Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Heatmap of IRF family members expression in NK cells throughout the course of MCMV infection represented as z-score of log2 normalized counts based on RNA-seq. b. Representative histogram of IRF4 expression in WT and Irf4−/− NK cells on day 2 PI (n = 4 biological replicates). c. Tracks (top) show chromatin accessibility dynamics of the Irf4 locus in Ly49H+ NK cells at days 0, 2, 4, and 7 PI as assessed by ATAC-seq. Graphs (bottom) show normalized counts for each peak indicated. d. Tables of enriched known motifs of highly accessible regions (log2FC > 1 & padj < 0.05) on day 4 PI versus day 2 PI from ATAC-seq data of Ly49H+ NK cells after MCMV infection. e. Heatmap of motif score from de novo motif analysis on highly accessible regions (log2FC > 1 & padj < 0.05) during day 0, 2, 4, and 7 transition based on ATAC-seq data of Ly49H+ NK cells after MCMV infection. f. IRF4 expression of sorted naïve splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as fold change of IRF4 MFI over unstimulated condition (n = 6 biological replicates per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. g. Representative histogram of IRF4 expression upon indicated stimulation gated on human CD56bright NK cells. Data are presented as paired fold change of IRF4 MFI compared to unstimulated condition (n = 8 donors per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. h. UMAP embedding of scATAC-seq data from in vitro stimulated human NK cells (sorted on CD3−CD14−CD19−CD7+NKG2C+) from HCMV− donors. i. Coverage plot of the IRF4 locus from in vitro stimulated human NK cells as in (H).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Control, Expressing, Infection, RNA Sequencing, In Vitro

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Scatter-plot comparing the expression of IRF family members that are differentially expressed on days 2 and 4 PI versus day 0 in Ly49H+ NK cells by RNA-seq analysis. Points highlighted in red and blue indicate genes that are upregulated and downregulated, respectively, on day 2 versus day 0 and day 4 versus day 0 (Padj < 0.05). b, Kinetics of RNA-seq normalized reads for Irf4 transcripts in Ly49H+ NK cells throughout the course of MCMV infection (n = 2 biological replicates per time point). c, Representative flow cytometric histogram of IRF4 expression in splenic Ly49H+ NK cells in MCMV-infected mice (left) and fold change of IRF4 mean fluorescent intensity (MFI) compared to uninfected mice (right) (n = 3 biological replicates for day 0, 2 and 3 and n = 2 biological replicates for day 4). d, UMAP embedding of scATAC-seq data from human NK cells of HCMV+ or HCMV− healthy donors. e, Coverage plot of the IRF4 locus from defined human NK cell subsets as in d. f, RNA-seq reads for IRF4 expression from human NK cell subsets represented as fragments per kilobase of transcript per million mapped reads (FPKM) (n = 5). CD56bright (CD56brightCD16dim), early CD56dim (CD56dimNKG2A+KIR−CD57−), CD56dim (CD56dimNKG2A−self-KIR+CD57+), adaptive NKG2C+ (CD56dimCD16+NKG2C+CD7lowNKp30lowCD57+). g, Representative histogram of IRF4 expression of sorted naive splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as FC of IRF4 MFI over unstimulated condition (n = 3 per group). h, Volcano plot of RNA-seq data from human NK cells stimulated with HCMV UL-40 peptide + IL-12/18 versus control peptide. Red and blue points represent DEGs (Padj < 0.05 and |log2FC| > 0.5) in stimulated and control conditions, respectively. Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. A two-tailed unpaired t-test was used for g. Statistical analysis was not derived from groups with n < 3.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, RNA Sequencing, Infection, Control, Two Tailed Test, Derivative Assay

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, GSEA pathway analysis of DEGs between WT and Irf4−/−Ly49H+ cells on day 7 PI, from scRNA-seq experiment in Fig. 4. b, Representative histograms of CD98 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). c, Representative histograms of CD71 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). d, Representative histogram and gMFI of kynurenine uptake by splenic WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI. Kyn + lysine (Lys) was added as a non-competitive control and Kyn + leucine (Leu) acted as a competitive control (n = 4 biological replicates in each condition). e, Representative histogram and gMFI of transferrin uptake assay from naive or day 5 PI splenic WT or Irf4−/−Ly49H+ NK cells (n = 11 biological replicates). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–e and adjusted for multiple comparisons for d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Control, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, b Representative histograms and quantification of IFN-γ production (A) and LAMP-1 (also known as CD107a) (B) from either naive WT (gray) or Irf4−/− (orange) NK cells upon in vitro stimulation with nothing, IL-12 + IL-18, PMA + Ionomycin, or PMA + Ionomycin + IL-12 for 3 hours. c–f. Representative histograms (upper panels) and quantifications (bottom panels) of CD25, Gzmb, CD69, and IFN-γ expression gated on Ly49H+ NK cells from MCMV-infected WT:Irf4−/− mBMC on day 2 PI. Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. n = 3 biological replicates per group. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: In Vitro, Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a–c. Number of NK cells in blood (A), liver (B), and spleen (C) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. d–f. Maturation status based on the expression of CD27 and CD11b of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 5 for blood, and n = 6 for liver and spleen) mice in the blood (D), liver (E), and spleen (F) at 8 weeks old. g. Percentage of Ly49H+ NK cells within NK cells (Lineage-NK1.1+ CD49b+) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. h. Experimental schematic of WT:Irf4−/− mixed bone-marrow chimera (mBMC) generation. i–k. Percentage of NK cells of WT or Irf4−/− mice from WT:Irf4−/− mBMC in the blood (n = 60 biological replicates) (I) and spleen (n = 21 biological replicates) ( J) after 8 weeks (I-J), 16 weeks (n = 3 biological replicates) or 6 months (n = 5 biological replicates) post-transplant (K). l. Representative of flow plots of CD62L versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 13 biological replicates in spleen). m. Representative of flow plots of CD11b versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 16 biological replicates in spleen). n. Histogram and percentage quantification of Ly49H+ NK cells within splenic NK cells of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC (n = 24 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Unpaired (A-G) and paired (I-N) two-tailed t-tests were performed.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Experimental schematic of adoptive transfer of WT and Irf4−/− NK cells into Rag2−/− Il2rg−/− mice. b. Fold change chimerism between WT and Irf4−/− NK cells on day 7 over day 0 (pre-transfer) in blood, liver, and spleen (n = 3 biological replicates in blood, and n = 6 biological replicates in liver and spleen). c. Representative histogram of IRF4 expression between transferred WT and Irf4−/− NK cells on day 7 post-transfer into Rag2−/− Il2rg−/−. d. Representative flow plots of CD27 and CD11b expression between WT and Irf4−/− NK cells on day 7 post-transfer. Data are represented as percentage of each subset within WT or Irf4−/− NK cells in indicated tissues (n = 3 biological replicates). e. Histogram of CD122 and quantification of CD122 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). f. Histogram of CD132 and quantification of CD132 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). g. Histogram of pSTAT5 and quantification of pSTAT5 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). H-J. h–j. Quantification of Ki-67 staining in the blood and spleen between WT and Irf4−/− NK cells (H, n = 5 biological replicates per group), BIM and BCL2 MFI from splenic NK cells (I-J, n = 9 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Adoptive Transfer Assay, Expressing, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Kaplan–Meier survival curves from Rag2−/− Il2rg−/− mice transferred with WT Ly49H+ NK, Irf4−/−Ly49H+ NK or no NK cells (n as indicated). b, Percentage of Ly49H+ NK cells from adoptive co-transfer of WT (CD45.1) and Irf4−/− (CD45.2) NK cells into Ly49H-deficient mice from the blood throughout the course of MCMV infection (left) and percent chimerism of WT versus Irf4−/− transferred Ly49H+ NK cells (right) (n = 4 biological replicates). c, Representative flow plots gated on transferred WT (CD45.1) and Irf4−/− (CD45.2) Ly49H+ NK cells on day 7 in different tissues (left) and the percent chimerism of WT and Irf4−/− NK cells within transferred Ly49H+ NK cells between different tissues (n = 4 biological replicates per group). d, Representative histogram of CTV dilution on day 0 and day 4 PI gated on transferred Ly49H+ NK cells (left). Dashed gray and orange lines represent WT and Irf4−/− NK cells on day 0, respectively; solid gray and orange lines represent WT and Irf4−/− NK cells on day 4 PI, respectively. Percentage of transferred NK cells that have divided at least once (n = 5 biological replicates per group) (right). e, Representative histogram of BIM expression on day 5 PI from splenic WT or Irf4−/−Ly49H+ NK cells (left) and BIM gMFI (right) (n = 4 biological replicates). gMFI, geometric mean fluorescence intensity. f, Percentage of FLICA+ (marking activated caspase) WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI WT:Irf4−/− mBMC splenocytes upon ex vivo treatment with ABT199 (BCL2 inhibitor) (n = 3 biological replicates in each condition). Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. For statistical testing, a log-rank test was used for a and a two-tailed paired t-test was used for b–f and adjusted for multiple comparisons for b and c.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Expressing, Fluorescence, Ex Vivo, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Representative flow plots showing CD62L and CD27 expression of transferred WT or Irf4−/−Ly49H+ NK cells on day 7 PI (left). Percentage of NK cell subset based on CD62L and CD27 expression of transferred cells on day 7 PI in the blood, liver and spleen (right) (n = 5 biological replicates in blood and spleen and n = 4 biological replicates in liver). b, Representative histogram and percentages of Ly6C, CX3CR1 and KLRG1 of transferred WT or Irf4−/− Ly49H+ NK cells on day 7 PI from spleens of MCMV-infected mice (n = 5 per group). c, Fold change of chimerism between splenic WT and Irf4−/−Ly49H+ NK cells upon infection of WT:Irf4−/− mBMC with MCMV from day 0, 2, 4, 5 and 7 PI (n = 6 biological replicates for day 0 and 7, n = 5 biological replicates for day 2, n = 7 and 8 biological replicates for day 4 and 5, respectively). d, Kinetics of different subsets of splenic WT and Irf4−/−Ly49H+ NK cells from WT:Irf4−/− mBMC during MCMV infection setting based on CD62L and CD27 expression from day 0 to 7 PI (n = 6 biological replicates for day 0 and 7, n = 7 biological replicates for day 2 and n = 8 biological replicates for day 4 and 5, respectively). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–d and adjusted for multiple comparisons for c and d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, WT and Irf4−/− Ly49H+ NK cells were sorted on day 0, 2, 4 and 7 following MCMV infection and single-cell RNA-sequencing performed on multiplexed samples. UMAP embedding of scRNA-seq colored by Louvain clusters (kn = 30). b, Top 50 DEGs between WT and Irf4−/−Ly49H+ NK cells during MCMV shown as z score of normalized reads. c, PAGA network analysis based on scRNA-seq data from WT versus Irf4−/−Ly49H+ NK cells scRNA-seq. d, UMAP embedding of WT and Irf4−/−Ly49H+ NK cells on day 7 from clusters 1 and 3 in a. e, Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/−Ly49H+ NK cells on day 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/−Ly49H+ NK cells, respectively. Highlighted genes are effector genes and/or factors known to be important for NK cell differentiation/maturation. f, Dot plot of scRNA-seq for maturation markers between WT and Irf4−/−Ly49H+ NK cells on day 7 PI shown as mean normalized expression and percent of cell fraction within the group. g, UMAP plot from d as MPEC or SLEC gene signature module scores.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, RNA Sequencing, Cell Differentiation, Expressing

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. UMAP embedding of WT (left) or Irf4−/− (right) of Ly49H+ NK cells colored by genotypes and days post-infection. b. Proportion of each genotype from each time points per Louvain clusters. c. Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/− Ly49H+ NK cells on day 0, 2, 4 and 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/− Ly49H+ NK cells, respectively. d. Number of differentially expressed genes between WT or Irf4−/− Ly49H+ NK cells from each time point as identified by MAST. e. Violin plot quantification of normalized reads for MYC transcript by scRNA-seq between WT and Irf4−/− Ly49H+ NK cells. f. Representative histogram of Myc protein by flow cytometry between WT (gray) and Irf4−/− (orange) Ly49H+ NK cells on day 5 PI. g. Violin plot quantification of MAGIC-imputed reads for CD71 (Tfrc), IRP1 (Aco1), and IRP2 (Ireb2) on day 7 PI between WT (blue) and Irf4−/− (yellow) Ly49H+ NK cells by scRNA-seq.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Flow Cytometry

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Normalized count heat map of IRF4 binding upon anti-NK1.1 + cytokines (IFN-α + IL-12/18 + IL-2/15) stimulation or IL-15 alone at +0.5 kb from the peak summit (n = 12,344 peaks). b, IGV tracks showing IRF4-binding signal between unstimulated (IL-15 alone, gray) or stimulated (anti-NK1.1 + cytokines, orange) conditions in Bcl2l11, Sell, Tfrc and Ireb2 loci. Regions highlighted in dashed boxes represent called IRF4 peaks by MACS2 in stimulated condition. c, Top: Venn diagram comparing IRF4-bound genes in stimulated condition and DEGs between WT versus Irf4−/−Ly49H+ NK cells on day 7 PI from scRNA-seq. Bottom: distribution of IRF4-bound DEGs between WT versus Irf4−/−Ly49H+ NK shown as fold change as analyzed by MAST. d, Proportion of IRF4 genome-wide occupancy within the exon, intergenic, intronic and promoter regions. e, Enriched de novo motifs found by HOMER on IRF4-bound regions upon stimulation. P values were calculated by HOMER (Methods). f, Metacoverage of IRF4-bound accessible regions that contain the indicated motifs in Ly49H+ NK cells upon MCMV infection as assessed by ATAC-seq. Data are shown as median of normalized counts. g, Top: the z score of DEGs that contain AICE motifs in Ly49H+ NK cells during MCMV infection. Bottom: average of scaled log-normalized counts of AICE-motif-containing genes enriched in either WT or Irf4−/−Ly49H+ NK cells (Methods) throughout the course of MCMV. Adjusted P values (Padj) were calculated by ‘geseca’ function of the fgsea package (Methods).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Binding Assay, Genome Wide, Infection

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Gating strategy for sorting WT or Irf4−/− Ly49H+ NK cells. b. Gating strategy for phenotypic analysis of Ly49H+ NK cells after lymphocytes, singlets and live cells cleanup (as depicted in Extended Data Fig. 7a). c. Gating strategy for human NK cells.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques:

Journal: Journal of Clinical Investigation

Article Title: TET repression and increased DNMT activity synergistically induce aberrant DNA methylation

doi: 10.1172/jci124070

Figure Lengend Snippet: Figure 3. Increased RELA binding levels at pro- moter regions of TET-targeting miRNAs. (A) Acti- vation of NF-κB signaling pathway in NUGC-3 cells by TNF-α. A downstream target gene of NF-κB signaling pathway, IL6, was upregulated by TNF-α treatment. Data represent mean ± SE. (B) Heat- map of RELA binding levels in NUGC-3 cells treated with TNF-α. RELA binding levels at genomic regions around TSSs of 44,112 transcripts were aligned according to the binding level after TNF-α treatment. Clear increase by the treatment was observed. Each row shows ± 2.5 kb centered on TSS. (C) Enriched motifs in RELA peaks detected in NUGC-3 cells treated with TNF-α. NF-κB binding motifs were most significantly enriched in NUGC-3 cells treated with TNF-α, showing successful detection of RELA binding sites. (D–F) RELA bind- ing status around the putative promoter regions of TET-targeting miRNAs. RELA binding levels at putative promoter regions of MIR26B (CTDSP1) (D) and MIR20A (MIR17HG) (E) were robustly increased by TNF-α treatment. RELA binding levels at these host genes were comparable to that at the IL6 promoter (F). Black boxes indicate genomic regions with peaks detected. The y axis represents the read pileup normalized to the total number of reads at a base pair position (rpm/bp).

Article Snippet: Crosslinked chromatin (100 μg) extracted from NUGC-3 cells with mock and TNF-α (30 ng/mL) treatment was immunoprecipitated using 5 μg antibody against RELA (R&D Systems, catalog AF5078).

Techniques: Binding Assay

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: Neurofilament antibody, anti-human/mouse, PE, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-119-496 RRID: AB_2857467.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Cell Reports

Article Title: Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells

doi: 10.1016/j.celrep.2020.107982

Figure Lengend Snippet:

Article Snippet: Anti-mouse IRF4, APC, REA201 , Miltenyi Biotec , Catalog # 130-100-913; RRID: AB_2652517.

Techniques: Recombinant, Electron Microscopy, Staining, Software

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for p-IRF3 (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: SDS Page, Transfection

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were seeded in 12-well plates to achieve ∼ 65% confluency in 24 hours, at which time cells were treated with either 8 ng/mL TNFα or a water vehicle control. Cells were harvested 24 hours after treatment, and RTqPCR was performed. Resulting levels of CCL22 mRNA are shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, two-tail t test; error bars represent standard deviations. B , HeLa cells were seeded and grown as for A and treated with either 10 ng/mL PMA or a 0.0004% DMSO vehicle control. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. C , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were harvested for RTqPCR; levels of RELA/p65 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. D , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. E , HeLa cells described in C and D were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the IRF3 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. F , HeLa cells carrying the shRNAs described above were transfected as in E in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Derivative Assay, Expressing, shRNA, Stable Transfection, SDS Page, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against IRF3 (IRF3-1 or IRF3-2) were harvested for RTqPCR; levels of IRF3 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. B , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. C , HeLa cells described in A and B were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the RELA/p65 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. D , HeLa cells carrying the shRNAs described above were transfected as in C in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1, 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP. E-F , HeLa cells ( E ) and MCF7 cells ( F ) were transfected with a mock control or 2 μg/mL of empty plasmid (EV) or the constitutively active IRF3-5D with Trans IT-LT1 (HeLa) at a 1:2 ratio or TransfeX (MCF7) at a 1:4 ratio. Cells were harvested 48 hours after transfection, and RTqPCR was performed. Resulting fold change of CCL22 mRNA relative to the mock control is shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, one-tailed t test; error bars represent standard deviations.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Expressing, shRNA, Stable Transfection, Derivative Assay, SDS Page, Transfection, Plasmid Preparation, One-tailed Test