMDMs. n = 4 per group. Data are presented as mean ± SD. I Representative fluorescent micrographs and quantification analysis of ARG1 (yellow), F4/80 (red) and EGFP-tag staining in I/R mice kidney with AAV-shScr and AAV-shCCN1 injection. J Flow cytometry of I/R mice kidney with CCN1 treatment. n = 4 mice per group. Data are presented as mean ± SD.

Article Snippet: After blocking with 10% normal goat serum, sections were incubated overnight at 4 °C with CCN1 (1:50, #39328), AQP1 (1:50, #69343), F4/80 (1:200, #30325) or ARG1 (1:50, #93668) primary antibodies (all from Cell Signaling Technology), followed by fluorophore-conjugated secondary antibodies [ ].

Techniques: RNA Sequencing, Comparison, Expressing, Flow Cytometry, Concentration Assay, Activity Assay, Staining, Injection

Journal: Cell Death & Disease

Article Title: Tubule-derived CCN1 drives renal repair via α v β 5 -STAT6-ARG1-dependent reprogramming of macrophages

doi: 10.1038/s41419-025-08340-2

Figure Lengend Snippet: A GSEA enrichment of STAT-related pathways in CCN1-treated BMDMs. B Protein levels of p-STAT6, STAT6 and ARG1 in BMDMs. C Co-IP and MS workflow for CCN1-interacting proteins. D Overlap of CCN1-interacting proteins identified by Co-IP/MS and BioGRID database. E Molecular docking of CCN1 with ITGAV and ITGB5. F Co-IP validation of CCN1 binding to ITGAV and ITGB5 in BMDMs. G Protein levels of p-STAT6, STAT6 and ARG1 after α v β 5 integrin inhibitor treatment. n = 3 per group. Data are presented as mean ± SD.

Article Snippet: After blocking with 10% normal goat serum, sections were incubated overnight at 4 °C with CCN1 (1:50, #39328), AQP1 (1:50, #69343), F4/80 (1:200, #30325) or ARG1 (1:50, #93668) primary antibodies (all from Cell Signaling Technology), followed by fluorophore-conjugated secondary antibodies [ ].

Techniques: Co-Immunoprecipitation Assay, Biomarker Discovery, Binding Assay