Journal: Nucleic Acids Research

Article Title: Analysis of epigenetic features characteristic of L1 loci expressed in human cells

doi: 10.1093/nar/gkac013

Figure Lengend Snippet: Promoters of L1 loci expressed in MCF7 cells are hypomethylated. Analysis of CpG methylation at promoters of expressed loci ( n = 160, orange), unexpressed loci ( n = 2,885, blue), total loci ( n = 4,815, purple), expressed loci with an ATAC peak ( n = 132), expressed loci without an ATAC peak ( n = 28), unexpressed loci with an ATAC peak ( n = 279), and unexpressed loci without an ATAC peak ( n = 2,606). A total of 158 loci were unmapped following bisulfite sequencing and thus excluded from this analysis. Significance between different groups of L1 loci was determined by Student's t -test (**** P < 0.0001).

Article Snippet: ATAC library preparation and PE sequencing were done by the Tulane Center for Translational Research in Infection and Inflammation NextGen Sequencing core according to their standard protocols seeding 2 million cells.

Techniques: CpG Methylation Assay, Methylation Sequencing

Journal: Nucleic Acids Research

Article Title: Analysis of epigenetic features characteristic of L1 loci expressed in human cells

doi: 10.1093/nar/gkac013

Figure Lengend Snippet: Long-distance interactions of specific L1-loci in MCF7 cells. Screenshots of Integrated Genomics Viewer of L1-2830 ( A ), L1-0650 ( B ) and L1-0238 ( C ) in MCF7 cells. L1-2830 is an expressed locus with the most mapped RNA-Seq reads in MCF7. L1-0650 is a ‘transitional’ locus, i.e. unexpressed locus with the second most ATAC-sequencing reads mapped. L1-0238 is a randomly-selected unexpressed locus. Information regarding the locus location (chromosome, start site, end site and orientation), RNA-Seq reads, presence of an ATAC peak, L1 sub-family, and number of reads from ATAC sequencing is shown in a table format at the top. Loops indicate CTCF binding sites (purple, shaded) within 500 kb of the L1 start site (black arrow). RNA polymerase II (Pol II) loops (red) are only shown if the pol II binding site overlaps within 500 bp of the L1 start site. Red arrows indicate magnification of the indicated genomic region that includes analysis of the activating histone marks from previously described CHIP-Seq data (Figure ). The status of each histone mark is examined in two experiments.

Article Snippet: ATAC library preparation and PE sequencing were done by the Tulane Center for Translational Research in Infection and Inflammation NextGen Sequencing core according to their standard protocols seeding 2 million cells.

Techniques: RNA Sequencing, Sequencing, Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Analysis of epigenetic features characteristic of L1 loci expressed in human cells

doi: 10.1093/nar/gkac013

Figure Lengend Snippet: Comparative analysis of full-length L1 loci identified within the TTC28 gene. ( A ) Schematic of the TTC28 gene (black) and exons (heavy black bars) transcription start site (green arrow), seven full-length L1 loci (blue arrows reflecting L1 orientation relative to the gene), and a CTCF binding site (orange box). The chart below the schematic contains information regarding each of the seven loci: L1-5212, L1-5214, L1-5215, L1-5216, L1-5217, L1-5218 and L1-5219. The information provided is L1 sub-family, RNA-Seq reads for expressed loci (Illumina or PacBio technologies ), and sequence status of YY1 and RUNX3 binding sites . Reads mapped to L1-5217 in HEK293 cells were manually determined not to primarily originate from an L1 promoter. ( B ) 3D interactions and epigenetic marks identified at L1 loci within the TTC28 gene in MCF7 cells. Loops indicate CTCF binding sites (purple, shaded) within 500 kb of the L1-5219 start site (black arrow). RNA polymerase II (Pol II) loops (red) are only shown if the pol II binding site directly overlaps with an L1 start site. DNAse Hypersensitivity Site (DHS) linkages (light red lines) and the surrounding genomic sequences (blue) are shown as well . A CTCF binding site 5′ of the L1-5219 is marked in red. Red arrows indicate magnification of the L1-5219 element and the associated enhancer region, respectively, showing the presence of activating histone marks at each genomic region as determined by previously described CHIP-Seq data (Figure ). The status of each histone mark is shown from two experiments.

Article Snippet: ATAC library preparation and PE sequencing were done by the Tulane Center for Translational Research in Infection and Inflammation NextGen Sequencing core according to their standard protocols seeding 2 million cells.

Techniques: Binding Assay, RNA Sequencing, Sequencing, Genomic Sequencing, ChIP-sequencing

Journal: Cancer immunology research

Article Title: Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable

doi: 10.1158/2326-6066.CIR-20-0638

Figure Lengend Snippet: Female C57BL/6-CEA-Tg mice (10–16 weeks old) were inoculated with MC38-CEA tumors. A. Treatment schema: injected tumor-bearing mice (n=10/group) with Ad-CEA (Vaccine, V) on days 7, 14, and 21 post-tumor implantation, with the IL15Rα superagonist N-803 on days 10 and 17, or each agent alone. Tumor growth and animal survival were monitored. Subsets of mice (n=7–8/group) were also treated with blocking anti-PD-L1 on days 14 and 21 post-tumor implantation, and tumor volumes were reported on day 25 post-tumor implantation (inset). The effect of CD8+ T-cell depletion on the survival of animals treated with the combination therapy was also monitored. On day 25 post-tumor inoculation, sera, spleens, and tumors were collected (n=3–5/group). B. Serum IFNγ quantified by ELISA. C. CEA-specific CD8+CD44+ splenocytes were identified via CEA tetramer staining. D-E. Genomic DNA was isolated from the tumors and were analyzed for (D) productive TCRβ rearrangement and (E) TCRβ clonality. F. Frequency of live tumor-infiltrating CD8+ T cells was determined by flow cytometry, and total CD8+ T cells per gram of tumor tissue was calculated. G. PD-1 and CTLA-4 expression on tumor-infiltrating CD8+ T cells was measured by flow cytometry. H. Tumor-infiltrating Tregs (CD4+CD25+FoxP3+) were quantified via flow cytometry. I. CD8+ T cell-to-Treg ratio in the tumor was determined. J. Immune-related transcriptome of the tumor samples was analyzed using the nCounter PanCancer Immune Profiling Panel. Heatmap showing select genes with data presented as fold change values on scale of −2 (blue) to +12 (red). One-way or two-way ANOVA with Tukey’s post hoc test for group analyses, t-test for comparison of two groups, and Kaplan-Meier survival analysis. *P<0.05; **P<0.01; ***P<0.005, ****P<0.001. Error bars represent mean±SEM. These studies were repeated 3–4 times with similar results.

Article Snippet: MHC class I-restricted (H2-Db) PE-labeled CEA-tetramer (sequence: EAQNTTYL) was purchased from MBL International Corporation (Woburn, MA).

Techniques: Injection, Tumor Implantation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable

doi: 10.1158/2326-6066.CIR-20-0638

Figure Lengend Snippet: Female C57BL/6-CEA-Tg mice (8–16 weeks old; n=10/group) were inoculated with 3×105 MC38-CEA cells on the flank (subcutaneous, s.c.). Tumor-bearing mice were either left untreated or administered with the pentatherapy combination using the treatment schedule described in Fig. 3A. A. Spleens and tumors were collected on day 28 post-tumor inoculation and assessed via cytometry for CD8+ T cells (gated on live, CD3+ cells). B. Tumor infiltration of CD8+ T cells was assessed via immunofluorescence. C-F. Using flow cytometry, CD8+ T cells were further examined for (C) Ki67+ cells, (D) CD44+ and CD44+CD62L+ cells, and (E) granzyme B+ cells, as well as (F) CEA-tetramer staining. Student t-test. G. Pentatherapy-treated mice were injected with CD8- or NK-depleting antibodies on days 3, 4, 5, 12, 19, and 26 post-tumor inoculation. Tumor growth was monitored. Two-way ANOVA with Tukey’s post hoc test; *P<0.05; **P<0.01; ****P<0.001. Error bars represent mean±SEM. These studies were repeated 3–4 times with similar results.

Article Snippet: MHC class I-restricted (H2-Db) PE-labeled CEA-tetramer (sequence: EAQNTTYL) was purchased from MBL International Corporation (Woburn, MA).

Techniques: Cytometry, Immunofluorescence, Flow Cytometry, Staining, Injection