Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a EZH2 ChIP peak profiles of gene Otx2 for WT, Mtf2 null, and Jarid2 null cells. Peaks were RPKM normalized and scaled across backgrounds. b Phase-contrast of Embryoid Bodies from different background and time point of differentiation. Each differentiation time point and background were performed in replicates. c , d Single-cell UMAPs of 4949 cells over all backgrounds and time points. e Single-cell UMAP of cells colored by predicted clusters. f – i Feature maps of selected lineage genes, color intensity based on normalized expression of individual gene.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Heatmap of differentially expressed genes comparing different mutants against WT in undifferentiated mESCs. Z-score normalized counts (row scaling) are shown in heatmap. b Venn diagram depicting overlap of a number of genes which were up/downregulated in Mtf2 null and Jarid2 null cells, with PRC2 targets (as determined from EZH2 ChIP targets; next panel). Significance of overlap calculated using hyper-geometrical test. c Heatmap of EZH2 binding peaks (RPKM normalized) for upregulated genes (from Mtf2 and Jarid2 null ESCs) for different genetic backgrounds. Heatmap depicts a window of +/−400 bp from the transcription start site (TSS) of the genes. d Dot plot showing the enriched Gene Ontology terms for the differentially expressed genes in different genetic backgrounds.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a ChIP peak profiles of histone mark H3K27me3 and H3K4me3 for selected upregulated ( Mtf2 null) lineage transcription factors for WT and Mtf2 null cells at pluripotent stage. ChIP profiles were RPKM normalized and scaled between two merged profiles per histone ChIP. b Boxplots depicting the RPKM values of promoter (as defined by +/−500 bp from TSS) H3K27me3, H3K4me3, and EZH2 for all PRC2 targets and the upregulated genes in Mtf2 null and Jarid2 null cells. Asterisk(*) represents a Two-sample Kolmogorov–Smirnov test p -value <0.05. n = 2878 for all PRC2 targets, n = 242 for upregulated Mtf2 null genes and n = 58 for upregulated Jarid2 null genes. Whisker ends of boxplot represent the maximum (top) and minimum values, respectively. Top and bottom of boxplots represent 75th and 25th percentile values, respectively, and finally, median values are shown as colored lines within the boxplots. c Transcription factor motif activity that explains part of the variance in transcript levels, based on motifs in the promoters (as defined by +/−500 bp from TSS) of all upregulated PRC2-bound genes (“Methods”; upregulated genes in all four cell lines, cf. Fig. ). Motifs are shown in aggregated z-scores. d – f Heatmaps showing the mRNA fold change, H3K27me3 and H3K4me3 levels for a set of transcription factors (left, identified in panel c ) and signaling factors (right, identified from Mtf2 and Jarid2 null DEGs list). Genes shown are all PRC2 targets. g Barplot depicting the GATA2 ChIP recovery relative to input. Control is a gene desert region (“Methods”). Dots in bars represent 4 replicates per sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Whisker Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Schematic of directed differentiation for monolayer cells from different genetic backgrounds (WT, Jarid2 null, and Mtf2 null). b Line plots showing the expression (normalized counts from RNA-seq) of selected temporally regulated genes during early lineage specification processes for different genetic backgrounds and differentiation directions. c , d Heatmap of differentially expressed genes across all time points and between genetic backgrounds. Data was k-means clustered and the normalized counts were shown. e , f Dot plots showing the enrichment of biological pathways for each cluster in ( c , d ), selected by p -value and gene-ratios of the terms. Top selected pathways were picked for each cluster and shown here. g , h Barplots of of H3K27me3 and H3K4me3 ChIP for selected targets ( Eomes and Gata6) . Each bar represents the average of the percentage of input recovered in the ChIP experiment of replicate experiments. Mtf2 WT and Jarid2 WT represent the background-matched wild-type lines of, respectively, Mtf2 null and Jarid2 null cells. Each dot represents a qPCR technical replicate for the sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing, RNA Sequencing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: Model of the role of PRC2 in the exit of pluripotency. PRC2 contributes to a repressive threshold for activation of key regulators of differentiation. This affects the exit of pluripotency and the differentiation to lineages of the three germ layers. One example is the upregulation of GATA2 upon MTF2 loss which results in increasing expression of other factors such as WNT7B and IRX3, as part of a feedforward loop that regulates the exit of pluripotency.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Activation Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium

doi: 10.3390/ijms20010022

Figure Lengend Snippet: Immunofluorescence measurement of LGR5 expression throughout the menstrual cycle in eutopic endometrium. ( A ) Epithelial and stromal expression of LGR5 in control and endometriosis tissue in five phases of the menstrual cycle. ( B ) Total tissue expression across five phases of the menstrual cycle in control and endometriosis tissues. ( C ) Differences in LGR5 expression throughout the menstrual cycle between control and endometriosis tissue groups ( t -test for each phase; proliferative: p = 0.0242; secretory: p = 0.0424; menstruation: p = 0.0121). (Control: n = 24; endometriosis: n = 24). ( D ) Example of immunofluorescence of LGR5 in eutopic endometrium (early secretory phase). In blue: DAPI; in red: LGR5. * p < 0.05.

Article Snippet: Samples stained with monoclonal rabbit anti-LGR5 primary antibody (1 μL per million cells was used; BioNova Scientific, Fremont, CA, USA) and goat Alexa647 anti-rabbit secondary antibody (Invitrogen) at a 1:500 dilution.

Techniques: Immunofluorescence, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium

doi: 10.3390/ijms20010022

Figure Lengend Snippet: In vitro analysis of LGR5 expression throughout the menstrual cycle in endometrial stromal fibroblast primary culture after treatment (E 2 and E 2 P 4 for six and six more days, respectively). ( A ) Left panels show expression of CYR61 (marker of the proliferative phase) and DKK1 (marker of the secretory phase) in the control group ( n = 4); right panels show expression of CYR61 and DKK1 in the endometriosis group ( n = 3). ( B ) Upper panel shows expression of LGR5 in proliferative (E 2 ) and secretory (E 2 P 4 ) phases of the control group; lower panel shows LGR5 expression in both phases of the endometriosis group (E 2 : estradiol; P 4 : progesterone).

Article Snippet: Samples stained with monoclonal rabbit anti-LGR5 primary antibody (1 μL per million cells was used; BioNova Scientific, Fremont, CA, USA) and goat Alexa647 anti-rabbit secondary antibody (Invitrogen) at a 1:500 dilution.

Techniques: In Vitro, Expressing, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium

doi: 10.3390/ijms20010022

Figure Lengend Snippet: Percentage of FACS-sorted LGR5 + cells. ( A ) Percentages of LGR5 + cells in different phases of the cycle (proliferative: n = 9; secretory: n = 4; ovulatory: n = 12; ND: n = 9). ( B ) Percentage of LGR5 + cells in eutopic endometrium of different types of endometriosis and controls (ovarian: n = 8; pelvic: n = 3; adenomyosis: n = 4; DIE: n = 10; control: n = 12). ( C ) Percentage of LGR5 + cells identified in eutopic endometrium of women with and without endometriosis (control: n = 12; endometriosis: n = 25). ( D ) Differences in LGR5 + cells between eutopic and ectopic endometrium. Left panel: ovarian endometriosis (eutopic: n = 8; ectopic: n = 4; p = 0.0286). Right panel: DIE (eutopic: n = 10; ectopic: n = 6; p = 0.0411). ( E ) Difference between ovarian and DIE ectopic endometrium (ovarian: n = 4; DIE: n = 6; p = 0.0381). ND: non-determined; DIE: deep infiltrating endometriosis. * p < 0.05, ** p < 0.03.

Article Snippet: Samples stained with monoclonal rabbit anti-LGR5 primary antibody (1 μL per million cells was used; BioNova Scientific, Fremont, CA, USA) and goat Alexa647 anti-rabbit secondary antibody (Invitrogen) at a 1:500 dilution.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium

doi: 10.3390/ijms20010022

Figure Lengend Snippet: Primers used for RT-qPCR.

Article Snippet: Samples stained with monoclonal rabbit anti-LGR5 primary antibody (1 μL per million cells was used; BioNova Scientific, Fremont, CA, USA) and goat Alexa647 anti-rabbit secondary antibody (Invitrogen) at a 1:500 dilution.

Techniques: Sequencing