Journal: The Journal of Biological Chemistry

Article Title: Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics

doi: 10.1016/j.jbc.2021.101147

Figure Lengend Snippet: WSP exposure induces inflammatory gene transcription in Beas-2B airway epithelial cells. Quantitative RT–PCR analysis of indicated gene expression in Beas-2B cells treated with ( A ) WSP at indicated concentrations for 2 h and ( B ) WSP at 100 μg/ml for 2, 4, and 24 h. Bars represent mean normalized C T values on a log 2 scale (±SD) relative to vehicle-treated controls (n = 4/group, ∗ p < 0.05 versus vehicle). WSP, wood smoke particle.

Article Snippet: Deidentified primary human small airway epithelial cells (smAECs; <2 mm diameter) were obtained from the National Jewish Health Biobank and plated onto an irradiated National Institutes of Health (NIH)/3T3 (American Type Culture Collection) fibroblast feeder layer in F-medium containing 1 μM Y-27632 (APEX Bio).

Techniques: Quantitative RT-PCR, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics

doi: 10.1016/j.jbc.2021.101147

Figure Lengend Snippet: Exposure to TCDD induces transcription of AHR-dependent WSP targets in Beas-2B and primary airway epithelial cells. Quantitative RT–PCR analysis of indicated gene expression in ( A ). Beas-2B cells treated with two concentrations of WSP or TCDD (10 nM) for 2 h and ( B ) primary human small airway epithelial cells treated with WSP (1000 μg/ml) or TCDD (10 nM) for 2 or 4 h. Bars represent mean normalized C T values on a log 2 scale (±SD) relative to vehicle-treated controls (n = 4/group, ∗ p < 0.05 versus vehicle). C , ChIP–quantitative PCR was performed in Beas-2B cells for novel early peak targets of AHR following stimulation with TCDD (10 nM). Bars represent AHR occupancy on a log 2 scale (±SD), expressed as the mean C T value at each target region relative to the geometric mean of C T values at three negative control regions (n = 4/group, ∗ p < 0.05 for indicated comparison). AHR, aryl hydrocarbon receptor; ChIP, chromatin immunoprecipitation; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; WSP, wood smoke particle.

Article Snippet: Deidentified primary human small airway epithelial cells (smAECs; <2 mm diameter) were obtained from the National Jewish Health Biobank and plated onto an irradiated National Institutes of Health (NIH)/3T3 (American Type Culture Collection) fibroblast feeder layer in F-medium containing 1 μM Y-27632 (APEX Bio).

Techniques: Quantitative RT-PCR, Gene Expression, Real-time Polymerase Chain Reaction, Negative Control, Comparison, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics

doi: 10.1016/j.jbc.2021.101147

Figure Lengend Snippet: Airway epithelial inflammatory responses to WSP are mediated in part through complex crosstalk between AHR and NFKB. A , Beas-2B cells transiently transfected with siRNA targeting AHR (si- AHR ) or a scrambled control construct (si-Ctrl) were treated with vehicle or WSP for 2 h and then assayed for indicated gene expression using quantitative RT–PCR. Bars represent mean normalized C T values on a log 2 scale (±SD) relative to si-Ctrl + vehicle-treated controls (n = 4/group, ∗ p < 0.05 for indicated comparison). B , Western blot verification of AHR knockdown by si- AHR transfection. C , si- RELA - or si-Ctrl-transfected Beas-2B cells were treated and assayed as described for ( A ). D , Western blot verification of RELA knockdown by si- RELA . AHR, aryl hydrocarbon receptor; WSP, wood smoke particle.

Article Snippet: Deidentified primary human small airway epithelial cells (smAECs; <2 mm diameter) were obtained from the National Jewish Health Biobank and plated onto an irradiated National Institutes of Health (NIH)/3T3 (American Type Culture Collection) fibroblast feeder layer in F-medium containing 1 μM Y-27632 (APEX Bio).

Techniques: Transfection, Control, Construct, Gene Expression, Quantitative RT-PCR, Comparison, Western Blot, Knockdown

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: Single cell force directed layouts (FDLs) are composites of twelve HGSC tumors. Single cell sub-sampling with generation of FDLs was repeated three times with comparable results. Panels for (A) E-cadherin and vimentin (top). EV clusters co-expressing E-cadherin and vimentin are encircled and numbered 1 to 7 , NKG2D activating receptor ligands (second row) and ADAM proteases (third row). (B) Nectin-family ligands (C) HLA-ABC and HLA-E inhibitory ligands and tumor associated antigens; CA125, mesothelin and HE4. (D) Box and whisker plots. Expression levels for 12 NK receptor ligands and two ADAM proteases across E, EV and V compartments of 12 HGSC tumors. Medians and interquartile ranges are shown. p-values: ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001. See also Table S4.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Sampling, Expressing, Whisker Assay

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: (A) Boolean logic computation (left panel). Combinatorial diversity of 12 NK receptor ligands and ADAM 10 and 17 proteases expressed by tumor cells. Each NK receptor ligand combination is a row (left hand side). The heat map (right panels) shows the frequency of tumor cells that express each ligand combination within the E, EV, and V compartments for each tumor sample (columns). Rows were ranked based on the highest (top) to lowest total cell frequency (bottom). (B) Venn diagram showing distinct and overlapping NK receptor ligand combinations across E, EV and V compartments. (C) The Simpson’s inverse index of diversity was significantly greater for the E versus V (p = 0.05) and EV versus V (p = 0.007) compartments across all HGSC tumors. Medians and inter quartile ranges are shown.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques:

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: OVCAR4 (E), Kuramochi (EV) and TYK-nu (V) cell lines exposed to vehicle or carboplatin at 0.5 or 1μg/mL for 1 week were processed for CyTOF with the tumor NK receptor ligand/ADAM antibody panel (Table S3). Parent populations (viable single cells negative for cisplatin and cPARP). The plots show frequencies of HGSC cells expressing activating and inhibitor NK receptor ligands (X-axis) gated out of the parent population (Y-axis). Plots show the mean of triplicates with standard deviations. *p ≤ 0.05, **p ≤ 0.005, for overall ANOVA.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Expressing

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: HGSC and NK-92 cell lines were cocultured at an effector (NK-92): target (OVCAR4) ratio of 1:1 for 6h unless otherwise indicated (STAR Methods). (A) Left panel. Frequency of CD9 expression in NK-92 cells post coculture with and without transwell, respectively. Mean with standard deviations are shown (n=4). Right panel. Exemplary 2D flow plots. Induction of CD9+ NK-92 cells after coculture. (B) Lack of extra- and intracellular CD9 protein expression in the NK-92 cell line but high extra- and intracellular CD9 protein levels in the OVCAR4 cell line. (C) RT-PCR of FACS purified CD9+ and CD9-NK-92 cells after coculture with OVCAR4 cells. Number of copies (upper plots) or fold gene expression changes (lower plots) after coculture compared to respective monocultures. (D) Pre-incubation of NK-92 cells with cytochalasin D (10μM), a trogocytosis inhibitor, before coculture with HGSC cell line results in partial inhibition of trogocytosis. (E) Transfer of membrane fragments with CD9 from OVCAR4 cells stained with PKH67 onto NK-92 cells. Cocultures shown at different target : effector ratios. PKH67 frequency changes (upper histograms) and CD9 percentage changes (lower histograms). (F) Visualization of trogocytosis by microscopy. OVCAR4 cells and NK-92 cells stained with PKH67 (green) and PKH26 (red) respectively, were cocultured for 3h and stained with antibodies against CD45 and CD9. Images (from Keyence BZ-X800 microscope) for cells grown in monoculture 20X and for coculture 60X. Arrows mark cells NK-92 cells (red), OVCAR4 cells (green) and NK-92 cells after trogocytosis (white). Images were enhanced for brightness and contrast to optimize the printed image. See also Figure S6.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Incubation, Inhibition, Staining, Microscopy

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: (A) Eleven HGSC and 15 non-HGSC tumor cell lines were screened by CyTOF for CD9 expression. (B) Cell lines ranked by level of CD9 expression. Cell lines selected for coculture with NK-92 cells; HGSC (E, EV and V) (magenta), non HGSC cell lines with high levels of CD9 (green) and non-HGSC cell lines with lower levels of CD9 (yellow). (C) Representative flow plots showing the frequency of CD9+ NK-92 cells after coculture with non-HGSC cell lines. OVCAR4 included as a control. (D) Preincubation of NK-92 cells with cytochalasin D (10μM) and coculture with cell lines as in (C) results in partial inhibition of trogocytosis.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: HGSC and NK-92 cells (1:1) were cocultured for 6h, treated with PMA/ionomycin or vehicle control and brefeldin A/monensin and processed for CyTOF with the NK cell antibody panel (Table S5 and STAR Methods). CD9+ and CD9-cells were manually gated from the CD45+ cell population. (A) Frequency of CD9+ and CD9-cells producing each cytokine as indicated. (B) Levels (raw median counts) of cytokine produced by CD9+ and CD9-NK-92 cells. Plots show means of triplicates with standard deviations. Student’s two-tailed t-tests determined statistically significant functional differences between CD9+ and CD9-NK-92 cells. p-values: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.0001, **** ≤ 0.00001. See also Figure S7 and Table S6.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Produced, Two Tailed Test, Functional Assay

Journal: bioRxiv

Article Title: High-Grade Serous Ovarian Tumor Cells Modulate NK Cell Function to Create an Immune-Tolerant Microenvironment

doi: 10.1101/2020.11.20.391706

Figure Lengend Snippet: Coculture HGSC cell lines (as indicated) were assayed for their susceptibility to NK-92 mediated cytotoxicity by the calcein release assay. (A) NK-92 cells have reduced cytotoxicity toward HGSC cell lines compared to the control K562 cell line, at the target: effector ratios shown. (B) Addition of a CD9 blocking antibody significantly increased NK-92 cytotoxicity. Data are shown for quadruplets performed with two antibody concentrations and different target : effector cell ratios. Statistical significance determined with two tailed t test: * p ≤ 0.05, ** p ≤ 0.01, p ≤ 0.001. (C) FACS-sorted CD9+ NK-92 after coculture have a reduced cytotoxicity function compared to CD9-NK-92 cells grown in monoculture. Statistical significance determined with two tailed t test: * p ≤ 0.03, (*) p = 0.06.

Article Snippet: Deidentified newly diagnosed chemo naïve HGSC tumors prepared as single cell suspensions for CyTOF analysis collected over a two-year period were purchased from Indivumed (Hamburg, Germany) (Table S2).

Techniques: Release Assay, Blocking Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Selection promotes age-dependent degeneration of the mitochondrial genome

doi: 10.1101/2024.09.27.615276

Figure Lengend Snippet: (A) Schematic of the plate-based single cell mtDNA sequencing workflow. (B) Histogram showing the percent of raw reads mapping to mtDNA as measured for 360 cells isolated from 24-month-old C57BL6/J WT mouse liver. The majority of reads map to mtDNA. (C) Average mtDNA coverage as measured for 360 cells isolated from 24-months-old C57BL6/J WT mouse liver, log10 scale. While this method allows profiling the whole mtDNA, coverage is not uniform across the genome. Coverage in NCR is notably lower than in other regions of mtDNA. (D) Control showing the assessed abundance of PWD-specific SNPs in a sample where equal number of C57BL6/J and mtPWD hepatocytes were mixed (n =< 5 cells per sample type, the uncertain cell number is due to inefficient FACS sorting). This experiment shows the level of precision of mutation abundance detection.

Article Snippet: Cryopreserved deidentified human hepatocytes were purchased from Xenotech, Lonza or UCSF Liver Center.

Techniques: Sequencing, Isolation, Control, Mutagenesis

Journal: bioRxiv

Article Title: Selection promotes age-dependent degeneration of the mitochondrial genome

doi: 10.1101/2024.09.27.615276

Figure Lengend Snippet: (A) Schematic of the proof-of-principal experiment. In the experiment we mixed hepatocytes from two mouse lines with distinct mtDNA sequences (mtC57BL6/J and mtPWD). Samples were fixed in 1% PFA and permeabilized. Each biological sample was split in two halves and each of the four resulting samples was labeled with a unique lipid-tagged oligo (MULTI-ATAC barcodes B1-4). Next, all samples were pooled together and tagmented. After tagmentation, the cell suspension was loaded on 10X chip to generate 10X-mtATAC and MULTI-ATAC barcode libraries. (B) Average mtDNA coverage (read depth) in single mtC57BL6/J cells, n= 2294, 10910 scale. (C) A histogram showing enrichment of mtDNA reads over total reads in single mtC57BL6/J cells. (D) A histogram of the number of occupied droplets carrying different percentages of PWD-specific SNPs when mtC57BL6/J and mtPWD liver cells were mixed. The peak on the left represent droplets carrying mtC57BL6/J cells, and the peak on the right represents droplets carrying mtPWD cells. A shallow and wide distribution in the middle represents droplets with more than one cell (multiplets) carrying both mtC57BL6/J and mtPDW cells. (E) Classification of droplets according to sample-specific MULTI-ATAC barcodes matches cells’ mtDNA genotype. Droplets marked only by B1 or B2 align at the left consistent with mtC57BL6/J cells, while droplets marked with B3 or B4 align at the right consistent with mtPWD cells. There are three kinds of multiplet droplets: homotypic ones carrying both B1 and B2, which have only mtC57BL6/J cells; homotypic ones carrying B3 and B4 which have only mtPWD cells; and heterotypic ones carrying either B1 or B2 together with either B3 or B4, which carry both types of cells. The heterotypic multiplets with mixed mtC57BL6/J and mtPWD genomes are distributed across the middle of the graph, while the homotypic multiplets align as expected on the left or the right. Homotypic multiplets on the sides of the graph are masking underlying single cell data. B1: n=1052, B2: n=1208, B3: n=465, B4: n=696, Multiplets: n=921. Mean PWD-specific SNPs abundance for mtC57BL6/J cells is 0.35%, mean PWD-specific SNPs abundance for mtPWD samples is 99.12%.

Article Snippet: Cryopreserved deidentified human hepatocytes were purchased from Xenotech, Lonza or UCSF Liver Center.

Techniques: Labeling, Suspension

Journal: bioRxiv

Article Title: Selection promotes age-dependent degeneration of the mitochondrial genome

doi: 10.1101/2024.09.27.615276

Figure Lengend Snippet: (A) Droplet digital PCR (ddPCR) measurements of mtDNA copy number (blue points) in single hepatocytes from young and old mice and a middle-aged human. Boxes indicate the 25th and 75th percentiles, red line marks the median. The whiskers extend to the most extreme data points not considered outliers (conventionally defined as outside 1.5 times the interquartile range above the upper quartile and bellow the lower quartile; red points). (B) Simulation of mtDNA mutations accumulation. MtDNAs were treated as individuals with a measured population size (n) in each cell, with other variables (blue) assigned. See methods section for full description of the parameters. (C) Dynamics of accumulation of simulated neutral de novo somatic mutations. The plot tracks the fate of a generic allele as mutants emerge in many simulations. 234 mutations (colored lines) emerged in 250 simulations. Most disappeared shortly after emergence. Only 2 persisted at the end and only one reached an abundance of 10% (black dotted line). Model parameters: mutation rate 3.16×10 - per base pair per replication and 10,000 genomes per cell. (D, E, F) Simulations illustrating the impact of variables on the abundance distribution of mutations: time (number of generations) (D), mtDNA copy number (E) and mutation rate (F). Grey wedges highlight the difference in X-axis scale for D-F panels. In the lifetime of a mouse (∼80 replacement generations of mtDNA) chance accumulation of a mutation to critically high levels (usually 60%) in a cell with high mtDNA copy number is exceedingly unlikely. Models’ parameters unless specified otherwise in the figure panel: 16,299bp genome, 10,000 genomes per cell, 3.16×10 - mutation rate, 80 generations, 10,000 simulated cells.

Article Snippet: Cryopreserved deidentified human hepatocytes were purchased from Xenotech, Lonza or UCSF Liver Center.

Techniques: Digital PCR, Mutagenesis

Journal: bioRxiv

Article Title: Selection promotes age-dependent degeneration of the mitochondrial genome

doi: 10.1101/2024.09.27.615276

Figure Lengend Snippet: (A) A spectrum of mtDNA mutations identified in 41-year-old human hepatocytes. Note that annotation of mouse and human mtDNAs differ with linearization of the human genome splitting the NCR in two. (B) AAA vs C# plot of 41-year-old human hepatocytes. Blue asterisk marks the 3243A>G allele. (C) A spectrum of mutations in the NCR of 4,942 hepatocytes from the 41-year-old human also shown in A. Colored bars indicate sites that meet our criteria for positively selected driver alleles. Mutations were classified as drivers if the allele was detected in at least 10 cells at levels above 50%, and there were more cells with >50% abundance than cells with <50% abundance. (D) Abundance-distribution of the driver mutations identified in NCR of 41-year-old human hepatocytes. (E) An example of a driver-passenger pair in a single liver cell from 41-year-old human. (F) Abundance distribution of the driver and the passenger alleles shown in (E) among the 4,942 sequenced cells of the sample. (G, H) The NS/S and STOP/S rises with increase in mutations abundance. (I) Local NS/S for each NS allele on AAA vs C# plot for 41-year-old human hepatocytes shown in (B). Data in this figure were generated with 10X-based approach.

Article Snippet: Cryopreserved deidentified human hepatocytes were purchased from Xenotech, Lonza or UCSF Liver Center.

Techniques: Generated

Journal: bioRxiv

Article Title: Selection promotes age-dependent degeneration of the mitochondrial genome

doi: 10.1101/2024.09.27.615276

Figure Lengend Snippet: (A) A spectrum of mtDNA mutations identified in 81-year-old human hepatocytes. (B) Abundance distribution of mutations in 24-month-old WT mice (3 mice form one experiment), 24-month-old heterozygous mutator mice (3 mice from one experiment) and 81-year-old human (one human) hepatocytes. Data from each sample were subsampled to equal number of reads per cell (100,000). For mouse data clones and mutations at p5171 (Oril) were excluded as they are very frequent and mask the signal from other mutations. Samples were normalized to have equal number of mutations: 1,829 mutations were randomly selected for each sample. These data indicate an increased mutation frequency has little influence on the proportion of mutations reaching high abundance, while age appears to have a large impact. (C) Impact of the mutation rate on the abundance distribution among cells of a neutral mutant allele. The results are shown for simulations that ran for 3,000 generations that we estimate to be equivalent to mtDNA turnover over −80 years of human life. Number of mtDNAs was set to 5,000. (D) The impact of the selection coefficient on abundance distribution among cells of a neutral mutant allele. Model parameters: mutaton rate 3.16×1a.a per base pair per generation, 3,000 generations, 5,000 mtDNAs per cell.

Article Snippet: Cryopreserved deidentified human hepatocytes were purchased from Xenotech, Lonza or UCSF Liver Center.

Techniques: Clone Assay, Mutagenesis, Selection