Journal: Genome Medicine

Article Title: A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high-grade serous ovarian cancer

doi: 10.1186/s13073-020-00786-7

Figure Lengend Snippet: OriPrint is a solid stratifier and establishes the tissue of origin as a major source of variance for HGSOC. a Diffusion map with pseudotime timeline performed on OriPrint CpGs for samples of all cohorts. The origin is situated either in the distal FI (purple origin) or OSE (orange origin) samples. b Diffusion maps showing the classification output for the three indicated clustering methods. The overlap plot shows in white the samples that are concordantly classified by all three methods and in green the samples that have a different classification in at least one of the three methods. c PCA analysis coupled to Gaussian Mixture Model (GMM) clustering of the Melbourne tumor cohort. Left: First two components of global variance in DNA methylation for the considered samples. Middle: The two probability distributions calculated by GMM. Right: Overlay of the OriPrint classification, showing a consistent overlap with GMM’s distributions

Article Snippet: For each sample, 500 ng of genomic DNA was bisulfite converted using the EZ-DNA methylation Gold Kit (Zymo research) according to the kit’s manual, except that the final elution volume was reduced to 12 μl.

Techniques: Diffusion-based Assay, DNA Methylation Assay

Journal: Genome Medicine

Article Title: A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high-grade serous ovarian cancer

doi: 10.1186/s13073-020-00786-7

Figure Lengend Snippet: PAX8, a defining marker of HGSOC, is differentially methylated and expressed in FI-like vs. OSE-like tumors. a Graphical representation of the methylation of CpGs in PAX8 promoter across the indicated sample groups . b Dotplot depicting the gene expression level of PAX8 by RNAseq (blue bars, left Y -axis) and the DNA methylation level of its promoter by array (orange bars, right Y -axis) in the considered categories. The table summarizes the results of Mann-Whitney U tests (two-tailed) performed in the indicated comparisons. Data are shown as mean ± standard deviation. c PAX8 IHC performed on a tissue microarray of FFPE HGSOC. Samples were divided according to staining intensity. FI-like and OSE-like tumors were compared for the enrichment in the indicated categories by chi-square testing. d Dotplot depicting the DNA methylation level of PAX8 promoter in TCGA samples stratified as FI-like and OSE-like tumors. Mann-Whitney U test (two-tailed) for significance

Article Snippet: For each sample, 500 ng of genomic DNA was bisulfite converted using the EZ-DNA methylation Gold Kit (Zymo research) according to the kit’s manual, except that the final elution volume was reduced to 12 μl.

Techniques: Marker, Methylation, Expressing, DNA Methylation Assay, MANN-WHITNEY, Two Tailed Test, Standard Deviation, Microarray, Staining

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, Schematic of the loss-of-function and NLS-LacZ/H2B-Venus transcriptional reporter allele ( Fltp ZV ) for Fltp . b, Laser scanning confocal microscopy (LSM) image of a representative small intestinal (SI) crypt isolated from a Fltp ZV/+ reporter mouse line depicting FVR hi/low/neg crypt cells. FVR (Venus, green), DAPI (blue, nuclei), E-cad (red, membrane). Scale bar, 25 μm. c, Experimental design for microarray analysis of the three small intestinal (SI) crypt cell populations distinguishable by FVR activity. The heatmap depicts the expression profiles of key stem-cell and intestinal lineage genes. Expression is scaled row-wise and the colours range from dark blue (low expression) to orange (high expression) and represent normalized expression (row z-score). ISC, intestinal stem cell. CBC, crypt base columnar cell. Sec. progenitor, secretory progenitor. EEC, enteroendocrine cell. d, LSM images showing FVR (Venus, green) expression in the secretory lineages in the adult intestine of Fltp ZV/+ mice (4 months) co-stained against ChgA (red, enteroendocrine cells), Lyz1 (white, Paneth cells), Muc2 (red, goblet cells), and Dclk1 (red, tuft cells). DAPI (blue) stains the nucleus. Scale bars, 75 μm. e, f, LSM image depicting a representative SI crypt isolated from adult Fltp ZV/+ reporter mice with indicated FVR neg/low/hi cells stained for DAPI (blue, nucleus), FVR (Venus, green), ChgA (red, enteroendocrine cells), and Lyz1 (white, Paneth cells) ( e ) and quantification of Lyz1 + FVR + Paneth cells (PCs) and ChgA + FVR + enteroendocrine cells (EECs) ( f ). For Paneth cells: n (mice) = 8 with 99 analysed crypts (95.58 % of PCs = FVR + ). For enteroendocrine cells: n (mice) = 5 with 76 analysed crypts (96.13 % of EECs = FVR + ). Scale bar, 25 μm. g, Relative abundance of Fltp + Lgr5 hi , FVR low and FVR hi cells determined by single-cell qRT-PCR. n = 145 Lgr5 hi cells; n = 112 FVR low cells; n = 126 FVR hi cells. n (mice) = 3 for Fltp ZV/+ , Lgr5-ki h, Fltp expression in crypts treated with indicated Wnt/PCP ligands for two days. n (independent experiments) = 4. Error bars represent SEM. Two-tailed Student’s t -test, * P <0.05, *** P <0.001.

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Confocal Microscopy, Isolation, Membrane, Microarray, Activity Assay, Expressing, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, LSM images of immune-stained flow-sorted FVR hi/low/neg SI crypt cells isolated from Fltp ZV/+ mice showing that three distinct crypt cell populations are distinguishable by FVR activity. FVR (Venus, green), DAPI (blue, nuclei). Scale bar, 75 μm. b, Relative mean fluorescent intensity of GFP (Venus) in FVR low and FVR hi cells isolated from Fltp ZV+ cells by flow cytometry. n (independent experiments) = 6. Error bar represent SD. c, Heatmap depicting the p-values determined by Fisher’s exact test of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways which were significantly enriched in at least one of the populations. Pathways associated with neuroendocrine (green), immuno-regulatory function (blue), actin cytoskeletal regulation and signalling (MAPK/ Insulin, red) as well as cellular metabolism (black) and the cell cycle (red) are indicated. d, FACS plot depicting the experimental strategy to obtain Lgr5 hi cells (intestinal stem cells, ISCs) from Lgr5-EGFP-ires-CreERT2 (Lgr5-ki) mice. e, f, Confirmation of the microarray results. Comparison of the crypt cell populations distinguishable by FVR activity with Lgr5 hi ISCs by qRT-PCR, for the expression of intestinal lineage markers ( Lyz1 , Paneth cells; Chga , enteroendocrine cells; Muc2 , goblet cells) ( e ) and proliferation markers ( Ccnd1 , Ki67) and cell-cycle exit marker ( Cdkn1a ) ( f ). n (independent experiments) = 3. Error bars represent SEM. Two-tailed Student’s t -test, * P <0.05, ** P <0.01, **** P <0.0001. g, h, i, Heatmaps depicting the enrichment of the enteroendocrine cell (EEC) gene signature in the FVR low population ( g ), and the enrichment of genes expressed in Paneth cells (PC) formed via Lgr5 hi ISCs ( h ) or via a Goblet/Paneth cell precursor ( i ) in FVR hi cells. Gene signatures are derived from Grün et al., 2016. FVR labels all reported EEC subsets . Expression is scaled row-wise and the colours range from dark blue (low expression) to orange (high expression) and represent normalized expression (row z-score).

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Staining, Isolation, Activity Assay, Flow Cytometry, Microarray, Comparison, Quantitative RT-PCR, Expressing, Marker, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, b , Flow cytometry analysis ( a ) and relative abundance ( b ) of Fltp lineage - (mT), Fltp + intermediate cells (mTmG) and Fltp lineage + (mG) crypt cells isolated from Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. n (mice) = 6. Error bars represent SD. c, qRT-PCR data comparing relative Fltp expression in mTmG crypt cells and Lgr5 hi intestinal stem cells. n (independent experiments) = 3 for Lgr5 hi cells. n (independent experiments) = 2 (4 mice) for mTmG cells. Error bars represent SD. Two-tailed Students’s t -test, * P <0.05. d, e, Representative LSM images of Fltp + intermediate mTmG cells at indicated positions. SI crypts from Fltp T2AiCre/+ ;Gt(ROSA)26 mTmG/+ mice were stained for DAPI (blue, nucleus), mT (Fltp lineage - , RFP, green), mG (Fltp + intermediate cells, green, GFP), and Lyz1 (white, Paneth cells). Scale bar, 25 μm. ( d ). Abundance of mTmG cells at indicated positions ( e ). n (mice) = 3. f, g, Representative LSM images of Lgr5 hi and mTmG cells isolated by flow cytometry and stained for active, phosphorylated Jun N-terminal kinase (pJnk, white) indicating active Wnt/PCP signalling and DAPI (blue, stains nuclei) ( f ). Quantification of the mean fluorescent intensity of pJnk ( g ). n (independent experiments) = 3 for Lgr5 hi cells. n (independent experiments) = 5 for mTmG cells. Error bars represent SD. Two-tailed Student’s t -test, * P <0.05. Scale bar, 50 μm. h, MA-plot comparing the expression of ISC signature genes in mTmG and Lgr5 hi (ISCs) cells. Differentially expressed genes (FDR < 0.01) are indicated in red. The y-axis indicates the fold change in log2 and the x-axis indicates the mean log2 expression value. Clca1 is the only significantly regulated gene. n (Lgr5 hi ISC microarray samples) = 6. n (mTmG microarray samples) = 4. i, MA-plot comparing the expression of lineage-specifying genes in mTmG and Lgr5 hi cells. The y-axis indicates the fold change in log2 and the x-axis indicates the mean log2 expression value. No gene is significantly regulated. n (microarray samples) = 6 for Lgr5 hi ISC. n (microarray samples) = 4 for mTmG.

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Flow Cytometry, Isolation, Quantitative RT-PCR, Expressing, Two Tailed Test, Staining, Microarray

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, Experimental setup to assess stress response of Fltp + cells (mTmG) compared to Lgr5 hi ISCs. Mice were treated with two consecutive doses of 5-FU (100 mg/kg/day). Lgr5 + cells and Fltp + mTmG cells were analysed by flow cytometry 48 h and 24 days after the final 5-FU dose. Proliferative activity was determined after a 4 h chase of 5-ethynyl-2’-deoxyuridine (EdU) 48h after the final 5-FU dose. b, c, Representative LSM images of duodenal sections from Lgr5-ki ( b ) and Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice ( c ) 48h post 5-FU treatment. Sections were stained for DAPI (blue, nucleus), GFP (Lgr5 + cells, green) and Lyz1 (white, Paneth cells) ( b ) or mT (Fltp lineage - , RFP, red), mG (Fltp lineage + , GFP, green), and Lyz1 (white, Paneth cells) ( c ). Scale bars, 75μm. d-g, Representative FACS plots of Lgr5-GFP cell populations ( d ) and mT/mTmG/mG cell populations ( e ) from untreated and 5-FU treated mice and quantification of frequencies referred to total crypt cells ( f, g ) 48h post 5-FU treatment. n (mice) = 6 for untreated Lgr5-ki mice. n (mice) = 4 for treated Lgr5-ki mice. n (mice) = 6 for untreated Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. n (mice) = 7 for treated Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. Error bars represent SEM. Two-tailed Student’s t -test, ** P <0.01, *** P <0.001. h, i, Representative LSM images ( h ) and quantification ( i ) of 5-ethynyl-2’-deoxyuridine (EdU) positive (white) Lgr5 hi and mTmG cells after a 4 h chase of 5-ethynyl-2’-deoxyuridine (EdU) 48h after the final 5-FU dose. DAPI (blue) stains the nucleus. n (mice) = 3 for untreated Lgr5-ki mice. n (mice) = 5 for treated Lgr5-ki mice. n (mice) = 3 for Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. n (mice) = 7 for treated Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. Error bars represent SEM. Two-tailed Student’s t -test, * P <0.1. Scale bars, 75μm. j, k, l, Representative LSM images showing immune-stained duodenal sections from untreated and 5-FU treated Fltp T2AiCre/+ ; Gt(ROSA)26 mTmG/+ mice. DAPI (blue) stains nuclei, mGFP (green) labels Fltp lineage + cells, mTomato (red) marks Fltp - cells, Lyz1 (Paneth cells, white) and ChgA (enteroendocrine cells, red) ( j ). Scheme showing the mGFP expression pattern in crypt-villi units that are considered as stem-cell tracings (mGFP expression in all intestinal lineages in crypt-villi units) ( k ) and quantification of stem-cell tracings from sections ( l ). n (mice) = 5 for untreated mice. n (mice) = 3 for 5-FU treated mice. Scale bar, 75 μm. Error bars represent SD.

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Flow Cytometry, Activity Assay, Staining, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, UMAP plot depicting the identified cell states during differentiation of ISCs towards the Paneth cell lineage. Our analysis revealed an ISC subpopulation that is primed towards PC fate (PC-primed ISC), a PC progenitor population (PC pro), a PC population characterized by absence of Lyz2 expression (PC1) and a PC population characterized by Lyz2 expression (PC2). b, Dotplot showing the expression of the stem-cell marker Lgr5 in PC progenitor populations and genes that distinguish the mature PC types PC1 and PC2. c, Smoothed gene expression heatmap of differentially expressed genes of the displayed cell states plotted along diffusion pseudotime from ISCs to mature PCs. d, ISC score and PC score along pseudotime shows high ISC score in PC-primed ISCs. Also, Paneth cell progenitors express both ISC and PC markers. e, Experimental scheme of the 5-bromo-2’-deoxyuridine (BrdU) pulse-chase experiment. To assess label-retention, Fltp ZV/+ mice were treated with BrdU for 14 days (d) and analysed after a chase period of 10 and 21 d. f, Representative LSM images of duodenal sections stained for FVR (Venus, green), BrdU (red) and Lyz1 (white, Paneth cells) after 14 d BrdU administration. Scale bar, 25μm. g, Relative proportion of FVR + /BrdU + cells of total FVR + cells at the indicated time points. 14 d BrdU: n (mice) = 4 with 1432 analysed FVR + cells; 10 d chase: n (mice) = 2 with 785 analysed FVR + cells; 21 d chase: n (mice) = 4 with 2401 analysed FVR + cells. Error bars represent SD. h, Relative proportion of Lyz1 + (long-living and newly formed Paneth cells) and Lyz1 - (LRC) cells of the total FVR + /BrdU + cells at the indicated time points. 14 d BrdU: n (mice) = 4 with 1432 analysed FVR + cells; 10 d chase: n (mice) = 2 with 785 analysed FVR + cells; 21 d chase: n (mice) = 4 with 2401 analysed FVR + cells. Error bars represent SD. Two-tailed Student’s t -test, * P <0.05, **** P <0.0001. i, j, k, Scheme depicting the compartmentalization of the SI crypt. Actively cycling stem cells (Lgr5 + ) and Paneth cells reside in the Paneth cell (PC) zone. Quiescent/label-retaining cells locate at position +4/ +5. The transit-amplifying zone (TA zone) contains mainly proliferative (absorptive) progenitors ( i ). Representative LSM images from duodenal sections stained for FVR (Venus, green), BrdU (red) and Lyz1 (white, Paneth cells) after 10 d and 21 d chase. The position (arrowhead) of the FVR + LRC (BrdU + /Lyz1 - ) is defined according to the scheme in ( i ). Quantification of FVR + LRCs (BrdU + /Lyz1 - ) at indicated positions (according to j). 14 d BrdU: n (cells) = 116 (in 4 mice), 10 d chase: n (cells) = 32 (in 2 mice), 21 d chase: n (cells) = 55 (in 4 mice) ( k ). Scale bar, 25 μm ( j ). Error bars represent SD.

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Expressing, Marker, Gene Expression, Diffusion-based Assay, Pulse Chase, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

doi: 10.1101/2020.08.12.247585

Figure Lengend Snippet: a, Heatmap showing Pearson correlation of the highly variable genes in all cell types present in Celsr1 crsh/+ ; Fltp ZV/ZV mutant and control samples (see Supplementary Table 2). In all cases, Celsr1 crsh/+ ; Fltp ZV/ZV and control samples of the respective cell type correlate the most. n (mice) = 10 for control mice. n (mice) = 4 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. b, Barplot depicting cell-cycle annotation for all cell types into G1 (non-cycling), S and G2M (proliferating) phase. Paneth progenitors in Celsr1 crsh/+ ; Fltp ZV/ZV mutant mice are less proliferative than in control mice. All other cell types are not affected in terms of proliferation. n (mice) = 4 for control mice. n (mice) = 4 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. c, Heatmap depicting differential expression of transcription factors between Celsr1 crsh/+ ; Fltp ZV/ZV mutant and control mice and between displayed progenitor groups, ordered by log-fold change in PC-primed ISCs and in PC progenitors. In the EEC progenitors, only Hmga1 was significantly different in Sox4 + early EEC progenitors and none was significantly different in Ngn3 + progenitors. Benjamin-Hochberg adjusted p-value < 10 -5 . n (mice) = 10 for control mice. n (mice) = 4 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. d, Heatmap depicting differential gene expression in specific signalling pathways. Grey boxes in the dotplot highlight significantly different genes. Benjamin-Hochberg adjusted p-value < 10 -5 . n (mice) = 10 for control mice. n (mice) = 4 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. e, Heatmap depicting expression of Wnt target genes. Expression of Axin2, Ascl2 and Lgr5 were significantly different in Celsr1 crsh/+ ; Fltp ZV/ZV PC-primed ISCs. Benjamin-Hochberg adjusted p-value < 10 -5 . n (mice) = 10 for control mice. n (mice) = 4 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. f, g, h, Representative LSM images of duodenal sections stained for ChgA (red, enteroendocrine cells), Lyz1 (white, Paneth cells), DAPI (blue, nucleus) ( f ) and quantification of Paneth cells ( g ) and ChgA + EECs ( h ) in control and Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. n (mice) = 6 for control mice. n (mice) = 7 for Celsr1 crsh/+ ; Fltp ZV/ZV compound mutant mice. Error bars represent SEM. Two-tailed Student’s t -test, * P <0.1. Scale bar, 75 μm.

Article Snippet: Taqman probes (Applied Biosystems): Lyz1 , Mm00657323_m1; Chga , Mm00514341_m1, Gapdh , Mm99999915_g1; RN18S , Mm03928990_g1; Mki67 , Mm01278617_m1; Cdkn1a (p21), Mm04205640_g1; Muc2 , Mm01276696_m1; Ccnd1 , Mm00432359_m1; Ror2 , Mm01341765_m1/ Mm00443470_m1; Fltp , Mm01290543_g1; Fltp , Mm01290541_m1 Prickle1 , Mm01297035_m1; Dvl2 , Mm00432899_m1; Celsr1 , Mm00464808_m1; Fzd6 , Mm00433387_m1; Jun , Mm00495062_s1.

Techniques: Mutagenesis, Control, Quantitative Proteomics, Gene Expression, Expressing, Staining, Two Tailed Test

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Fate tracing experiment design b . Col1a1 in-situ hybridization in a PdgfrbCreER;tdTomato kidney. Scale bar 1000 μm. c . Percentage of Col1a1-mRNA expressing cells that co-express tdTomato at day10 after (unilateral ureteral obstruction, UUO, n = 3, shown mean). d . Time-course UUO experiment design. e . UMAP embedding of the mouse Pdgfrb+ cells. Labels refer to a cell-types identified. f . Percent of cells per cell type and time-point. g . Expression of selected genes on the UMAP embedding from e. h . Scheme of the PDGFRa/PDGFRb isolation UUO experiment. i . Quantification of Pdgfra+/Pdgfrb+ cells by flow cytometry (n=5 per group). *p<0.05; **p<0.01 by one-way ANOVA with post-hoc Bonferroni correction. Data shown as mean ± s.e.m. j . Left: UMAP embedding of the Pdgfra+/Pdgfrb+ cells Right: Percent of cells per cluster. k . Expression of selected genes in each of the cell clusters from j. n., o . UMAP and diffusion map embedding of fibroblasts and myofibroblasts. p . Computational cell ordering (pseudotime) and expression of selected genes on the embedding in n. q . Enrichment of transcription factor motif occurrence in fibroblasts and myofibroblasts. TF motifs were identified from Pdgfra + /Pdgfrb + day 10 UUO ATAC-Seq data (see ). For details on statistics and reproducibility, please see .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: In Situ Hybridization, Expressing, Isolation, Flow Cytometry, Diffusion-based Assay

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Representative image of Col1a1 in-situ hybridization in a PdgfrbCreER;tdTomato kidney after UUO surgery. Scale bar 10 μm b-c . Quantification of aSMA + cells in PDGFRbtdtom + kidneys from UUO day 10. n=3. Mean± SD. d . Scaled expression of the top 10 genes by specificity in each cell cluster depicted in . All cells from each cell cluster are averaged in one column. e . Expression of select genes in all 10 cell clusters from . f . ECM score visualized on the same UMAP embedding from . g . Distribution of ECM score, collagen score, glycoprotein score and proteoglycan score per cell cluster. h . Immunofluorescence (IF) staining in sham and UUO (day 10) mouse kidney showing Pdgfra expression in a subset of PDGFRbCreER;tdTomato positive cells (arrows). i . RNA in-situ hybridization showing colocalization of Col1a1 expression in PDGFRa/PDGFRb double-positive cells. Col1a1/PDFGRa/PDFRb triple-positive cells (arrows) occur solely in the kidney interstitium. j . Left: Col1a1 expression and ECM score in CD10 negative cells stratified according to PDGFRa and PDGFRb expression. Right: Percent of Col1a1 positive and negative cells in the same data, stratified in the same way. Col1a1 negative cells occur mostly in PDGFRa/b double-negative cells while Col1a1 positive cells occur predominantly in PDGFRa/b double-positive cells (n=51,849). Group comparisons: (other genes) vs. (a/b): p~0, (a - ) vs. (a/b): p~0, (b) vs. (a/b): p~0, (other genes) vs. (a): p~0, (b) vs. (a): p~0, (other genes) vs. (b):p~0. Bonferroni corrected p-values based on a two-sided Wilcoxon rank sum test. k . Distribution of IF/TA-Score over 62 patients and representative image of a trichrome stained human kidney tissue microarray (TMA) stained by multiplex RNA in-situ hybridization using PDGFRa, PDGFRb and Col1a1 probes with nuclear counterstain (DAPI) of 62 kidneys (patient data in Extended Data Table 2) (left), average scaled Col1a1 expression in the in-situ hybridization data stratified by PDGFRa/PDGFRb detection in the same data (middle) and percent of Col1a1 positive and negative cells in the same data stratified in the same way (right). Group comparison: (a/b) vs. (col1α1): p~0, (a/b) vs. (b): p~0, (a/ - ) vs. (a): p~0. Bonferroni corrected p-values based on a two-sided Wilcoxon rank sum test. l-p . A Diffusion Map embedding of pericytes and matrix producing cells with annotation of the different time points in m, cell cluster annotation in n and scaled expression of selected genes in o-q. q. The surgery type per cell (sham versus UUO) visualized on the same UMAP embedding from (top), or with colors representing the cell types/states (bottom). r . Expression of select genes on the same UMAP embedding from 3j. s . ECM and collagens score distribution for the 4 major cell types (top) and for mesenchymal clusters (bottom). Scale bars h+j 50 μm, in k 10 μm. For details on statistics and reproducibility, please see .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: In Situ Hybridization, Expressing, Immunofluorescence, Staining, RNA In Situ Hybridization, Microarray, Multiplex Assay, Comparison, Diffusion-based Assay

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Scaled expression of the top 10 genes by specificity in each of the mesenchymal cell clusters depicted in . Each 100 cells are averaged in one column. b . Cell doublet score (see ) of mouse PDGFRa/b + dataset per cell cluster. c . A violin plot of Col15a1 expression per cell cluster. Only mesenchymal cells are shown. Bonferroni corrected p-values based on a two-sided Wilcoxon rank sum test in Supplemental File 4. d . A UMAP embedding of Meg3 as in and multiplex in-situ staining of Meg3 on human kidney tissue. Scale bars d1 30 μm, d2+3 40 μm e . Representative image of multiplex RNA in-situ hybridization for PDGFRa, PDGFRb and Meg3 in n=34 human kidneys (Patient Data in Extended Data Table 2). Meg3 colocalizes with PDGFRa and PDGFRb. Scale bar 10 μm f . Percent of Meg3 - cells out of PDGFRa/b double-positive cells, quantified from RNA in-situ hybridization. n=34. Tukey box-whisker plot. g . Expression of select genes on the same UMAP embedding from . For details on statistics and reproducibility, please see .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: Expressing, Multiplex Assay, In Situ, Staining, RNA In Situ Hybridization, Whisker Assay

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Classification tree of human PDGFRb dataset derived by the CHETAH algorithm based on single cell expression and clustering information. b . Supervised classification of mouse PDGFRa + /b + cells using human PDGFRb + cells as a reference (see classification tree in a.). Heatmap displays percentage of mouse PDGFRa + /b + cells in each mouse cell cluster. Fibroblasts 1 in mice are largely classified as Fibroblasts 1 according to human data. Mouse myofibroblasts are classified as Node 15 and myofibroblasts 2b in humans indicating variability between mouse and human with myofibroblast states. c . Schematic of proposed cellular origin of fibrosis. d . Scaled gene expression of transcription factors discovered by ATAC-Seq (see ) in six fibroblasts and myofibroblast cell populations. e . ATAC-Seq signal for motif matches inside open chromatin regions for five selected transcription factors. f . Genome browser snapshots for select genes. ATAC-Seq signal and motif matches in open chromatin regions are shown. Multiz Align is conservation scores between mouse and human, ClinVar lift is clinical variants lift to mouse genome coordinates. Nrf, Irf8, Elf/Ets and Klf motifs are located in promoter and enhancer open chromatin regions of myofibroblast associated genes such as Col1a1, Col15a1, Tgfb and Nkd2. Creb5_Atf3 is found in genes associated to Fib1. cluster, such as Tmeff2. g . Expression of some of the genes investigated in g-i. Visualized on the same UMAP embedding from . i . Scaled expression of genes that correlate or anti-correlate with injury time across matrix producing cells (mouse PDGFRb + data). Note the expression of Ogn, Scara5 and Pcolce2 is largely specific to day0-day2 cells while the expression Nkd2, Fbn2 and Nkd1 is specific is increased in day 10 after UUO. h . Signaling pathway enrichment in the same mesenchymal cell clusters depicted in .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: Derivative Assay, Expressing, Gene Expression

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a . Expression of Nkd2 visualized on the UMAP embedding from . b . Percent of Col1a1+/- cells in mouse Pdgfra+/Pdgfrb+ cells ( , stratified by Pdgfra and Nkd2 expression). c . Scaled gene expression of Nkd2 correlating or anti-correlating genes in human Pdgfrb+ cells . d.-e . RNA in-situ hybridization (ISH) of PDGFRa, PDGFRb and NKD2 in human kidneys and quantification of triple positive cells (n=36, Patient data in Extended Data Table 2). n=20 and 16. Two-tailed Mann-Whitney test. Tukey box whisker plot. IF-score = interstitial fibrosis score. Scale bar 10μm. f.-g . Representative Western blots of Nkd2 overexpression and KO cells. For gel source data, see . h . GSEA (Gene set enrichment analysis) of ECM genes in Nkd2-perturbed PDGFRb - kidney cells. n=3 each. * P < 0.05, **p< 0.01, and ***p < 0.001 as determined by FGSEA-multilevel method after adjusting p-values for multiple testing correction (Benjamini & Hochberg). i . ISH of Pdgfra, Pdgfrb and Nkd2 in human iPSC derived kidney organoids. j . Quantification of Nkd2 RNA expression in organoids. n=4 each. Two-tailed unpaired t-test. k.-l . Immunofluorescence staining and quantification of Col1a1 in organoids. n=4 each. * P < 0.05, **p< 0.01, and ***p < 0.001 by 1-way ANOVA followed by Bonferroni’s correction. Scale bar in i+k 50 μm. Data shown as mean±SD. For details on statistics and reproducibility, please see .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: Expressing, Gene Expression, RNA In Situ Hybridization, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Western Blot, Over Expression, Derivative Assay, RNA Expression, Immunofluorescence, Staining

Journal: Nature

Article Title: Decoding myofibroblast origins in human kidney fibrosis

doi: 10.1038/s41586-020-2941-1

Figure Lengend Snippet: a-b . Gene ontology Biological Process terms for genes that correlate or anti-correlate with Nkd2 + expression across single cells in pericytes fibroblasts and myofibroblasts in mouse PDGFRa + /b + data (a) and human PDGFRb + data (b). Genes correlated with Nkd2 + expression are related to ECM expression, integrin signaling and focal adhesion. c . Pathway activity as estimated by the PROGENy algorithm in NKD2 + vs. NKD2 - cells from the human PDGFRb + dataset. p>0.05 n.s., *p<0.05, **p<0.01, ***p<0.001, p values were adjusted for multiple testing using Benjamin/Hochberg method (FDR) (c). d . Scaled gene expression of top 100 genes whose expression is correlated or anti-correlated with Nkd2 expression across single cells in human PDGFRb + data (see also b.) e. Gene regulatory network predicted based on the expression of cells and genes depicted in l. using the GRNBoost2 + algorithm. Connection between genes indicate putative direct or indirect regulatory interactions. Colors indicate clustering of the gene regulatory network using the Louvain algorithm and highlights the regulatory network of ECM expression (module 2, Nkd2 + ) and fibroblast and pericyte maintenance (module 4 and 3) f . Module 2 from l. Depicted separately, connections of Nkd2 are highlighted in red. g . Expression of genes highlighted in e. and f. including Etv1 transcription factor and Lamp5 which are both directly connected to Nkd2 in e. and f. h . Expression of Col1a1, Fibronectin (Fn) and Acta2 (aSMA) by qPCR after Nkd2 over-expression in human immortalized PDGFRb + cells treated with transforming growth factor beta (TGFb) or vehicle (PBS). n=3 per group. 1-way ANOVA followed by Bonferroni’ post-hoc test. Data represent the mean ± SD. i . Expression of NKD2 by RNA qPCR in NKD2 KO cells. ****P <0.0001 by 1-way ANOVA followed by Bonferroni’ post-hoc test. Data represent the mean ± SD. j . Expression of Col1a1, Fibronectin (Fn) and Acta2 by RNA qPCR after Nkd2 knock-out in the same clones depicted in h. n=3 per group. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 (vs. control NTG); ****p <0.0001 (vs. TGFb NTG) by 2-way ANOVA followed by Sidak’s post-hoc test. Data represent mean ± SD. k . PID signaling pathways enriched in PDGFRb + NKD2-KO clones and overexpression (up indicates up-regulated genes in indicated condition, and down indicates down regulated genes). l . Gene ontology Biological Process terms enriched in PDGFRb + NKD2-KO clones (up indicates up-regulated genes in KO condition, and down indicates down regulated genes). m . Scaled gene expression of WNT pathway receptors and ligands in Nkd2-perturbed human kidney PDGFRb + cells.*p< 0.05, **p< 0.01, and ***p < 0.001 as determined by the empirical Bayes from the test for differential expression after adjusting p-values for multiple testing correction (Benjamini & Hochberg) n . Representative image of multiplex RNA in-situ hybridization of PDGFRa, PDGFRb and NKD2 in human iPSC derived kidney organoids. o . Immunofluorescence stainings of human iPSC derived kidney organoids (day 7+18). LTA and HNF4a mark proximal tubular like-cells. pan-CK (Cytokeratin) marks epithelial-like cells. ERG (ETS regulated-gene) marks endothelial-like cells. Dach1 and Nephs1 mark podocyte-like cells. Col1a1 marks fibroblast/myofibroblasts. p . Immunofluorescence stainings of Col1a1 in IL1b treated kidney organoids. Scale bar in n, o, p=50 μm. For details on statistics and reproducibility, please see .

Article Snippet: The following antibodies were used: anti-mouse PDGFRa (AF1062, 1:100, R&D), anti-CD10 human (clone HI10a, 1:100, biolegend), anti-HNF4a (clone C11F12, 1:100, Cell Signalling), anti-Pan-Cytokeratin TypeI/II (Invitrogen, Ref. MA1-82041), anti-Dach1 (Sigma, HPA012672, 1:100), anti-Col1a1 (Abcam, ab34710, 1:100), anti-ERG (abcam, ab92513, 1:100), anti-CXCL12/SDF-1 (R&D, MAB350, 1:100), AF488 donkey anti goat (1:200, Jackson Immuno Research), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research)

Techniques: Expressing, Activity Assay, Gene Expression, Over Expression, Knock-Out, Clone Assay, Control, Protein-Protein interactions, Quantitative Proteomics, Multiplex Assay, RNA In Situ Hybridization, Derivative Assay, Immunofluorescence

Journal: Frontiers in Endocrinology

Article Title: Thyroseq v3, Afirma GSC, and microRNA Panels Versus Previous Molecular Tests in the Preoperative Diagnosis of Indeterminate Thyroid Nodules: A Systematic Review and Meta-Analysis

doi: 10.3389/fendo.2021.649522

Figure Lengend Snippet: Synthesis of the molecular tests’ diagnostic performance.

Article Snippet: Afirma Gene Expression Classifier (GEC) from Veracyte Inc. is a microarray-based test with a proprietary algorithm that analyses the mRNA expression of a panel of 167 genes ( ).

Techniques: Diagnostic Assay

Journal: Frontiers in Endocrinology

Article Title: Thyroseq v3, Afirma GSC, and microRNA Panels Versus Previous Molecular Tests in the Preoperative Diagnosis of Indeterminate Thyroid Nodules: A Systematic Review and Meta-Analysis

doi: 10.3389/fendo.2021.649522

Figure Lengend Snippet: Forest plot of sensitivity and specificity for Afirma GSC panel.

Article Snippet: Afirma Gene Expression Classifier (GEC) from Veracyte Inc. is a microarray-based test with a proprietary algorithm that analyses the mRNA expression of a panel of 167 genes ( ).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Thyroseq v3, Afirma GSC, and microRNA Panels Versus Previous Molecular Tests in the Preoperative Diagnosis of Indeterminate Thyroid Nodules: A Systematic Review and Meta-Analysis

doi: 10.3389/fendo.2021.649522

Figure Lengend Snippet: Forest plot of sensitivity and specificity for Afirma GEC panel.

Article Snippet: Afirma Gene Expression Classifier (GEC) from Veracyte Inc. is a microarray-based test with a proprietary algorithm that analyses the mRNA expression of a panel of 167 genes ( ).

Techniques:

Journal: BMC Research Notes

Article Title: RNA isolation method for single embryo transcriptome analysis in zebrafish

doi: 10.1186/1756-0500-3-73

Figure Lengend Snippet: Validation of the RNA isolation method from single zebrafish embryos . (a) RNA RIN values and (b) yields from: Single, individual embryos (4); Semi-single, homogenized, pooled, and split embryo material (4, see text); Developmental, embryos from the 16-cell to 8-somite stage (8); Unfertilized, unfertilized eggs (30); Epibolic, embryos from dome stage to 90% epiboly (186). Stages and RNA yield could not be linked. Note that RIN values show overlap because of a single decimal place measurement accuracy. (c) Mean, unnormalized log 2 signal intensities from microarray analysis (smooth bar, foreground signal and scatter board bar, background signal). (d) Principal component analysis (PCA) of the unnormalized log 2 ratios (test/reference) from the Single and Semi-single samples. (e) Spearman correlations showing the similarity of the unnormalized microarray data from Single and Semi-single samples.

Article Snippet: Each hybridization mixture was made up from 300 ng 'Test' and 300 ng 'Reference' sample according to the Two-Color Microarray-Based Gene Expression Analysis Manual version 5.5 (Agilent Technologies).

Techniques: Isolation, Microarray

Journal: BMC Research Notes

Article Title: RNA isolation method for single embryo transcriptome analysis in zebrafish

doi: 10.1186/1756-0500-3-73

Figure Lengend Snippet: Dissecting zebrafish development with microarray analysis . (a) Eight selected embryos ranging from the 16-cell to 8-somite stage. (b) Mean, unnormalized log 2 signal intensities from microarray analysis (smooth bars, foreground signal and scatter board bars, background signal). (c) Principal component analysis (PCA) on unnormalized log 2 ratio data (test/reference) showing a 'developmental' curve starting at the 16-cell stage and ending at the 8-somite stage. (d) Spearman correlations between the samples reflect the developmental distance.

Article Snippet: Each hybridization mixture was made up from 300 ng 'Test' and 300 ng 'Reference' sample according to the Two-Color Microarray-Based Gene Expression Analysis Manual version 5.5 (Agilent Technologies).

Techniques: Microarray

Journal: Journal of Neuroinflammation

Article Title: Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca 2+ -dependent PKC/p38MAPK/NF-κB pathway

doi: 10.1186/s12974-019-1398-3

Figure Lengend Snippet: The changes of expression of NR2B in CatC-stimulated microglia in vitro. Primary cultured microglia form WT mice were stimulated with 100 ng/ml active CatC for 24 h, then microarray-based gene expression analysis was performed. A The heat map showed 25 differentially expressed genes, including 10 upregulated genes and 15 downregulated genes. B The mRNA expression of NR2B was verified by qRT-PCR with 100 ng/ml active CatC stimulation and/or cathepsin inhibitor E-64 (50μΜ) for 12 h (a). Phosphorylated NR2B (p-NR2B) and total NR2B (t-NR2B) expressions were quantified in CatC-stimulated microglia by western blot analysis (b, c). C The same experiments were performed in BV2 cells. GAPDH as a loading control. The data represent the increased folds of treatment groups relative to the control. The data represent mean ± SEM, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The Whole Mouse Genome Oligo Microarray was generated for test and control samples according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology).

Techniques: Expressing, In Vitro, Cell Culture, Microarray, Quantitative RT-PCR, Western Blot