ionomycin calcium salt from streptomyces conglobatus  (Millipore)

 
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    Structured Review

    Millipore ionomycin calcium salt from streptomyces conglobatus
    Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation <t>(PMA/ionomycin).</t> Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).
    Ionomycin Calcium Salt From Streptomyces Conglobatus, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Integrative dissection of gene regulatory elements at base resolution"

    Article Title: Integrative dissection of gene regulatory elements at base resolution

    Journal: bioRxiv

    doi: 10.1101/2022.10.05.511030

    Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation (PMA/ionomycin). Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).
    Figure Legend Snippet: Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation (PMA/ionomycin). Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).

    Techniques Used: Functional Assay, Sequencing, Expressing, Flow Cytometry, Activation Assay

    2) Product Images from "Reevaluation of Piezo1 as a gut RNA sensor"

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    Journal: bioRxiv

    doi: 10.1101/2022.09.23.509216

    ssRNA40 does not activate Piezo1 -transfected HEK293 cells (A) Fluo-4 calcium imaging of HEK293 cells, with or without transfection of mouse Piezo1 , representative of ≥ 3 independent recordings for each condition. Treatment concentrations are 10 µg/mL ssRNA40 or ssRNA41, 30 µM Yoda1, and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of Piezo1 -transfected HEK293 cells during different treatments. Yoda1 was applied 90 seconds after any given RNA sample, and only cells that responded to Yoda1 (presumably Piezo1-transfected) were analyzed. Transfection efficiency was generally > 60% of the cell culture. n = 50 cells plotted as mean ± 95% CI. (C) Quantification of HEK293 cell calcium responses. n = 50 cells per condition plotted as mean ± 95% CI. One-way ANOVA with Bonferroni correction: n.s. p ≥ 0.05, **** p
    Figure Legend Snippet: ssRNA40 does not activate Piezo1 -transfected HEK293 cells (A) Fluo-4 calcium imaging of HEK293 cells, with or without transfection of mouse Piezo1 , representative of ≥ 3 independent recordings for each condition. Treatment concentrations are 10 µg/mL ssRNA40 or ssRNA41, 30 µM Yoda1, and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of Piezo1 -transfected HEK293 cells during different treatments. Yoda1 was applied 90 seconds after any given RNA sample, and only cells that responded to Yoda1 (presumably Piezo1-transfected) were analyzed. Transfection efficiency was generally > 60% of the cell culture. n = 50 cells plotted as mean ± 95% CI. (C) Quantification of HEK293 cell calcium responses. n = 50 cells per condition plotted as mean ± 95% CI. One-way ANOVA with Bonferroni correction: n.s. p ≥ 0.05, **** p

    Techniques Used: Transfection, Imaging, Cell Culture

    RNA activates RIN14B cells independently of Trpa1 (A) Calcium imaging of ssRNA40 and ssRNA41 responses in RIN14B cells loaded with Fluo-4 AM, representative of ≥ 3 independent recordings for each condition. Cells were stimulated with 25 µg/mL ssRNA40 or ssRNA41 and 10 µM ionomycin. The mean Δ F/F 0 ± 95% CI is shown for a single recording each of n = 50 cells. (B) To block Trpa1, 10 µM A-967079 was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. 10 µM AITC was used as a positive control for Trpa1 activation. Bar graphs represent n = 50 – 200 cells from 1 – 4 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and A-967079-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p
    Figure Legend Snippet: RNA activates RIN14B cells independently of Trpa1 (A) Calcium imaging of ssRNA40 and ssRNA41 responses in RIN14B cells loaded with Fluo-4 AM, representative of ≥ 3 independent recordings for each condition. Cells were stimulated with 25 µg/mL ssRNA40 or ssRNA41 and 10 µM ionomycin. The mean Δ F/F 0 ± 95% CI is shown for a single recording each of n = 50 cells. (B) To block Trpa1, 10 µM A-967079 was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. 10 µM AITC was used as a positive control for Trpa1 activation. Bar graphs represent n = 50 – 200 cells from 1 – 4 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and A-967079-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p

    Techniques Used: Imaging, Blocking Assay, Incubation, Positive Control, Activation Assay, Fluorescence

    related to Figure 3 Fecal and dietary extracts activate HEK293 Piezo1-KO cells (A) FLIPR assays on HEK293 Piezo1-KO cells, with or without transfection of human Piezo1 . Each treatment condition was followed up with ionomycin to elicit maximum response for normalization (not shown). Treatment concentrations are 5 mg/mL fecal or dietary extract, 5 µM Yoda1, and 10 µM ionomycin. n = 4 wells per condition plotted as mean ± SEM. (B) Quantification of FLIPR calcium recordings for different treatments. n = 4 wells per condition plotted as mean ± SEM. Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, ** p
    Figure Legend Snippet: related to Figure 3 Fecal and dietary extracts activate HEK293 Piezo1-KO cells (A) FLIPR assays on HEK293 Piezo1-KO cells, with or without transfection of human Piezo1 . Each treatment condition was followed up with ionomycin to elicit maximum response for normalization (not shown). Treatment concentrations are 5 mg/mL fecal or dietary extract, 5 µM Yoda1, and 10 µM ionomycin. n = 4 wells per condition plotted as mean ± SEM. (B) Quantification of FLIPR calcium recordings for different treatments. n = 4 wells per condition plotted as mean ± SEM. Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, ** p

    Techniques Used: Transfection

    RNA activates RIN14B cells independently of Piezo1 (A – C) Calcium imaging of RIN14B cell activity during application of negative control vehicle with and without gadolinium inhibition of Piezo1. Gadolinium visibly reduced spontaneous calcium transients. (D – F) RIN14B cell calcium influx in response to fecal RNA, with and without gadolinium. (G – I) RIN14B cell calcium influx in response to the positive control Piezo1 agonist Yoda1, which is blocked by gadolinium. The calcium imaging was performed on GCaMP6s-transfected cells. GCaMP6s calcium responses were measured during stimulation with 25 µg/mL fecal RNA, 15 µM Yoda1, and 10 µM ionomycin. To block Piezo1, 30 µM gadolinium was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. Line graphs represent mean ± 95% CI of a single recording each of n = 50 cells. Bar graphs represent n = 100 – 150 cells from ≥ 2 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and gadolinium (Gd3+)-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: * p
    Figure Legend Snippet: RNA activates RIN14B cells independently of Piezo1 (A – C) Calcium imaging of RIN14B cell activity during application of negative control vehicle with and without gadolinium inhibition of Piezo1. Gadolinium visibly reduced spontaneous calcium transients. (D – F) RIN14B cell calcium influx in response to fecal RNA, with and without gadolinium. (G – I) RIN14B cell calcium influx in response to the positive control Piezo1 agonist Yoda1, which is blocked by gadolinium. The calcium imaging was performed on GCaMP6s-transfected cells. GCaMP6s calcium responses were measured during stimulation with 25 µg/mL fecal RNA, 15 µM Yoda1, and 10 µM ionomycin. To block Piezo1, 30 µM gadolinium was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. Line graphs represent mean ± 95% CI of a single recording each of n = 50 cells. Bar graphs represent n = 100 – 150 cells from ≥ 2 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and gadolinium (Gd3+)-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: * p

    Techniques Used: Imaging, Activity Assay, Negative Control, Inhibition, Positive Control, Transfection, Blocking Assay, Incubation, Fluorescence

    ssRNA40 does not alter calcium activity or mechanotransduction in N2a cells (A) Fluo-4 calcium imaging of N2a cells during exposure to different treatments, representative of ≥ 3 independent recordings for each condition. The magnitude of the change in fluorescence (Δ F ) is represented on a fire color scale and is superimposed on a grayscale baseline fluorescence image. Cells were exposed to buffer only (vehicle) or 10 µg/mL ssRNA40 or ssRNA41 for up to 3 minutes, followed by 30 µM Yoda1 and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of ssRNA40 and ssRNA41, each followed by Yoda1 and ionomycin control treatments. Only cells that responded to Yoda1 (functionally expressing Piezo1) were analyzed. Fluorescence values are shown as Δ F normalized to the initial fluorescence (Δ F / F 0 ). n = 50 cells plotted as mean ± 95% confidence interval (CI). (C) Quantification of calcium responses to different treatments. n = 50 cells per condition. Error bars indicate mean ± 95% CI. One-way ANOVA with Bonferroni correction: not significant (n.s.) p ≥ 0.05, **** p
    Figure Legend Snippet: ssRNA40 does not alter calcium activity or mechanotransduction in N2a cells (A) Fluo-4 calcium imaging of N2a cells during exposure to different treatments, representative of ≥ 3 independent recordings for each condition. The magnitude of the change in fluorescence (Δ F ) is represented on a fire color scale and is superimposed on a grayscale baseline fluorescence image. Cells were exposed to buffer only (vehicle) or 10 µg/mL ssRNA40 or ssRNA41 for up to 3 minutes, followed by 30 µM Yoda1 and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of ssRNA40 and ssRNA41, each followed by Yoda1 and ionomycin control treatments. Only cells that responded to Yoda1 (functionally expressing Piezo1) were analyzed. Fluorescence values are shown as Δ F normalized to the initial fluorescence (Δ F / F 0 ). n = 50 cells plotted as mean ± 95% confidence interval (CI). (C) Quantification of calcium responses to different treatments. n = 50 cells per condition. Error bars indicate mean ± 95% CI. One-way ANOVA with Bonferroni correction: not significant (n.s.) p ≥ 0.05, **** p

    Techniques Used: Activity Assay, Imaging, Fluorescence, Expressing

    Fecal and dietary extracts induce cell line-specific activity independently of Piezo1 (A) Calcium imaging of N2a Piezo1-KO cells and HEK293 cells, with or without Piezo1 transfection, representative of ≥ 2 independent recordings for each condition. Fluo-4 or GCaMP6s were used to image the N2a cells or HEK293 cells, respectively. Treatment concentrations are 5 mg/mL fecal or dietary extract, 30 µM Yoda1, or 10 µM ionomycin. Scale bar is 200 µm. (B) Quantification of calcium responses. n = 25 cells per condition plotted as mean ± 95% CI. Pairwise comparisons between untransfected and transfected recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p
    Figure Legend Snippet: Fecal and dietary extracts induce cell line-specific activity independently of Piezo1 (A) Calcium imaging of N2a Piezo1-KO cells and HEK293 cells, with or without Piezo1 transfection, representative of ≥ 2 independent recordings for each condition. Fluo-4 or GCaMP6s were used to image the N2a cells or HEK293 cells, respectively. Treatment concentrations are 5 mg/mL fecal or dietary extract, 30 µM Yoda1, or 10 µM ionomycin. Scale bar is 200 µm. (B) Quantification of calcium responses. n = 25 cells per condition plotted as mean ± 95% CI. Pairwise comparisons between untransfected and transfected recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p

    Techniques Used: Activity Assay, Imaging, Transfection

    3) Product Images from "Functional inference of gene regulation using single-cell multi-omics"

    Article Title: Functional inference of gene regulation using single-cell multi-omics

    Journal: Cell genomics

    doi: 10.1016/j.xgen.2022.100166

    High-throughput single-cell epigenomic and transcriptional profiling of resting and stimulated human blood cells (A) Schematic highlighting design of stimulation experiment. Human peripheral blood mononuclear cells (PBMCs) were stimulated with DMSO control, lipopolysaccharide (LPS), interferon gamma (IFN-Ɣ), or phorbol myristate acetate (PMA) plus ionomycin for 1 or 6 h with or without a Golgi inhibitor (GI) for the 6-h treatment condition. Cells were then split and profiled using scATAC-seq and scRNA-seq for each condition and time point considered. (B) Total number of cells profiled per condition passing quality control filtering for scATAC and scRNA-seq. (C) Uniform manifold approximation and projection (UMAP) of scATAC-seq cells based on latent semantic indexing (LSI) dimensionality reduction, with cells colored by treatment condition. (D) UMAP of scRNA-seq cells based on principal-component analysis (PCA) dimensionality reduction, with cells colored by treatment condition. (E) UMAPs of scATAC-seq cells (top) and scRNA-seq cells (bottom), highlighting individual conditions under control (6 h) and PMA (1 and 6 h) conditions. (F) Aggregate accessibility profiles for scATAC-seq monocyte cells around genes IFITM3 and HES4 . (G) Distribution of single-cell expression levels based on the imputed scRNA-seq counts for stimulation-specific gene markers shown in (F) per condition for scRNA-seq monocyte cells.
    Figure Legend Snippet: High-throughput single-cell epigenomic and transcriptional profiling of resting and stimulated human blood cells (A) Schematic highlighting design of stimulation experiment. Human peripheral blood mononuclear cells (PBMCs) were stimulated with DMSO control, lipopolysaccharide (LPS), interferon gamma (IFN-Ɣ), or phorbol myristate acetate (PMA) plus ionomycin for 1 or 6 h with or without a Golgi inhibitor (GI) for the 6-h treatment condition. Cells were then split and profiled using scATAC-seq and scRNA-seq for each condition and time point considered. (B) Total number of cells profiled per condition passing quality control filtering for scATAC and scRNA-seq. (C) Uniform manifold approximation and projection (UMAP) of scATAC-seq cells based on latent semantic indexing (LSI) dimensionality reduction, with cells colored by treatment condition. (D) UMAP of scRNA-seq cells based on principal-component analysis (PCA) dimensionality reduction, with cells colored by treatment condition. (E) UMAPs of scATAC-seq cells (top) and scRNA-seq cells (bottom), highlighting individual conditions under control (6 h) and PMA (1 and 6 h) conditions. (F) Aggregate accessibility profiles for scATAC-seq monocyte cells around genes IFITM3 and HES4 . (G) Distribution of single-cell expression levels based on the imputed scRNA-seq counts for stimulation-specific gene markers shown in (F) per condition for scRNA-seq monocyte cells.

    Techniques Used: High Throughput Screening Assay, Expressing

    4) Product Images from "DLL4 and VCAM1 enhance the emergence of T cell–competent hematopoietic progenitors from human pluripotent stem cells"

    Article Title: DLL4 and VCAM1 enhance the emergence of T cell–competent hematopoietic progenitors from human pluripotent stem cells

    Journal: Science Advances

    doi: 10.1126/sciadv.abn5522

    PSC-derived T cells are functional and display a diverse TCR repertoire. ( A ) Schematic overview and timeline of experiments to assess T cell phenotype and function. ( B ) Flow cytometry analysis of mature, PSC-derived T cells followed CD8SP maturation. Plots are representative of n = 3 differentiation replicates. ( C ) High-throughput sequencing of TCR β chains from PSC-derived T cell differentiation cultures ( n = 3 independent differentiation wells). Data for UCB, peripheral blood–derived T cells, and primary thymus were generated and first presented by Edgar et al. ( 61 ) and shown again here for comparison to the PSC-derived cell sequencing, which we report here for the first time. Error bars, ±SD. ( D ) Left: Flow cytometry analysis of Vδ1, Vδ2, and Vγ9 expression of TCRγδ T cells differentiated in vitro from UCB or PSCs. Right: Flow cytometry quantification of CD3 − , CD56 + cells from PSC-derived cells at day 28 of in vitro T cell maturation. Plots are representative of n = 3 differentiation replicates. ( E ) Expansion of sorted CD3 + , TCRαβ + PSC-derived T cells. Two expansion phases were carried out. Each expansion phase begins with 24-hour stimulation with immobilized αCD3 and soluble αCD28 antibodies on RetroNectin-coated plates in the presence of IL-12, IL-15, IL-7, IL-18, and IL-21. After 24 hours, cells were transferred to fresh RetroNectin-coated plates and cultured for 9 days in the presence of IL-15 and IL-7, with the remaining cytokines and αCD3 αCD28 antibodies removed. ( F ) Following 7 days of expansion, cells were assayed for production of effector proteins in the presence or absence of nonspecific PMA/ionomycin stimulation. Intracellular flow cytometry is representative of three stimulation replicates. ( G ) Enzyme-linked immunosorbent assay (ELISA) analysis of the growth medium from cells stimulated with PMA/ionomycin and unstimulated controls. Mean ± SD, n = 3 replicates, P values from unpaired t tests.
    Figure Legend Snippet: PSC-derived T cells are functional and display a diverse TCR repertoire. ( A ) Schematic overview and timeline of experiments to assess T cell phenotype and function. ( B ) Flow cytometry analysis of mature, PSC-derived T cells followed CD8SP maturation. Plots are representative of n = 3 differentiation replicates. ( C ) High-throughput sequencing of TCR β chains from PSC-derived T cell differentiation cultures ( n = 3 independent differentiation wells). Data for UCB, peripheral blood–derived T cells, and primary thymus were generated and first presented by Edgar et al. ( 61 ) and shown again here for comparison to the PSC-derived cell sequencing, which we report here for the first time. Error bars, ±SD. ( D ) Left: Flow cytometry analysis of Vδ1, Vδ2, and Vγ9 expression of TCRγδ T cells differentiated in vitro from UCB or PSCs. Right: Flow cytometry quantification of CD3 − , CD56 + cells from PSC-derived cells at day 28 of in vitro T cell maturation. Plots are representative of n = 3 differentiation replicates. ( E ) Expansion of sorted CD3 + , TCRαβ + PSC-derived T cells. Two expansion phases were carried out. Each expansion phase begins with 24-hour stimulation with immobilized αCD3 and soluble αCD28 antibodies on RetroNectin-coated plates in the presence of IL-12, IL-15, IL-7, IL-18, and IL-21. After 24 hours, cells were transferred to fresh RetroNectin-coated plates and cultured for 9 days in the presence of IL-15 and IL-7, with the remaining cytokines and αCD3 αCD28 antibodies removed. ( F ) Following 7 days of expansion, cells were assayed for production of effector proteins in the presence or absence of nonspecific PMA/ionomycin stimulation. Intracellular flow cytometry is representative of three stimulation replicates. ( G ) Enzyme-linked immunosorbent assay (ELISA) analysis of the growth medium from cells stimulated with PMA/ionomycin and unstimulated controls. Mean ± SD, n = 3 replicates, P values from unpaired t tests.

    Techniques Used: Derivative Assay, Functional Assay, Flow Cytometry, Next-Generation Sequencing, Cell Differentiation, Generated, Sequencing, Expressing, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Gut microbiota-derived metabolites confer protection against SARS-CoV-2 infection"

    Article Title: Gut microbiota-derived metabolites confer protection against SARS-CoV-2 infection

    Journal: Gut Microbes

    doi: 10.1080/19490976.2022.2105609

    SCFAs enhance adaptive immunity against rVSV/Spikeβ-GFP via GPR41/GPR43 in a sex-dependent manner. (A–B) hACE2 mice were given control or SCFA water for two weeks before intranasal inoculation with replication-competent rVSV/Spike-nLuc. 48h later, (A) viral burdens were determined by measuring luciferase activity; and (B) lung T cells were stimulated with PMA/ionomycin for two hours before flow cytometry analysis. (C–K) The impact of SCFA treatment on adaptive immunity to rVSV/Spikeβ-GFP. (C,G) Overview of experiment. hACE2 (C) or Gpr41 −/− Gpr43 −/− (G) mice were given control or SCFA water for two weeks before intranasal infection with 6x10 4 IU of replication-competent rVSV/Spikeβ-GFP. Blood was collected two and four weeks post infection for analysis of immune cells. Four weeks post infection, mice were reinfected with 5x10 5 IU of rVSV/Spikeβ-GFP and sacrificed after 3 d. (D–E, H–I) Two weeks following the primary infection, (D, H) blood immune cells were analyzed via flow cytometry, and purified IgG (E) or plasma (i) preincubated with rVSV/Spikeβ-GFP before infecting Vero-TMPRSS2 cells. 24 hours later, GFP+ cells were measured via flow cytometry. Results from only the male mice are shown. Data are shown as percentage of the no IgG/plasma condition. (F, J-K) Lung B and T cells ( J ) and GFP+ cells (F, K) in the lung 72 hours after the secondary infection. Error bars indicate mean±SEM. For (E) and (I), significance was determined using one-way ANOVA with Tukey’s test for multiple comparisons; for all other panels, significance was determined using unpaired t-test. All data represent 2 independent experiments. * p
    Figure Legend Snippet: SCFAs enhance adaptive immunity against rVSV/Spikeβ-GFP via GPR41/GPR43 in a sex-dependent manner. (A–B) hACE2 mice were given control or SCFA water for two weeks before intranasal inoculation with replication-competent rVSV/Spike-nLuc. 48h later, (A) viral burdens were determined by measuring luciferase activity; and (B) lung T cells were stimulated with PMA/ionomycin for two hours before flow cytometry analysis. (C–K) The impact of SCFA treatment on adaptive immunity to rVSV/Spikeβ-GFP. (C,G) Overview of experiment. hACE2 (C) or Gpr41 −/− Gpr43 −/− (G) mice were given control or SCFA water for two weeks before intranasal infection with 6x10 4 IU of replication-competent rVSV/Spikeβ-GFP. Blood was collected two and four weeks post infection for analysis of immune cells. Four weeks post infection, mice were reinfected with 5x10 5 IU of rVSV/Spikeβ-GFP and sacrificed after 3 d. (D–E, H–I) Two weeks following the primary infection, (D, H) blood immune cells were analyzed via flow cytometry, and purified IgG (E) or plasma (i) preincubated with rVSV/Spikeβ-GFP before infecting Vero-TMPRSS2 cells. 24 hours later, GFP+ cells were measured via flow cytometry. Results from only the male mice are shown. Data are shown as percentage of the no IgG/plasma condition. (F, J-K) Lung B and T cells ( J ) and GFP+ cells (F, K) in the lung 72 hours after the secondary infection. Error bars indicate mean±SEM. For (E) and (I), significance was determined using one-way ANOVA with Tukey’s test for multiple comparisons; for all other panels, significance was determined using unpaired t-test. All data represent 2 independent experiments. * p

    Techniques Used: Mouse Assay, Luciferase, Activity Assay, Flow Cytometry, Infection, Purification

    6) Product Images from "Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division"

    Article Title: Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E22-01-0005

    Enrichment of endogenous NMII heavy chains to CIA network. Immunofluorescence microscopy of U2OS cells that were first permeabilized for 1 min in the presence of TRITC-phalloidin to stabilize actin filaments and then fixed with PFA followed by anti-NMII staining. For each NMII, an example of control treatment (DMSO) and ionomycin treatment (45 s) is shown. Insets emphasize CIA network near the nucleus, where it is most prominent and where other actin-based structures are sparse. Scale bar is 10 µm in overview images and 3 µm in insets.
    Figure Legend Snippet: Enrichment of endogenous NMII heavy chains to CIA network. Immunofluorescence microscopy of U2OS cells that were first permeabilized for 1 min in the presence of TRITC-phalloidin to stabilize actin filaments and then fixed with PFA followed by anti-NMII staining. For each NMII, an example of control treatment (DMSO) and ionomycin treatment (45 s) is shown. Insets emphasize CIA network near the nucleus, where it is most prominent and where other actin-based structures are sparse. Scale bar is 10 µm in overview images and 3 µm in insets.

    Techniques Used: Immunofluorescence, Microscopy, Staining

    NMII deletion reduces the intensity of the actin network in CIA. (A) Fixed-cell imaging of WT and NMII KO U2OS cell lines for actin filaments (TRITC-phalloidin) in either unstimulated (DMSO) or ionomycin-stimulated (45 s) state, with zooms to the right denoting boxed regions of full-field views. INF2 KD cells also shown. Bar is 20 µm in overview images and 3 µm in insets. (B) Plot of actin filament intensity from fixed-cell images as in panel A. Western blot at right shows efficiency of INF2 KD. Unpaired t test: p values
    Figure Legend Snippet: NMII deletion reduces the intensity of the actin network in CIA. (A) Fixed-cell imaging of WT and NMII KO U2OS cell lines for actin filaments (TRITC-phalloidin) in either unstimulated (DMSO) or ionomycin-stimulated (45 s) state, with zooms to the right denoting boxed regions of full-field views. INF2 KD cells also shown. Bar is 20 µm in overview images and 3 µm in insets. (B) Plot of actin filament intensity from fixed-cell images as in panel A. Western blot at right shows efficiency of INF2 KD. Unpaired t test: p values

    Techniques Used: Imaging, Western Blot

    Increased cytoplasmic calcium triggers rapid myosin II activation. (A) Time-lapse montage of U2OS cell transfected with GFP-F-tractin and stimulated with ionomycin for the indicated times (seconds). Medial cell section imaged. Asterisks highlight the cytoplasmic CIA network. Bar is 10 µm. (B) Western blots of U2OS cell extracts after the indicated times of ionomycin treatment. (C) Plot of time course for changes in cytoplasmic actin polymerization as well as phosphorylated MRLC and total MRLC changes after ionomycin addition in U2OS cells. Error bars are SEM. (D) Images of fixed U2OS cells stained for phosphorylated MRLC, actin filaments (TRITC-phalloidin), and mitochondria (Tom20) after 45 s stimulation with DMSO (top) or ionomycin (bottom). Images at right are color merged zooms of boxed regions. Bar is 10 µm in overview images and 3 µm in zooms. (E) Western blots of U2OS cell extracts after the indicated times of histamine treatment.
    Figure Legend Snippet: Increased cytoplasmic calcium triggers rapid myosin II activation. (A) Time-lapse montage of U2OS cell transfected with GFP-F-tractin and stimulated with ionomycin for the indicated times (seconds). Medial cell section imaged. Asterisks highlight the cytoplasmic CIA network. Bar is 10 µm. (B) Western blots of U2OS cell extracts after the indicated times of ionomycin treatment. (C) Plot of time course for changes in cytoplasmic actin polymerization as well as phosphorylated MRLC and total MRLC changes after ionomycin addition in U2OS cells. Error bars are SEM. (D) Images of fixed U2OS cells stained for phosphorylated MRLC, actin filaments (TRITC-phalloidin), and mitochondria (Tom20) after 45 s stimulation with DMSO (top) or ionomycin (bottom). Images at right are color merged zooms of boxed regions. Bar is 10 µm in overview images and 3 µm in zooms. (E) Western blots of U2OS cell extracts after the indicated times of histamine treatment.

    Techniques Used: Activation Assay, Transfection, Western Blot, Staining

    NMII proteins incorporate into the CIA network. (A) Western blots of actin from experiments in which U2OS cells were stimulated with either DMSO or ionomycin for 45 s, extracted with nonionic detergent, and then separated into supernatant and pellet fractions by ultracentrifugation. For the LatA sample, cells were pretreated with 2 µM LatA 20 min before ionomycin treatment. (B) Ratio of actin in the pellet vs. supernatant for experiments in panel A (four replicates). Unpaired t test with p values > 0.05 considered not significant (n.s.), p values for ionomycin-treated INF2 KO = 0.0038 (**) and for LatA = 0.0005 (***). (C) Numerical data for panel B. n.d., not determined. (D) Western blots of several proteins in assays described in panel A, to determine whether their distribution changes upon ionomycin stimulation. Actin shown as a control for each protein (from the same blot as the protein of interest).
    Figure Legend Snippet: NMII proteins incorporate into the CIA network. (A) Western blots of actin from experiments in which U2OS cells were stimulated with either DMSO or ionomycin for 45 s, extracted with nonionic detergent, and then separated into supernatant and pellet fractions by ultracentrifugation. For the LatA sample, cells were pretreated with 2 µM LatA 20 min before ionomycin treatment. (B) Ratio of actin in the pellet vs. supernatant for experiments in panel A (four replicates). Unpaired t test with p values > 0.05 considered not significant (n.s.), p values for ionomycin-treated INF2 KO = 0.0038 (**) and for LatA = 0.0005 (***). (C) Numerical data for panel B. n.d., not determined. (D) Western blots of several proteins in assays described in panel A, to determine whether their distribution changes upon ionomycin stimulation. Actin shown as a control for each protein (from the same blot as the protein of interest).

    Techniques Used: Western Blot

    NMII KO inhibits CIA-stimulated mitochondrial calcium influx. (A) Live-cell imaging of WT or NMII KO U2OS cell lines transfected with the mito-R-GECO probe to monitor mitochondrial calcium levels. One time point before stimulation (pre) and three time points (in seconds) after ionomycin stimulation are shown. Bar is 10 µm. (B) Plot of mitochondrial calcium changes upon ionomycin stimulation for the indicated cell lines. Error bars are SEM. (C) Plot of maximal change in mitochondrial calcium levels from the data in panel B. Unpaired t test with p values
    Figure Legend Snippet: NMII KO inhibits CIA-stimulated mitochondrial calcium influx. (A) Live-cell imaging of WT or NMII KO U2OS cell lines transfected with the mito-R-GECO probe to monitor mitochondrial calcium levels. One time point before stimulation (pre) and three time points (in seconds) after ionomycin stimulation are shown. Bar is 10 µm. (B) Plot of mitochondrial calcium changes upon ionomycin stimulation for the indicated cell lines. Error bars are SEM. (C) Plot of maximal change in mitochondrial calcium levels from the data in panel B. Unpaired t test with p values

    Techniques Used: Live Cell Imaging, Transfection

    7) Product Images from "The transcriptional regulator Sin3A balances IL-17A and Foxp3 expression in primary CD4 T cells"

    Article Title: The transcriptional regulator Sin3A balances IL-17A and Foxp3 expression in primary CD4 T cells

    Journal: bioRxiv

    doi: 10.1101/2022.04.19.488789

    Sin3A inactivation favors IL-2 upregulation, which neutralization halts Foxp3 and rescues IL-17A expression. A-B) Cells were cultured as depi cted in Figure 2 and then stimulated with PMA and Ionomycin for 4h. A . Representative contour plots and B. percentages of IL-2 + cells, N=7. C. RT-PCR analysis of Il2 expression, N=7. D-E ) Cells were cultured as depicted in Figure 2 in the absence or the presence of a neutralizing anti-IL-2 mAb. D. Bar plot depicting the frequency of Foxp3 + , N=5 and E. relative IL-17A + cells, N=10. F. Relative increase in IL-17A + Sin3A sufficient and deficient cells with respect to the dose of anti-IL-2 mAb, N=3. G . Correlation of the relative increase in IL-17A + cells in the presence of anti-IL-2 antibody and the percentage of IL-2 + cells in TAM-treated cells. Bars: Mean. Error bars: SD. Two-tailed paired Student’s T-test.
    Figure Legend Snippet: Sin3A inactivation favors IL-2 upregulation, which neutralization halts Foxp3 and rescues IL-17A expression. A-B) Cells were cultured as depi cted in Figure 2 and then stimulated with PMA and Ionomycin for 4h. A . Representative contour plots and B. percentages of IL-2 + cells, N=7. C. RT-PCR analysis of Il2 expression, N=7. D-E ) Cells were cultured as depicted in Figure 2 in the absence or the presence of a neutralizing anti-IL-2 mAb. D. Bar plot depicting the frequency of Foxp3 + , N=5 and E. relative IL-17A + cells, N=10. F. Relative increase in IL-17A + Sin3A sufficient and deficient cells with respect to the dose of anti-IL-2 mAb, N=3. G . Correlation of the relative increase in IL-17A + cells in the presence of anti-IL-2 antibody and the percentage of IL-2 + cells in TAM-treated cells. Bars: Mean. Error bars: SD. Two-tailed paired Student’s T-test.

    Techniques Used: Neutralization, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    STAT3 and RORγt are expressed in Sin3A-deficient cells lacking IL17A. A . Schematic representation of STAT3 and RORγt-driven events over Th17 differentiation. B-E. Representative western blot of total ( B ) and nuclear ( C ) STAT3 and pSTAT3 (ser727) in control (NT) and Sin3A-deleted Th17-skewed CD4 + T cells (TAM), and quantification of relative representation over independent experiments ( D-E ), N=5. Bars: Mean. Error bars: SD.Two-tailed paired Student’s T-test, except in C. pSTAT3/β-ACTIN: Wilcoxon matched-pairs signed rank test (not normally distributed according to Shapiro-Wilk test). F . Contour plots depicting IL-17A and RORγt levels in viable PMA and Ionomycin stimulated control (NT) and TAM-treated CD4+ T cells. G. Representative histograms of RORγt protein levels and H. relative expression (Relative Fluorescence Intensity, TAM relative to NT), N=8. I. RT-PCR analysis of Rorc (N=6) and Rora (N=7) gene levels. Relative expression is shown.
    Figure Legend Snippet: STAT3 and RORγt are expressed in Sin3A-deficient cells lacking IL17A. A . Schematic representation of STAT3 and RORγt-driven events over Th17 differentiation. B-E. Representative western blot of total ( B ) and nuclear ( C ) STAT3 and pSTAT3 (ser727) in control (NT) and Sin3A-deleted Th17-skewed CD4 + T cells (TAM), and quantification of relative representation over independent experiments ( D-E ), N=5. Bars: Mean. Error bars: SD.Two-tailed paired Student’s T-test, except in C. pSTAT3/β-ACTIN: Wilcoxon matched-pairs signed rank test (not normally distributed according to Shapiro-Wilk test). F . Contour plots depicting IL-17A and RORγt levels in viable PMA and Ionomycin stimulated control (NT) and TAM-treated CD4+ T cells. G. Representative histograms of RORγt protein levels and H. relative expression (Relative Fluorescence Intensity, TAM relative to NT), N=8. I. RT-PCR analysis of Rorc (N=6) and Rora (N=7) gene levels. Relative expression is shown.

    Techniques Used: Western Blot, Two Tailed Test, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction

    Sin3A deficient T cells fail to differentiate in Th17 skewing conditions. Sin3A F/F Rosa26-CreER T2 CD4 T cells treated or not with TAM were kept for 3 days in Th17-skewing conditions as described in Figure 2 . Cells were then left untreated or stimulated with PMA/ionomycin (PI) for 4h, and analyzed by intracellular cytokine staining ( A-B ) or RT-PCR ( C-D ). A. Representative dot plots and B. bar plots showing the percentage of cells expressing IL-17A + and IFNγ + (N=5). C-D . RT-PCR analysis of indicated gene expression after PI stimulation. Relative levels are depicted: IL17a, ifng , Il17f, Il21 (N=7), Il22 (N=4). Il23r levels were analyzed without re-stimulation (N=7). Bars: Mean. Error bars: SD. Two-tailed paired Student’s T-test.
    Figure Legend Snippet: Sin3A deficient T cells fail to differentiate in Th17 skewing conditions. Sin3A F/F Rosa26-CreER T2 CD4 T cells treated or not with TAM were kept for 3 days in Th17-skewing conditions as described in Figure 2 . Cells were then left untreated or stimulated with PMA/ionomycin (PI) for 4h, and analyzed by intracellular cytokine staining ( A-B ) or RT-PCR ( C-D ). A. Representative dot plots and B. bar plots showing the percentage of cells expressing IL-17A + and IFNγ + (N=5). C-D . RT-PCR analysis of indicated gene expression after PI stimulation. Relative levels are depicted: IL17a, ifng , Il17f, Il21 (N=7), Il22 (N=4). Il23r levels were analyzed without re-stimulation (N=7). Bars: Mean. Error bars: SD. Two-tailed paired Student’s T-test.

    Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test

    Sin3A inactivation leads to the upregulation of Foxp3, able to limit IL-17A. Cells were cultured as depicted in Figure 2 and analyzed after PMA and Ionomycin stimulation for 4h. A. Representative contour plots and B. percentages of IL-17A + and Foxp3 + cells, N=10. C . RT-PCR analysis of Foxp3 expression N=7. D . Cells were cultured as depicted in Figure 2 in the absence or the presence of the P60 Foxp3 inhibitor. Cells were then harvested and stimulated with PMA and ionomycin for 4h. Bar plots report the percentage of IL-17A+ cells relative to untreated controls, N=6. Error bars: SD. Two-tailed paired Student’s T-test.
    Figure Legend Snippet: Sin3A inactivation leads to the upregulation of Foxp3, able to limit IL-17A. Cells were cultured as depicted in Figure 2 and analyzed after PMA and Ionomycin stimulation for 4h. A. Representative contour plots and B. percentages of IL-17A + and Foxp3 + cells, N=10. C . RT-PCR analysis of Foxp3 expression N=7. D . Cells were cultured as depicted in Figure 2 in the absence or the presence of the P60 Foxp3 inhibitor. Cells were then harvested and stimulated with PMA and ionomycin for 4h. Bar plots report the percentage of IL-17A+ cells relative to untreated controls, N=6. Error bars: SD. Two-tailed paired Student’s T-test.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test

    8) Product Images from "Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair"

    Article Title: Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair

    Journal: Nature Communications

    doi: 10.1038/s41467-022-29481-4

    Damage-induced SM translocation is mediated by a calcium-activated lipid scramblase. a Time-lapse images of wild-type (WT) or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 1500 U/ml SLO for the indicated time in medium containing (+Ca 2+ ) or lacking Ca 2+ (–Ca 2+ / +EGTA). b Percentage of cells displaying EqtSM-positive puncta after 30 min of SLO treatment as in ( a ). Data are means ± SD. n = 29 cells for +Ca 2+ , 35 cells for –Ca 2+ /+EGTA, 35 cells for WT, 32 cells for TMEM16F-KO over three independent experiments. P values were calculated by unpaired two-tailed t test. c Time-lapse images of WT or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 5 μM ionomycin for the indicated time in medium containing (+Ca 2+ ) or lacking Ca 2+ (–Ca 2+ /+EGTA). d Percentage of cells displaying EqtSM-positive puncta after 30 min of ionomycin treatment as in ( a ). Data are means ± SD. n = 41 cells for +Ca 2+ , 25 cells for –Ca 2+ /+EGTA, 41 cells for WT, 41 cells for TMEM16F-KO over three independent experiments. P values were calculated by unpaired two-tailed t test. e Time-lapse images of WT or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 1 mM LLOMe after preincubation with or without BAPTA-AM (100 μM, 45 min). f Time-course plotting EqtSM-positive puncta per 100 μm 2 cell area in wild-type (WT) cells treated as in ( e ). Data are means ± SD. n = 24 cells for WT –BAPTA, 21 cells for WT + BAPTA over three independent experiments. P values were calculated by unpaired two-tailed t test. g Time-course plotting EqtSM-positive puncta per 100 μm 2 cell area in wild-type or TMEM16F-KO HeLa cells treated with LLOMe in the absence of BAPTA-AM as in ( f ). Data are means ± SD. n = 25 cells for WT, 15 cells for TMEM16F-KO over three independent experiments. h Schematic illustration of how membrane damage triggers SM scrambling. PM plasma membrane, CaPLSase calcium-activated phospholipid scramblase. Scale bar, 10 µm. Source data including exact P values are provided as a Source Data file.
    Figure Legend Snippet: Damage-induced SM translocation is mediated by a calcium-activated lipid scramblase. a Time-lapse images of wild-type (WT) or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 1500 U/ml SLO for the indicated time in medium containing (+Ca 2+ ) or lacking Ca 2+ (–Ca 2+ / +EGTA). b Percentage of cells displaying EqtSM-positive puncta after 30 min of SLO treatment as in ( a ). Data are means ± SD. n = 29 cells for +Ca 2+ , 35 cells for –Ca 2+ /+EGTA, 35 cells for WT, 32 cells for TMEM16F-KO over three independent experiments. P values were calculated by unpaired two-tailed t test. c Time-lapse images of WT or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 5 μM ionomycin for the indicated time in medium containing (+Ca 2+ ) or lacking Ca 2+ (–Ca 2+ /+EGTA). d Percentage of cells displaying EqtSM-positive puncta after 30 min of ionomycin treatment as in ( a ). Data are means ± SD. n = 41 cells for +Ca 2+ , 25 cells for –Ca 2+ /+EGTA, 41 cells for WT, 41 cells for TMEM16F-KO over three independent experiments. P values were calculated by unpaired two-tailed t test. e Time-lapse images of WT or TMEM16F-KO HeLa cells expressing GFP-tagged EqtSM and treated with 1 mM LLOMe after preincubation with or without BAPTA-AM (100 μM, 45 min). f Time-course plotting EqtSM-positive puncta per 100 μm 2 cell area in wild-type (WT) cells treated as in ( e ). Data are means ± SD. n = 24 cells for WT –BAPTA, 21 cells for WT + BAPTA over three independent experiments. P values were calculated by unpaired two-tailed t test. g Time-course plotting EqtSM-positive puncta per 100 μm 2 cell area in wild-type or TMEM16F-KO HeLa cells treated with LLOMe in the absence of BAPTA-AM as in ( f ). Data are means ± SD. n = 25 cells for WT, 15 cells for TMEM16F-KO over three independent experiments. h Schematic illustration of how membrane damage triggers SM scrambling. PM plasma membrane, CaPLSase calcium-activated phospholipid scramblase. Scale bar, 10 µm. Source data including exact P values are provided as a Source Data file.

    Techniques Used: Translocation Assay, Expressing, Two Tailed Test

    9) Product Images from "The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes"

    Article Title: The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2022.810611

    Effect of C2IIa, C2II and C2I on Ca (2+) mobilization and morphological changes in primary human PMNs. (A) Treatment with C2IIa induces rapid Ca 2+ influx into PMNs. Primary human PMNs were loaded with indo-1-AM. After pre-warming to 37°C, the cells were loaded into the flow cytometer and Ca 2+ baseline was determined for 2 min. Afterwards, the cells were treated for 4 min with C2IIa (5 μg/ml), C2II (5 μg/ml) or C2I (1 μg/ml) and subsequently for 3 min with ionomycin. The individual points of the curves correspond to time in full seconds versus the medians of all fluorescence ratios representing [Ca 2+ ] i , following their normalization according to the maximum values reached after addition of 2 μm ionomycin. Data is representative for three independent experiments. (B) Treatment with C2IIa but not with C2II induces changes in PMN morphology. Primary human PMNs were treated at 37°C with either C2IIa (5 μg/ml) or C2II (5 μg/ml) or without any protein as control (Ctrl). Pictures from the cells were taken after 1 h and quantitative analysis of morphological changes of PMNs was performed. Data show the results of 3 independent experiments, each with 3 technical replicates per condition. Values represent the mean ± SEM (n = 9). Significance was tested against the untreated control (Ctrl) using Kruskal–Wallis test with Dunn’s correction for multiple comparison (**** p
    Figure Legend Snippet: Effect of C2IIa, C2II and C2I on Ca (2+) mobilization and morphological changes in primary human PMNs. (A) Treatment with C2IIa induces rapid Ca 2+ influx into PMNs. Primary human PMNs were loaded with indo-1-AM. After pre-warming to 37°C, the cells were loaded into the flow cytometer and Ca 2+ baseline was determined for 2 min. Afterwards, the cells were treated for 4 min with C2IIa (5 μg/ml), C2II (5 μg/ml) or C2I (1 μg/ml) and subsequently for 3 min with ionomycin. The individual points of the curves correspond to time in full seconds versus the medians of all fluorescence ratios representing [Ca 2+ ] i , following their normalization according to the maximum values reached after addition of 2 μm ionomycin. Data is representative for three independent experiments. (B) Treatment with C2IIa but not with C2II induces changes in PMN morphology. Primary human PMNs were treated at 37°C with either C2IIa (5 μg/ml) or C2II (5 μg/ml) or without any protein as control (Ctrl). Pictures from the cells were taken after 1 h and quantitative analysis of morphological changes of PMNs was performed. Data show the results of 3 independent experiments, each with 3 technical replicates per condition. Values represent the mean ± SEM (n = 9). Significance was tested against the untreated control (Ctrl) using Kruskal–Wallis test with Dunn’s correction for multiple comparison (**** p

    Techniques Used: Flow Cytometry, Fluorescence

    10) Product Images from "Amphibian mucus triggers a developmental transition in the frog-killing chytrid fungus"

    Article Title: Amphibian mucus triggers a developmental transition in the frog-killing chytrid fungus

    Journal: bioRxiv

    doi: 10.1101/2022.01.21.477224

    Encystation is driven by calcium signaling, not protein translation. ( A ) Cells exposed to buffer (control) or 10 mg/mL mucin and DMSO or cycloheximide (CHX) stained for cell wall (calcofluor white), flagella (tubulin tracker), and actin (phalloidin). ( B ) Percent cells encysted after DMSO (left) or cycloheximide (CHX; right) treatment upon exposure to control buffer or mucin. ( C-D ) Percent encysted cells treated with the indicated (C) calcium chelator, (D) ionophore, or (C-D) DMSO carrier control five minutes after adding mucin or control buffer while adhered to a ConA coated surface. ( E ) Percent cells encysted after addition of control buffer, mucin, or ionomycin while adhered to a ConA coated surface or in suspension. Filled shapes represent mucin (blue), open shapes represent the control (gray) and ionomycin (green), and each shape corresponds to data from three independent biological replicates. Significance (bold font or p
    Figure Legend Snippet: Encystation is driven by calcium signaling, not protein translation. ( A ) Cells exposed to buffer (control) or 10 mg/mL mucin and DMSO or cycloheximide (CHX) stained for cell wall (calcofluor white), flagella (tubulin tracker), and actin (phalloidin). ( B ) Percent cells encysted after DMSO (left) or cycloheximide (CHX; right) treatment upon exposure to control buffer or mucin. ( C-D ) Percent encysted cells treated with the indicated (C) calcium chelator, (D) ionophore, or (C-D) DMSO carrier control five minutes after adding mucin or control buffer while adhered to a ConA coated surface. ( E ) Percent cells encysted after addition of control buffer, mucin, or ionomycin while adhered to a ConA coated surface or in suspension. Filled shapes represent mucin (blue), open shapes represent the control (gray) and ionomycin (green), and each shape corresponds to data from three independent biological replicates. Significance (bold font or p

    Techniques Used: Staining

    Calcium can induce encystation of Bd but not Sp . ( A ) Representative DIC microscopy of Bd zoospores grown for 72 hours in 1% tryptone media supplemented with cycloheximide (CHX). Note that control cells have developed into mature sporangia (no CHX, left), while cells treated with CHX have ceased development after encystation (middle, right) ( B ) The percentage of cells in original Bonner’s Salts, modified Bonner’s made with MgCl2 instead of CaCl2, and Bonner’s lacking both divalent cations (NDC) that encyst upon exposure to buffer alone (open shapes; gray) or 10 mg/mL mucin resuspended the same buffer (filled shapes; blue). This is a control experiment for the calcium chelator experiments shown in Fig. 2C . ( C ) The percentage of cells that encyst upon exposure to control buffer (circles), mucin (squares), DMSO (triangles), or ionomycin (diamonds) in Bd and Spizellomyces punctatus , Sp . (B-C) Three biological replicates with mean and standard error.
    Figure Legend Snippet: Calcium can induce encystation of Bd but not Sp . ( A ) Representative DIC microscopy of Bd zoospores grown for 72 hours in 1% tryptone media supplemented with cycloheximide (CHX). Note that control cells have developed into mature sporangia (no CHX, left), while cells treated with CHX have ceased development after encystation (middle, right) ( B ) The percentage of cells in original Bonner’s Salts, modified Bonner’s made with MgCl2 instead of CaCl2, and Bonner’s lacking both divalent cations (NDC) that encyst upon exposure to buffer alone (open shapes; gray) or 10 mg/mL mucin resuspended the same buffer (filled shapes; blue). This is a control experiment for the calcium chelator experiments shown in Fig. 2C . ( C ) The percentage of cells that encyst upon exposure to control buffer (circles), mucin (squares), DMSO (triangles), or ionomycin (diamonds) in Bd and Spizellomyces punctatus , Sp . (B-C) Three biological replicates with mean and standard error.

    Techniques Used: Microscopy, Modification

    11) Product Images from "DJ‐1 depletion prevents immunoaging in T‐cell compartments"

    Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

    Journal: EMBO Reports

    doi: 10.15252/embr.202153302

    Dj‐1 depletion also reduced signs of immunoaging in CD4 T‐cell compartments Expression level of CD31 among total blood or splenocyte CD4 T cells of 45‐week‐old (left) and young (right) mice. Representative flow‐cytometry plots of CD44 and CD62L expression on total CD4 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments; of note, more than one pLNs might be taken from several mice). Percentages of CD44 low CD62L high (Tn) (C) and CD44 high CD62L low (Tem) (D) cells among total CD4 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates. Representative histogram overlay of PD‐1 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD4 T cells (right panel). Representative histogram overlay of CTLA‐4 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of CTLA‐4 + cells among total CD4 T cells (right panel). Percentages of Ki‐67 + cells among total CD4 T cells. IFN‐γ production in CD4 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. The selected significantly enriched GO‐terms and pathways among the downregulated genes in CD4 Tconv cells from 45‐week‐old Dj‐1 KO mice versus the age‐ and gender‐matched WT littermates from microarray analysis (upper panel). Lower panel, volcano plot shows both downregulated and upregulated differentially expressed genes in splenic CD4 T cells from three 45‐week‐old Dj‐1 KO mice versus three age‐matched WT littermates. The dashed line in y axis corresponds to the value of 1.3 ( P = 0.05), while the two dashed lines in x ‐axis correspond to −1 and 1 (change fold = 2). A two‐tailed Student t ‐test was used to calculate the P values (for detailed microarray analysis method, refer to Materials and Methods). Comparison of naive CD4 (Tn) mitochondrial mass (mito mass, J) and membrane potential (mito potential, K) of young and 45‐week‐old Dj‐1 KO and WT mice. Comparison of CD4 Tem mitochondrial mass (mito mass, L) and membrane potential (mito potential, M) of young and 45‐week‐old Dj‐1 KO and WT mice. SP and LN represent spleen and lymph nodes, respectively. Data information: results represent at least four (B–G) and three (J–M) independent experiments. Data are mean of biological replicates ± SD. Each biological replicate indicates the measurement from one individual mouse. The P ‐values are determined by a two‐tailed un‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.
    Figure Legend Snippet: Dj‐1 depletion also reduced signs of immunoaging in CD4 T‐cell compartments Expression level of CD31 among total blood or splenocyte CD4 T cells of 45‐week‐old (left) and young (right) mice. Representative flow‐cytometry plots of CD44 and CD62L expression on total CD4 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments; of note, more than one pLNs might be taken from several mice). Percentages of CD44 low CD62L high (Tn) (C) and CD44 high CD62L low (Tem) (D) cells among total CD4 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates. Representative histogram overlay of PD‐1 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD4 T cells (right panel). Representative histogram overlay of CTLA‐4 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of CTLA‐4 + cells among total CD4 T cells (right panel). Percentages of Ki‐67 + cells among total CD4 T cells. IFN‐γ production in CD4 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. The selected significantly enriched GO‐terms and pathways among the downregulated genes in CD4 Tconv cells from 45‐week‐old Dj‐1 KO mice versus the age‐ and gender‐matched WT littermates from microarray analysis (upper panel). Lower panel, volcano plot shows both downregulated and upregulated differentially expressed genes in splenic CD4 T cells from three 45‐week‐old Dj‐1 KO mice versus three age‐matched WT littermates. The dashed line in y axis corresponds to the value of 1.3 ( P = 0.05), while the two dashed lines in x ‐axis correspond to −1 and 1 (change fold = 2). A two‐tailed Student t ‐test was used to calculate the P values (for detailed microarray analysis method, refer to Materials and Methods). Comparison of naive CD4 (Tn) mitochondrial mass (mito mass, J) and membrane potential (mito potential, K) of young and 45‐week‐old Dj‐1 KO and WT mice. Comparison of CD4 Tem mitochondrial mass (mito mass, L) and membrane potential (mito potential, M) of young and 45‐week‐old Dj‐1 KO and WT mice. SP and LN represent spleen and lymph nodes, respectively. Data information: results represent at least four (B–G) and three (J–M) independent experiments. Data are mean of biological replicates ± SD. Each biological replicate indicates the measurement from one individual mouse. The P ‐values are determined by a two‐tailed un‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Transmission Electron Microscopy, In Vitro, Microarray, Two Tailed Test

    Dj‐1 depletion reduces signs of immunoaging in murine CD8 T cells Representative flow‐cytometry plots of CD44 and CD62L expression on total CD8 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. Percentages of CD44 low CD62L high (Tn) (B) and CD44 high CD62L low (Tem) (C) cells among total CD8 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments). Representative histogram overlay of PD‐1 expression among total CD8 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD8 T cells (right panel). Percentages of Ki‐67 + cells among total CD8 T cells. Percentage of Ki‐67 + cells among splenic CD8 Tn, Tem, and Tcm in 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. Representative flow‐cytometry plots of Ki‐67 and PD‐1 among splenic CD8 Tem of 45‐week‐old mice. IFN‐γ production in CD8 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. Representative flow‐cytometry plots of CD44 and CD49d among blood CD8 T cells of 60‐week‐old mice. Percentages of Tn (top), Tvm (middle), and Tmem (bottom) among 60‐week‐old or young mice (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). Representative histogram of CD31 among blood CD8 T cells of 60‐week‐old mice. CD31 MFI of total CD8 T cells (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). Data information: SP and LN represent spleen and lymph nodes, respectively. Results represent at least four (B‐H) or two (I‐L) independent experiments. Data are mean of biological replicates ± SD. Each dot/symbol represents the measurement from one mouse. The P ‐values are determined by a two‐tailed non‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source data are available online for this figure.
    Figure Legend Snippet: Dj‐1 depletion reduces signs of immunoaging in murine CD8 T cells Representative flow‐cytometry plots of CD44 and CD62L expression on total CD8 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. Percentages of CD44 low CD62L high (Tn) (B) and CD44 high CD62L low (Tem) (C) cells among total CD8 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments). Representative histogram overlay of PD‐1 expression among total CD8 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD8 T cells (right panel). Percentages of Ki‐67 + cells among total CD8 T cells. Percentage of Ki‐67 + cells among splenic CD8 Tn, Tem, and Tcm in 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. Representative flow‐cytometry plots of Ki‐67 and PD‐1 among splenic CD8 Tem of 45‐week‐old mice. IFN‐γ production in CD8 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. Representative flow‐cytometry plots of CD44 and CD49d among blood CD8 T cells of 60‐week‐old mice. Percentages of Tn (top), Tvm (middle), and Tmem (bottom) among 60‐week‐old or young mice (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). Representative histogram of CD31 among blood CD8 T cells of 60‐week‐old mice. CD31 MFI of total CD8 T cells (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). Data information: SP and LN represent spleen and lymph nodes, respectively. Results represent at least four (B‐H) or two (I‐L) independent experiments. Data are mean of biological replicates ± SD. Each dot/symbol represents the measurement from one mouse. The P ‐values are determined by a two‐tailed non‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source data are available online for this figure.

    Techniques Used: Flow Cytometry, Expressing, Mouse Assay, Transmission Electron Microscopy, In Vitro, Two Tailed Test

    12) Product Images from "Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice"

    Article Title: Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27860-x

    Lysosomal exocytosis, pH and TRPML1 activity in WT and Trpml3 − / − AMΦ. a Lysosomal exocytosis experiments measuring hexosaminidase release from WT and Trpml3 − / − AMΦ. Maximum effects were obtained with ionomycin (4 µM). TRPML3 activator ML3-SA1 elicited no significant effects in both WT and Trpml3 − / − AMΦ. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. b Cartoon illustrating LAMP1 translocation assay shown in c , d . Upon lysosomal exocytosis the lysosomal protein LAMP1 is detected on the plasma membrane (PM) by anti-LAMP1 followed by Alexa Fluor 488-conjugated secondary antibody. c Representative images of LAMP1 translocation assay using WT and Trpml3 − / − AMΦ. Shown are results obtained after 120 min treatment with DMSO, ML3-SA1 (30 µM), or 10 min treatment with ionomycin (4 µM). Scale bar 10 µm. d Quantification of experiments as shown in c (mean ± SEM from 3 biologically independent experiments, each). e Fura-2 calcium imaging experiments using HEK293 cells expressing human or murine TRPML1(NC), TRPML2 or TRPML3, respectively, indicating the specific levels of activation. Channels were stimulated with either ML1-SA1 ( = EVP-169) or ML-SA1 (10 µM, each). Shown are average values (mean ± SEM). Each dot represents one biologically independent experiment with 10–20 cells, each. **** p
    Figure Legend Snippet: Lysosomal exocytosis, pH and TRPML1 activity in WT and Trpml3 − / − AMΦ. a Lysosomal exocytosis experiments measuring hexosaminidase release from WT and Trpml3 − / − AMΦ. Maximum effects were obtained with ionomycin (4 µM). TRPML3 activator ML3-SA1 elicited no significant effects in both WT and Trpml3 − / − AMΦ. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. b Cartoon illustrating LAMP1 translocation assay shown in c , d . Upon lysosomal exocytosis the lysosomal protein LAMP1 is detected on the plasma membrane (PM) by anti-LAMP1 followed by Alexa Fluor 488-conjugated secondary antibody. c Representative images of LAMP1 translocation assay using WT and Trpml3 − / − AMΦ. Shown are results obtained after 120 min treatment with DMSO, ML3-SA1 (30 µM), or 10 min treatment with ionomycin (4 µM). Scale bar 10 µm. d Quantification of experiments as shown in c (mean ± SEM from 3 biologically independent experiments, each). e Fura-2 calcium imaging experiments using HEK293 cells expressing human or murine TRPML1(NC), TRPML2 or TRPML3, respectively, indicating the specific levels of activation. Channels were stimulated with either ML1-SA1 ( = EVP-169) or ML-SA1 (10 µM, each). Shown are average values (mean ± SEM). Each dot represents one biologically independent experiment with 10–20 cells, each. **** p

    Techniques Used: Activity Assay, Translocation Assay, Imaging, Expressing, Activation Assay

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    Millipore ionomycin calcium salt from streptomyces conglobatus
    Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation <t>(PMA/ionomycin).</t> Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).
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    Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation (PMA/ionomycin). Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).

    Journal: bioRxiv

    Article Title: Integrative dissection of gene regulatory elements at base resolution

    doi: 10.1101/2022.10.05.511030

    Figure Lengend Snippet: Integrative analysis of the CD69 regulatory landscape. A) Gene regulatory landscape characterization by successive functional assays and deep learning. B) Genomic tracks depict accessibility of the CD69 locus in primary CD4+ T cells and Jurkat cells, without or with stimulation (PMA/ionomycin). Enformer signal track shows the predicted contribution of underlying sequence to CD69 expression (magnitude of the model gradient at each position with respect to CD69 promoter signal, summed over 128 bp bins)in Jurkat. Grey bars depict regions with differential accessibility in stimulated Jurkat cells, relative to resting (FDR=0.2). CRISPRi sgRNA positions are also indicated. ATAC signal corresponds to reads per genomic content (RPGC). C) Flow cytometry of CD69 expression in Jurkat cells targeted with the indicated CRISPRi sgRNA following a stimulation time course. Samples gated from the lentiviral transduced population (mCherry+). D) Expanded view of Enformer signal at single base resolution over RE-4, as denoted in panel b. E) Enrichment/depletion plot of dCas9 sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (y-axis; Log 2 Odds Ratio of normalized sgRNA reads). sgRNAs along the x-axis according to their 5’ starting position on the positive strand. Each data point represents mean±s.e.m. F) Enrichment/depletion plot of Cytidine Base Editor (CBE) sgRNAs in CD69+ Jurkat cells, relative to CD69-cells (as in panel e). For C,E,F, data represent 2-3 biological independent experiments. A 170 bp region critical for CD69 activation is denoted (D-F, light red).

    Article Snippet: Stimulation of Jurkat cells for 2-7 hour experiments was achieved with 50ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma-Alrich, P8139) and 500ng/ml ionomycin calcium salt from Streptomyces conglobatus (ionomycin, Sigma-Alrich, I0634).

    Techniques: Functional Assay, Sequencing, Expressing, Flow Cytometry, Activation Assay

    ssRNA40 does not activate Piezo1 -transfected HEK293 cells (A) Fluo-4 calcium imaging of HEK293 cells, with or without transfection of mouse Piezo1 , representative of ≥ 3 independent recordings for each condition. Treatment concentrations are 10 µg/mL ssRNA40 or ssRNA41, 30 µM Yoda1, and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of Piezo1 -transfected HEK293 cells during different treatments. Yoda1 was applied 90 seconds after any given RNA sample, and only cells that responded to Yoda1 (presumably Piezo1-transfected) were analyzed. Transfection efficiency was generally > 60% of the cell culture. n = 50 cells plotted as mean ± 95% CI. (C) Quantification of HEK293 cell calcium responses. n = 50 cells per condition plotted as mean ± 95% CI. One-way ANOVA with Bonferroni correction: n.s. p ≥ 0.05, **** p

    Journal: bioRxiv

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    doi: 10.1101/2022.09.23.509216

    Figure Lengend Snippet: ssRNA40 does not activate Piezo1 -transfected HEK293 cells (A) Fluo-4 calcium imaging of HEK293 cells, with or without transfection of mouse Piezo1 , representative of ≥ 3 independent recordings for each condition. Treatment concentrations are 10 µg/mL ssRNA40 or ssRNA41, 30 µM Yoda1, and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of Piezo1 -transfected HEK293 cells during different treatments. Yoda1 was applied 90 seconds after any given RNA sample, and only cells that responded to Yoda1 (presumably Piezo1-transfected) were analyzed. Transfection efficiency was generally > 60% of the cell culture. n = 50 cells plotted as mean ± 95% CI. (C) Quantification of HEK293 cell calcium responses. n = 50 cells per condition plotted as mean ± 95% CI. One-way ANOVA with Bonferroni correction: n.s. p ≥ 0.05, **** p

    Article Snippet: The following commercially available compounds were used in the study: ssRNA40 (Invivogen, A40-41-02), ssRNA41 (Invivogen, A41-41-02), Yoda1 (Sigma-Aldrich, SML558-5MG), AITC (Sigma-Aldrich, 377430), gadolinium(III) chloride (Sigma-Aldrich, 439770), A-967979 (Sigma-Aldrich, SML0085), and ionomycin (Sigma-Aldrich, I0634).

    Techniques: Transfection, Imaging, Cell Culture

    RNA activates RIN14B cells independently of Trpa1 (A) Calcium imaging of ssRNA40 and ssRNA41 responses in RIN14B cells loaded with Fluo-4 AM, representative of ≥ 3 independent recordings for each condition. Cells were stimulated with 25 µg/mL ssRNA40 or ssRNA41 and 10 µM ionomycin. The mean Δ F/F 0 ± 95% CI is shown for a single recording each of n = 50 cells. (B) To block Trpa1, 10 µM A-967079 was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. 10 µM AITC was used as a positive control for Trpa1 activation. Bar graphs represent n = 50 – 200 cells from 1 – 4 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and A-967079-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p

    Journal: bioRxiv

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    doi: 10.1101/2022.09.23.509216

    Figure Lengend Snippet: RNA activates RIN14B cells independently of Trpa1 (A) Calcium imaging of ssRNA40 and ssRNA41 responses in RIN14B cells loaded with Fluo-4 AM, representative of ≥ 3 independent recordings for each condition. Cells were stimulated with 25 µg/mL ssRNA40 or ssRNA41 and 10 µM ionomycin. The mean Δ F/F 0 ± 95% CI is shown for a single recording each of n = 50 cells. (B) To block Trpa1, 10 µM A-967079 was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. 10 µM AITC was used as a positive control for Trpa1 activation. Bar graphs represent n = 50 – 200 cells from 1 – 4 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and A-967079-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, **** p

    Article Snippet: The following commercially available compounds were used in the study: ssRNA40 (Invivogen, A40-41-02), ssRNA41 (Invivogen, A41-41-02), Yoda1 (Sigma-Aldrich, SML558-5MG), AITC (Sigma-Aldrich, 377430), gadolinium(III) chloride (Sigma-Aldrich, 439770), A-967979 (Sigma-Aldrich, SML0085), and ionomycin (Sigma-Aldrich, I0634).

    Techniques: Imaging, Blocking Assay, Incubation, Positive Control, Activation Assay, Fluorescence

    related to Figure 3 Fecal and dietary extracts activate HEK293 Piezo1-KO cells (A) FLIPR assays on HEK293 Piezo1-KO cells, with or without transfection of human Piezo1 . Each treatment condition was followed up with ionomycin to elicit maximum response for normalization (not shown). Treatment concentrations are 5 mg/mL fecal or dietary extract, 5 µM Yoda1, and 10 µM ionomycin. n = 4 wells per condition plotted as mean ± SEM. (B) Quantification of FLIPR calcium recordings for different treatments. n = 4 wells per condition plotted as mean ± SEM. Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, ** p

    Journal: bioRxiv

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    doi: 10.1101/2022.09.23.509216

    Figure Lengend Snippet: related to Figure 3 Fecal and dietary extracts activate HEK293 Piezo1-KO cells (A) FLIPR assays on HEK293 Piezo1-KO cells, with or without transfection of human Piezo1 . Each treatment condition was followed up with ionomycin to elicit maximum response for normalization (not shown). Treatment concentrations are 5 mg/mL fecal or dietary extract, 5 µM Yoda1, and 10 µM ionomycin. n = 4 wells per condition plotted as mean ± SEM. (B) Quantification of FLIPR calcium recordings for different treatments. n = 4 wells per condition plotted as mean ± SEM. Kruskal-Wallis with Dunn’s multiple comparisons test: n.s. p ≥ 0.05, ** p

    Article Snippet: The following commercially available compounds were used in the study: ssRNA40 (Invivogen, A40-41-02), ssRNA41 (Invivogen, A41-41-02), Yoda1 (Sigma-Aldrich, SML558-5MG), AITC (Sigma-Aldrich, 377430), gadolinium(III) chloride (Sigma-Aldrich, 439770), A-967979 (Sigma-Aldrich, SML0085), and ionomycin (Sigma-Aldrich, I0634).

    Techniques: Transfection

    RNA activates RIN14B cells independently of Piezo1 (A – C) Calcium imaging of RIN14B cell activity during application of negative control vehicle with and without gadolinium inhibition of Piezo1. Gadolinium visibly reduced spontaneous calcium transients. (D – F) RIN14B cell calcium influx in response to fecal RNA, with and without gadolinium. (G – I) RIN14B cell calcium influx in response to the positive control Piezo1 agonist Yoda1, which is blocked by gadolinium. The calcium imaging was performed on GCaMP6s-transfected cells. GCaMP6s calcium responses were measured during stimulation with 25 µg/mL fecal RNA, 15 µM Yoda1, and 10 µM ionomycin. To block Piezo1, 30 µM gadolinium was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. Line graphs represent mean ± 95% CI of a single recording each of n = 50 cells. Bar graphs represent n = 100 – 150 cells from ≥ 2 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and gadolinium (Gd3+)-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: * p

    Journal: bioRxiv

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    doi: 10.1101/2022.09.23.509216

    Figure Lengend Snippet: RNA activates RIN14B cells independently of Piezo1 (A – C) Calcium imaging of RIN14B cell activity during application of negative control vehicle with and without gadolinium inhibition of Piezo1. Gadolinium visibly reduced spontaneous calcium transients. (D – F) RIN14B cell calcium influx in response to fecal RNA, with and without gadolinium. (G – I) RIN14B cell calcium influx in response to the positive control Piezo1 agonist Yoda1, which is blocked by gadolinium. The calcium imaging was performed on GCaMP6s-transfected cells. GCaMP6s calcium responses were measured during stimulation with 25 µg/mL fecal RNA, 15 µM Yoda1, and 10 µM ionomycin. To block Piezo1, 30 µM gadolinium was pre-incubated on the cells for 5 minutes and included throughout the calcium imaging recording. Line graphs represent mean ± 95% CI of a single recording each of n = 50 cells. Bar graphs represent n = 100 – 150 cells from ≥ 2 independent recordings for each condition, with fluorescence values normalized to the response to ionomycin = 1.0, and the bars indicate mean ± 95% CI. Pairwise comparisons between untreated and gadolinium (Gd3+)-treated recordings using Kruskal-Wallis with Dunn’s multiple comparisons test: * p

    Article Snippet: The following commercially available compounds were used in the study: ssRNA40 (Invivogen, A40-41-02), ssRNA41 (Invivogen, A41-41-02), Yoda1 (Sigma-Aldrich, SML558-5MG), AITC (Sigma-Aldrich, 377430), gadolinium(III) chloride (Sigma-Aldrich, 439770), A-967979 (Sigma-Aldrich, SML0085), and ionomycin (Sigma-Aldrich, I0634).

    Techniques: Imaging, Activity Assay, Negative Control, Inhibition, Positive Control, Transfection, Blocking Assay, Incubation, Fluorescence

    ssRNA40 does not alter calcium activity or mechanotransduction in N2a cells (A) Fluo-4 calcium imaging of N2a cells during exposure to different treatments, representative of ≥ 3 independent recordings for each condition. The magnitude of the change in fluorescence (Δ F ) is represented on a fire color scale and is superimposed on a grayscale baseline fluorescence image. Cells were exposed to buffer only (vehicle) or 10 µg/mL ssRNA40 or ssRNA41 for up to 3 minutes, followed by 30 µM Yoda1 and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of ssRNA40 and ssRNA41, each followed by Yoda1 and ionomycin control treatments. Only cells that responded to Yoda1 (functionally expressing Piezo1) were analyzed. Fluorescence values are shown as Δ F normalized to the initial fluorescence (Δ F / F 0 ). n = 50 cells plotted as mean ± 95% confidence interval (CI). (C) Quantification of calcium responses to different treatments. n = 50 cells per condition. Error bars indicate mean ± 95% CI. One-way ANOVA with Bonferroni correction: not significant (n.s.) p ≥ 0.05, **** p

    Journal: bioRxiv

    Article Title: Reevaluation of Piezo1 as a gut RNA sensor

    doi: 10.1101/2022.09.23.509216

    Figure Lengend Snippet: ssRNA40 does not alter calcium activity or mechanotransduction in N2a cells (A) Fluo-4 calcium imaging of N2a cells during exposure to different treatments, representative of ≥ 3 independent recordings for each condition. The magnitude of the change in fluorescence (Δ F ) is represented on a fire color scale and is superimposed on a grayscale baseline fluorescence image. Cells were exposed to buffer only (vehicle) or 10 µg/mL ssRNA40 or ssRNA41 for up to 3 minutes, followed by 30 µM Yoda1 and 10 µM ionomycin. Scale bar is 200 µm. (B) Example calcium imaging traces of ssRNA40 and ssRNA41, each followed by Yoda1 and ionomycin control treatments. Only cells that responded to Yoda1 (functionally expressing Piezo1) were analyzed. Fluorescence values are shown as Δ F normalized to the initial fluorescence (Δ F / F 0 ). n = 50 cells plotted as mean ± 95% confidence interval (CI). (C) Quantification of calcium responses to different treatments. n = 50 cells per condition. Error bars indicate mean ± 95% CI. One-way ANOVA with Bonferroni correction: not significant (n.s.) p ≥ 0.05, **** p

    Article Snippet: The following commercially available compounds were used in the study: ssRNA40 (Invivogen, A40-41-02), ssRNA41 (Invivogen, A41-41-02), Yoda1 (Sigma-Aldrich, SML558-5MG), AITC (Sigma-Aldrich, 377430), gadolinium(III) chloride (Sigma-Aldrich, 439770), A-967979 (Sigma-Aldrich, SML0085), and ionomycin (Sigma-Aldrich, I0634).

    Techniques: Activity Assay, Imaging, Fluorescence, Expressing

    High-throughput single-cell epigenomic and transcriptional profiling of resting and stimulated human blood cells (A) Schematic highlighting design of stimulation experiment. Human peripheral blood mononuclear cells (PBMCs) were stimulated with DMSO control, lipopolysaccharide (LPS), interferon gamma (IFN-Ɣ), or phorbol myristate acetate (PMA) plus ionomycin for 1 or 6 h with or without a Golgi inhibitor (GI) for the 6-h treatment condition. Cells were then split and profiled using scATAC-seq and scRNA-seq for each condition and time point considered. (B) Total number of cells profiled per condition passing quality control filtering for scATAC and scRNA-seq. (C) Uniform manifold approximation and projection (UMAP) of scATAC-seq cells based on latent semantic indexing (LSI) dimensionality reduction, with cells colored by treatment condition. (D) UMAP of scRNA-seq cells based on principal-component analysis (PCA) dimensionality reduction, with cells colored by treatment condition. (E) UMAPs of scATAC-seq cells (top) and scRNA-seq cells (bottom), highlighting individual conditions under control (6 h) and PMA (1 and 6 h) conditions. (F) Aggregate accessibility profiles for scATAC-seq monocyte cells around genes IFITM3 and HES4 . (G) Distribution of single-cell expression levels based on the imputed scRNA-seq counts for stimulation-specific gene markers shown in (F) per condition for scRNA-seq monocyte cells.

    Journal: Cell genomics

    Article Title: Functional inference of gene regulation using single-cell multi-omics

    doi: 10.1016/j.xgen.2022.100166

    Figure Lengend Snippet: High-throughput single-cell epigenomic and transcriptional profiling of resting and stimulated human blood cells (A) Schematic highlighting design of stimulation experiment. Human peripheral blood mononuclear cells (PBMCs) were stimulated with DMSO control, lipopolysaccharide (LPS), interferon gamma (IFN-Ɣ), or phorbol myristate acetate (PMA) plus ionomycin for 1 or 6 h with or without a Golgi inhibitor (GI) for the 6-h treatment condition. Cells were then split and profiled using scATAC-seq and scRNA-seq for each condition and time point considered. (B) Total number of cells profiled per condition passing quality control filtering for scATAC and scRNA-seq. (C) Uniform manifold approximation and projection (UMAP) of scATAC-seq cells based on latent semantic indexing (LSI) dimensionality reduction, with cells colored by treatment condition. (D) UMAP of scRNA-seq cells based on principal-component analysis (PCA) dimensionality reduction, with cells colored by treatment condition. (E) UMAPs of scATAC-seq cells (top) and scRNA-seq cells (bottom), highlighting individual conditions under control (6 h) and PMA (1 and 6 h) conditions. (F) Aggregate accessibility profiles for scATAC-seq monocyte cells around genes IFITM3 and HES4 . (G) Distribution of single-cell expression levels based on the imputed scRNA-seq counts for stimulation-specific gene markers shown in (F) per condition for scRNA-seq monocyte cells.

    Article Snippet: 50 ng/mL Phorbol 12-myristate 13-acetate (PMA) (P8139, MilliporeSigma) + 250 ng/mL Ionomycin calcium salt (I0634, MilliporeSigma),.

    Techniques: High Throughput Screening Assay, Expressing