Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Validation of Firre knockout in MEFs. a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Biomarker Discovery, Knock-Out, Quantitative RT-PCR, Expressing, RNA Sequencing, ChIP-sequencing, Binding Assay, Hi-C

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Firre KO does not result in disruption of TAD boundaries. a-d Hi-C heatmaps showing ± 5 Mb of the Firre gene locus (mm9, chr.X: 45–51 Mb) in female wild-type and Firre KO MEFs, ( b ) male wild-type and Firre KO MEFs, ( c ) allele-specific haploid chromosomes for female Cast (wild type) and C57BL6 ( Firre KO), and ( d ) male C57BL6 ( Firre KO). The TAD boundaries and the insulation plot for each Hi-C dataset is depicted below. e Dot plots showing the boundary strength of the Firre -centered and the neighboring TAD boundaries in wildtype (gray) and Firre KO (red) samples. The TAD insulation scores of all TAD boundaries on chromosome X is shown as boxplots on the right panel. f Hi-C heatmaps from wild-type (grown on 2i) and Firre KO mouse embryonic stem cells (mESCs) (grown on feeders + 2i) showing ± 5 Mb of the Firre locus. g , h Boxplot showing the inter-TAD interaction frequency between the TAD domains neighboring the Firre locus in wild-type (gray) and Firre KO (red) cells ( g ) in female MEFs and ( h ) in mESCs. Error bars: s.d. (* p -value: Wilcoxon rank-sum test)

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Disruption, Hi-C, Insulation

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: TAD boundaries are preserved upon ectopic Firre cDNA insertion and its induced expression at target sites. a Cartoon depicting the approach for the generation of the transgenic MEFs with endogeous Firre KO and ectopic Firre cDNA insertions. b qRT-PCR showing the induced expression of the Firre cDNA in wild type, DOX − , and DOX + conditions. Error bars: s.e.m. (* p -value: t -test). c Chromosome ideograms showing the Firre cDNA insertion sites on four different chromosomes. d CTCF ChIP-seq signal from DOX − transgenic MEFs for each of the exons of Firre cDNA at randomly inserted loci. As the transgenic MEFs harbor an endogenous Firre deletion, the intronic regions did not harbor any ChIP-seq signal. e-h Hi-C heatmaps showing the TAD organization, TAD boundary position, and the insulation plots for male Firre KO, DOX − , and DOX + samples ± ~ 5 Mb of Firre cDNA insertion sites on ( e ) chr 8, ( f ) chr 10, ( g ) chr 11, and ( h ) chr 15

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, ChIP-sequencing, Hi-C, Insulation

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Firre KO results in a loss of Firre -DXZ4, and changes in super-loop interactions in female MEFs. a Hi-C heatmaps at 40 kb resolution in male and female wildtype and Firre KO MEFs showing the interaction frequency between Firre and DXZ4 ± 1 Mb. b Forty-two windows of 40 kb bins were slid across the entire heatmap (2 Mb × 2 Mb in size) to compare the interaction frequency at each position of female and male WT versus KO conditions to the Firre -DXZ4 interaction by t -tests. The p -value distribution in the female or male wild-type samples indicate a drastically enriched significance of Firre -DXZ4 interactions when compared with either the female Firre KO or male samples (* p < 0.05, one-way ANOVA). c Hi-C heatmap at 100 kb resolution showing the zoomed-in interactions between the mouse Firre , DXZ4, x75, and ICCE regions ± 100 kb in female and male wild-type and Firre KO MEFs. d Boxplots showing the interactions among the super-loop regions in wild-type (gray) and Firre KO (red) female (top) and male (bottom) MEF samples. The sample sizes of the boxplots are n = 12 for Firre -DXZ4, DXZ4-x75, and DXZ4-ICCE interactions, and n = 9 for all other combinations. p -value: t -test. Error bars: s.d

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Hi-C

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: CRISPR live-cell imaging and 3C validates the changes in super-loop interactions. a Four-color CRISPR live-cell imaging (CLING) from female wild-type and Firre KO MEFs. Firre (red), DXZ4 (green), and x75 (white) loci were simultaneously visualized with Hoechst staining (blue). Pseudo-coloring was used for visual simplicity. Scale bar: 5 μm. b Quantification of the colocalization percentages between Firre -DXZ4, DXZ4-x75, and Firre -x75 between wild-type (black) and Firre KO (red) MEFs. (* p -value: χ 2 -test, n > 80 nuclei). Error bars: s.e.m. c Chromosome conformation capture (3C) analysis showing the interaction frequency ratios of Firre -DXZ4, DXZ4-x75, and Firre -x75 in female Firre KO vs. wild-type MEFs (* p -value: t -test, n = 3). The black arc indicates 3C enrichment in the wild-type samples, whereas the red arc represents enrichment in the Firre KO samples

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: CRISPR, Live Cell Imaging, Staining

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: (A) Phase-contrast images of mESCs treated with LIF (1,000 U/mL), or 10, 15, and 20 µM SB-216763 for more than a month. All doses of SB-216763 maintained mESCs as compact, elliptical or circular colonies characteristic of pluripotent cells. Morphology for mESC colonies maintained with SB-216763 was comparable to those in LIF supplemented medium. Scale bars represent 100 µm. (B) Twenty thousand mESCs from each treatment were seeded in a 6-well chamber at each passage and monitored for the percentage of pluripotent-like colonies two days after each passage. Data are expressed as the mean of five fields analyzed ± SEM. The percentage of pluripotent-like colonies in vehicle-treated cultures was significantly different from 15 µM and 20 µM SB-216763 samples at passage 4 (p<0.05) and from all treated samples from passage 5 onwards (p<0.05). (C) The average number of the total colonies and total pluripotent-like colonies observed between passages 4–7. The number of pluripotent-like colonies in vehicle-treated cultures was significantly lower than that for all other treatments (p<0.05). None of the SB-216763-treated samples were significantly different from LIF-treated mESCs for either the total number of colonies or the total number of pluripotent-like colonies.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: The graph also shows expression of these markers in EBs derived from month-old LIF and SB-216763 mESCs. SYBR Green qPCR results for these four genes were normalized to β-actin housekeeping gene using qbasePLUS software. SB-216763 treated mESCs expressed Lrh-1 and Sox2 about 3-fold higher than LIF treated mESCs. Nanog expression was also moderately higher in the SB-216763-treated mESCs, whereas Oct-4 expression was nearly 50% higher in LIF-treated mESC. Expression of these genes in differentiated cells was generally lower than the mESCs maintained with either LIF or 10 µM SB-216763. The data are average ± S.D. of technical replicates from one of the two experiments.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques: Expressing, Derivative Assay, SYBR Green Assay, Software

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: MESCs maintained with either LIF or 10 µM SB-216763 for more than a month yielded pluripotent-like colonies. Immunostaining revealed that Nanog expression (green fluorescence) was homogeneous in mESC colonies maintained with SB-216763, whereas LIF-treated mESC colonies often displayed incomplete Nanog staining. Three representative colonies from each treatment are shown. Scale bars represent 40 µm.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques: Immunostaining, Expressing, Fluorescence, Staining

Journal: PLoS pathogens

Article Title: Virion assembly factories in the nucleus of polyomavirus-infected cells.

doi: 10.1371/journal.ppat.1002630

Figure Lengend Snippet: Figure 6. Tubular structures contain VP1. PyV-infected 3T3 cells or PML2/2 MEFs (MOI of 10–20 pfu/cell) at 32 hpi were frozen by high pressure and processed by cryo-substitution for immunoelectron microscopy. Thin sections (45 nm or 70 nm) of Lowicryl-embedded samples were stained with either anti-PML or anti-VP1 antibodies followed by a secondary antibody conjugated to 10 or 15 nm colloidal gold. Top panels: 45 nm sections stained for the PML protein; white arrows, anti-PML staining, black arrow, tubular structures. Bottom panels: 70 nm sections stained for VP1; white arrows, anti-VP1 staining, black arrows, tubular structures. doi:10.1371/journal.ppat.1002630.g006

Article Snippet: C57 mouse embryonic fibroblasts (MEF) were obtained from ATCC (SCRC-1008; Manassas, VA) and served as a wild-type MEF control.

Techniques: Infection, Immuno-Electron Microscopy, Staining

Journal: PLoS pathogens

Article Title: Virion assembly factories in the nucleus of polyomavirus-infected cells.

doi: 10.1371/journal.ppat.1002630

Figure Lengend Snippet: Figure 7. PyV DNA and T-antigen localization in PyV-infected PML2/2 MEFs. PML2/2 MEFs were infected with PyV at an MOI of 30–40 pfu/ cell. At 22 or 24 hpi cells were fixed, permeabilized, and co-stained with either anti-Tag and/or anti-MRE11a antibodies followed by AlexaFluor- conjugated secondary antibodies, a fluorescently-labeled PyV DNA FISH probe, and DAPI staining of nuclei. A) FISH for PyV DNA at 24 hpi followed by antibody staining for Tag. B) Infected cells were stained by FISH for PyV DNA at 22 hpi followed by antibody staining for MRE11 or co-stained for MRE11 and Tag. All images represent a 0.1 mm z-stack slice. doi:10.1371/journal.ppat.1002630.g007

Article Snippet: C57 mouse embryonic fibroblasts (MEF) were obtained from ATCC (SCRC-1008; Manassas, VA) and served as a wild-type MEF control.

Techniques: Infection, Staining, Labeling

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Conditional expression of death inducing proteins . (A) Schematic overview of the constructs used to establish the regulatory system. The vector pWHE644 represents the regulator construct. A human EF1α promoter constitutively transcribes a tricistronic mRNA. This mRNA contains the reverse transactivator rtTA2 S -M2 (blue arrow), the transsilencer tTS D -PP (yellow arrow), and a selection marker (puromycin resistance; gray arrow). Translation of the latter two genes is mediated by internal ribosome entry sites (IRES; open boxes) from polio-virus (PV) and encephalomyocarditis- virus (EMCV). The vector pWHE655 contains the response unit used for stable transfections. It features the target gene (red arrow) driven by the Tet-responsive promoter TRE tight (open box, broken arrow) and flanked by two repeats each of a 250 bp sequence from the chicken HS4 insulator (blue triangles). A murine phosphoglycerate kinase 1 promoter (PGK; broken arrow) drives expression of a gene mediating G418-resistance. PolyA sites in all vectors are marked by a “ ⊥.” (B) Schematic representation of the cytotoxic test proteins. The residues that border the active domains expressed in the experiment are indicated above their respective closed box. A methionine added to allow translation is represented by a star. (C) Schematic overview of the regulatory system. In the OFF-State, a transsilencer (white) binds to the minimal promoter (open boxes, broken arrow) and actively suppresses transcription (cross). In the ON-State, doxycycline (blue circles) binds to both transsilencer and reverse transactivator (black). The former dissociates from, the latter binds to the minimal promoter and activates transcription (gray arrow). (D) Response of the regulatory system to different doxycycline concentrations. The B16F10-tBid transfected cell line was incubated for 24 h with various concentrations of doxycycline and mortality was measured, shown for one representative experiment out of three performed. Concentrations between 5 and 10 μg/ml showed the highest extent of cell death. An additional control at 10 μg/ml Doxy with the parental stably transfected cell line B16F10-644 was included to discard doxycycline toxicity at higher concentrations as cause of cell death (dark green diamond). Cell viability at time point “0” is shown as light green diamond.

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Construct, Plasmid Preparation, Selection, Marker, Virus, Transfection, Sequencing, Incubation, Control, Stable Transfection

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Six parameter classification by flow cytometry of the cell death phenotype of dying and dead B16F10 cells . Cell death analysis is based on morphological features (FSc and SSc), on the exposure of PS (annexin A5-FITC) and plasma membrane ion selectivity (PI), on the mitochondrial membrane potential [DiIC1(5)] and on nuclear DNA content (Hoechst 33342) detected by flow cytometry. Note: after proper gating, up to eight physiologically different subpopulations can be recorded. Dot plots exemplarily show B16F10-revCasp-3 cells after 18 h of doxycycline (5 μg/ml) treatment (A) . Rapid cell death occurred after 6 h in tBid-expressing cells and more than 95% cell death was observed after 24 h. In the presence of various caspase inhibitors [z-VAD-fmk, z-DEVD-fmk (caspase-3 inhibitor) and Ac-LEHD-cmk (caspase-9 inhibitor); all 50 μM], a significant increase in the stressed cell fraction displaying low-mitochondrial potential was observed (B) . Expression of revCasp-3 in B16F10 cells induced cell death after 24 h in more than 80% of the cells. z-VAD-fmk (50 μM) completely inhibited doxycycline-driven apoptosis. Note: stressed cells do not arise in this type of cell death induction (C) . Expression of CpnT CTD induced cell death in more than 90% of the cells after 18 h. Note: primary necrosis was the most common type of cell death observed and death occurred independently of caspase activity (50 μM z-VAD-fmk) (D) . Lethal UVB irradiation (240 mJ/cm 2 ) of parental B16F10 cells causes a rather slow progressing kind of cell death. Note: in the presence of z-VAD-fmk (50 μM), a significant increase of the stressed cell fraction displaying low-mitochondrial potential was observed (E) . Heat shock (56°C, 30 min) caused immediate necrosis in 100% of cells independent of caspase activity (F) . Displayed are the mean values from three independent experiments of relative percentages of each cell phenotype during 48 h of culture (B–F) .

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Flow Cytometry, Clinical Proteomics, Membrane, Expressing, Activity Assay, Irradiation

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Reactive oxygen species (ROS) production by dying B16F10 melanoma cells . Cells were induced to die by conditional expression of the death proteins tBid, revCasp-3, and CpnT CTD or by UVB irradiation, stained with the ROS sensor DCFH and with PI and analyzed by flow cytometry (A) . Inhibition of ROS production was performed by treatment with N -acetyl-cysteine (NAC) or mitoTEMPO, 100 μM, respectively, and recorded at 9 h after death induction (B) . Mean and SEM values of the mean fluorescence intensities of FL1 in viable cells (PI-negative) are displayed for different time points. At least three independent experiments were performed (Two and one stars indicate statistical significance at the p < 0.001 and p < 0.05 levels, respectively).

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Irradiation, Staining, Flow Cytometry, Inhibition, Fluorescence

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Growth of B16F10 melanoma cells in the allogeneic host and concomitant immunity . Four million viable B16F10 cells (VC) were implanted s.c. in the right flank of BALB/c mice. Mice developed tumors reaching their maximum size after 2–3 weeks, followed by rejection [ (A) , black line]. Mice implanted with 4 million UVB-irradiated cells did not develop primary tumors [ (A) , purple line]. After challenge with 2 million viable cells s.c. on the left flank, those mice bearing primary tumors did not develop secondary tumors [ (B) , black line], while mice primarily inoculated with irradiated cells developed tumors similar to those of the naïve group [ (B) , purple and green lines, respectively]. Mean values ( n = 8) and the SEM are displayed. Time points showing statistical significance when compared to the group of mice implanted with VC are highlighted. Two stars and one star indicate statistical significance at the p < 0.01 and p < 0.05 levels, respectively. The two way ANOVA test corrected by Bonferroni was applied in this experiment.

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Irradiation

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Immune response against dead or dying allogeneic tumor cells . BALB/c mice ( n = 5) were immunized in the right flank s.c. (single dose) with B16F10 dying/dead cells. After 10 days, mice were challenged s.c. in the left flank with 2 million viable cells of the parental cell line B16F10-644. Tumor growth was monitored for 30 further days (A) . Cell death was induced by UVB irradiation; heat shock; doxycycline-controlled expression of death proteins tBid, revCasp-3, and CpnT CTD . Displayed are the mean values ( n = 5) of relative tumor volumes and SEM [ (B) , * p < 0.05 after Mann–Whitney U test] and the integral of tumor size [ (C) , total tumor mass]. Inverse association between ROS production and total tumor mass developed in the allogeneic host (D) .

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Irradiation, Expressing, MANN-WHITNEY