Journal: Oncology reports

Article Title: Growth inhibition and transcriptional effects of ribavirin in lymphoma.

doi: 10.3892/or.2019.7240

Figure Lengend Snippet: Figure 1. Viability of Pfeiffer (A) and Toledo (B), diffuse large B cell lymphoma cells, and Hut78 (C), a cutaneous T‑cell lymphoma cell line. Cells were treated with ribavirin at different concentrations and for different time periods. 3‑Deazaneplanocin A (DZNep) was used as a positive control. Statistically significant difference, *P<0.05. To note, the Pfeiffer cell line carries the A677G mutation in EZH2, while Toledo cells are wild‑type.

Article Snippet: The following secondary antibodies were used: for EZH2, anti-rabbit (cat. no. sc2370) and for STAT-1 and SCD, anti-mouse (cat. no. sc2371), which were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Positive Control, Mutagenesis

Journal: Oncology reports

Article Title: Growth inhibition and transcriptional effects of ribavirin in lymphoma.

doi: 10.3892/or.2019.7240

Figure Lengend Snippet: Figure 3. Effects of ribavirin upon EZH2 expression (A) and H3K27m3 (B) in Pfeiffer and Toledo, diffuse large B cell lymphoma cells, and Hut78, a cutaneous T‑cell lymphoma cell line. The expression of EZH2 was mostly unchanged but showed a small (non‑significant) decrease with DZNep in mutant Pfeiffer cells. No changes in H3K27m3 were observed. To note, the Pfeiffer cell line carries the A677G mutation in EZH2, while Toledo cells are wild‑type. EZH2, enhancer of zeste homolog 2; H3K27m3, trimethylation status of histone 3, lysine 27 trimethylated.

Article Snippet: The following secondary antibodies were used: for EZH2, anti-rabbit (cat. no. sc2370) and for STAT-1 and SCD, anti-mouse (cat. no. sc2371), which were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Mutagenesis

Journal: Oncology reports

Article Title: Growth inhibition and transcriptional effects of ribavirin in lymphoma.

doi: 10.3892/or.2019.7240

Figure Lengend Snippet: Figure 2. Clonogenicity of Pfeiffer (A) and Toledo (B), diffuse large B cell lymphoma cells, and Hut78 (C), a cutaneous T‑cell lymphoma cell line. Cells were treated with ribavirin at different concentrations and for different time periods. 3‑Deazaneplanocin A (DZNep) was used as a positive control. Statistically significant difference, *P<0.05. To note, the Pfeiffer cell line carries the A677G mutation in EZH2, while Toledo cells are wild‑type.

Article Snippet: The following secondary antibodies were used: for EZH2, anti-rabbit (cat. no. sc2370) and for STAT-1 and SCD, anti-mouse (cat. no. sc2371), which were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Positive Control, Mutagenesis

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A) MC3T3 cells were differentiated to mineralized osteoblasts for 9 days of culture (DIV) using Ascorbic Acid (AA) 50ug/mL. Nuclear extracts were collected at indicated the days and analyzed by western blot using specific antibodies against the indicated epigenetic regulators. TFIIB or RNA-polymerase II (RNAPII) was used to control for equal protein loading. (B-C and E-F) Binding of chromatin regulators to the Runx2 P1 promoter at the indicated differentiation days were analyzed by ChIP using antibodies against: (B) Wdr5, (C) Utx, (E) Ezh2, (F) Prmt5, and (G) Jarid1b. (D) Re-ChIP assay performed in chromatin samples obtained from differentiated cells (5 DIV) using first an antibody against Utx and subsequently an antibody against Wdr5. Results and statistical analyses are shown as described in figure legend 2. ***p<0.001, *p<0.05, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Western Blot, Binding Assay

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A-B and D-E) Differentiating MC3T3 cells (3DIV) were infected with lentiviral particles containing shRNAs against the chromatin modifiers: (A) Wdr5, (B) Utx, (D) Ezh2 and (E) Prmt5. Knockdown efficiencies were confirmed by RT-qPCR and western blot (A-D, left panels) analyses, 48 h post-infection (5 DIV). TFIIB or RNAPII proteins were used to control for equal protein loading. (C) Effect of knocking down Wdr5 (left) and Utx (right) expression on Bglap gene transcription. mRNA expression values were normalized against Gapdh mRNA levels. Statistical analyses were assessed with respect to the mRNA levels obtained in cells infected with an empty vector (EV). *p<0.05, ***p<0.001, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation

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