Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: A graphic representation of exon allocation for CA8-201 and alternatively spliced CA8-204 G . CA8-204 harbors a cryptic splice site in the 3’UTR produced by the “G” allele at rs6471859 acting as an eQTL to produce tissue-specific alternative splicing (APA = Alternative polyadenylation).

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Produced

Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: Proteins were overexpressed through transfection with V5-CA8-201 and FLAG-CA8-204 G in HEK293 cells and Immunoblotted against anti-V5, anti-FLAG (Sigma Aldrich) and anti-β tubulin (control) (Cell Signalling) antibodies, bands normalized with β-tubulin. The immunoblots on left panel represent V5-CA8-201, The right panel represent FLAG-CA8-204 G , size comparisons are facilitated by markers (middle). The truncated product from CA8-204 G (~27 kDa) distinguished in size from the WT CA8-201. We were unable to find expression of CA8-204 G in NBL cells.

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Transfection, Western Blot, Expressing

Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: Immunoblot of pITPR1 (forskolin-induced phosphorylation; 10μM forskolin) in HEK293 cells, transfected with empty vector, CA8-201 (WT) or CA8-204 G . Immunoblots suggest inhibition of pITPR1 expression through CA8-204 G overexpression, similar to CA8-201. Quantitation of pITPR1 expression was performed using ImageJ software. Data were normalized with vinculin. N = 3, ***P<0.001, **P<0.01. Quantitative analysis was performed using the one-way ANOVA followed by Bonferroni post–hoc test for each possible comparison (GraphPad software).

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Western Blot, Transfection, Plasmid Preparation, Inhibition, Expressing, Over Expression, Quantitation Assay, Software

Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: (A) ATP dependent free calcium release (monitored by Fura-2) in HEK293 cells transfected with empty vector (negative control), CA8-201 and CA8-204 G are shown in images. Results from calcium imaging suggest the regulation of calcium release by CA8-201 and CA8-204 G in HEK293 cells, through inhibition of intracellular calcium release. Quantitation for intracellular calcium concentrations in HEK293 was performed using one-way ANOVA, post-hoc test comparisons between three groups were carried out by Bonferroni’s comparison (Graphpad Prism software) N = 6, done in triplicates, ***P <0.001, **P<0.01, scale = 40μm. (B) ATP dependent free calcium release (monitored by Fura-2) in NBL cells transfected with empty vector, CA8-201 and CA8-204 G shown in images (left). Results from NBL cells suggest negative regulation of calcium release by CA8-201, through inhibition of ITPR1. Quantitation for intracellular calcium concentrations in NBL cells was performed using one-way ANOVA, post-hoc test comparisons between three groups were carried out by Bonferroni’s comparison (GraphPad Prism software) N = 6, done in triplicates, ***P <0.001, **P<0.01.

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Transfection, Plasmid Preparation, Negative Control, Imaging, Inhibition, Quantitation Assay, Software

Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: Gene transfer of AAV8-FLAG-CA8-204 G (CA8-204 G ); AAV8-V5-CA8-201 WT (CA8-201 WT ); AAV8-V5-CA8-201 MT (CA8-201 MT ); or empty vector were injected via the sciatic nerve injections before carrageenan injection to produce an inflammatory pain model. Paw withdrawal thermal latencies were measured daily after baseline, after virus injection and/o and after carrageenan injection (left paw). Mice that received AAV8-FLAG-CA8-204 G (1.5μl, 1E13 genome copies/ml) after day 7 had a significantly higher threshold for pain compared to CA8-201 MT , especially on days 13 and 15. Increasing paw withdrawal latencies reached a maximum of 10–12 seconds on day 14–15, similar to CA8-201 WT . CA8-201 MT produced no similar increase in withdrawal latencies. After carrageenan injections on Day 15, all groups showed reduced paw withdrawal latencies through day 18. However, mice in the CA8-201 WT and CA8-204 G groups showed a significantly enhanced paw withdrawal latency on days 20 and 22 compared to the CA8-201 MT , indicating the anti-hyperalgesia protection was provided by the CA8-204 G . On days 13, 15, 20, and 22 mark the differences in paw withdrawal latency significantly greater than baseline, in CA8-201 and CA8-204 G compared with CA8-201 MT . Mice in the negative control group showed paw withdrawal latency either at baseline or below baseline on these days. N = 8, ***P<0.001, **P<0.001 Two-way (repeated measures ANOVA) followed by Bonferroni’s post-hoc test statistical group test (GraphPad Prism).

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Plasmid Preparation, Injection, Produced, Negative Control

Journal: PLoS Genetics

Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis -eQTL

doi: 10.1371/journal.pgen.1008226

Figure Lengend Snippet: Immunostaining was performed in DRG sections in mice receiving SN of AAV8-V5-CA8-201 MT , AAV8-V5-CA8-201 and AAV8-FLAG-CA8-204 G . (A) Immunostaining was done using antibodies anti-V5 for CA8-201 (V5, green) and anti-S100 (glial marker) (S100, red), and anti-V5 with anti-S100 antibodies, respectively from the merged image (Merge). (B) Immunostaining was performed using antibodies anti-S100 (S100, red), anti-FLAG for CA8-204 G (FLAG, green) and anti-FLAG and anti-S100 from the merged figures (merge); ( C) Immunostaining using antibodies anti-Advillin (Advillin, red), anti-V5 (V5-CA8-201, green) and anti-Advillin and anti-V5 (Merge) from the merged figures was carried out in DRG sections; ( D) Using antibodies anti-Advillin (Advillin, red), anti-FLAG for CA8-204 G (FLAG-CA8-204 G , green) and anti-FLAG and anti-Advillin from the merged figures (merge). (i) Histogram of the IF results demonstrates CA8-201 and CA8-204 G co-localize with DRG neuroglia and neuronal populations. The % overexpression was calculated from the positive cells overlapping with advillin and S100 markers, divided by DAPI as counter-stain which was used. (ii) Overlap (%) between CA8-204 G and CA8-201 were calculated separately from the total population of DRG glial and neuronal cells. IF was done using DRG tissues retrieved from animals after 4 weeks after SN injection. Percentage of overexpression of CA8-201 and CA8-204 G and overlap in total DRG cells were quantified using one-way ANOVA with Bonferroni's post-hoc test (***P<0.001, **P<0.01, *P<0.1, ns = non-significant, N = 8 each gene group). (IF = Immunofluorescence) (Scale = 50μm).

Article Snippet: Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056).

Techniques: Immunostaining, Marker, Over Expression, Staining, Injection, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy

doi: 10.1016/j.xcrm.2021.100449

Figure Lengend Snippet:

Article Snippet: Human CD14 MicroBeads Kit , Miltenyi Biotec , CAT#130-050-201.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Purification, Control, Virus, Recombinant, Cell Culture, Saline, Cell Isolation, RNA Sequencing, Gene Expression, Sequencing, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Annexin V magnetic microbeads , Miltenyi , 130-090-201.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages

doi: 10.7554/eLife.52535

Figure Lengend Snippet:

Article Snippet: CD14 + monocytes were then isolated by positive selection magnetic sorting, using human CD14 Microbeads and LS columns (Miltenyi Biotec).

Techniques: Derivative Assay, Cell Culture, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Recombinant, Isolation, Western Blot, Fluorescence, Software, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TL1A Enhances the Activation of MAIT Cells Suboptimally Stimulated with IL-12 and IL-18 CD8 + T cells were enriched from healthy peripheral blood mononuclear cells (PBMCs) and stimulated overnight with different combinations of cytokines: IL-12 at 2 ng/mL, IL-18 at 50 ng/mL, IL-15 at 25 ng/mL, and TL1A from 0.01 to 100 ng/mL as indicated. (A–C) Proportions of CD8 + MAIT/CD161 + or CD161 − cells producing IFN-γ (A), TNF-α (B), or CD69 (C) following overnight stimulation with suboptimal concentrations of IL-12 and IL-18, plus varying concentrations of TL1A. (D) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different combinations of cytokines. (E–H) Frequency of MAIT cells expressing IFN-γ (E), TNF-α (F), GrB (G), and CD69 (H) upon stimulation with the indicated cytokines. Data were acquired from seven donors in 2–3 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Activation Assay, Expressing, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR and Cytokine Signaling Combine to Promote MAIT Cell Effector Functions (A–J) Magnetic-activated cell sorting (MACS)-enriched CD8 T cells from the blood were cultured overnight in the presence of the indicated cytokines, together with the THP1 cell pulsed with DMSO or the MAIT-antigen 5-OP-RU (A–E) or with αCD3/CD28 beads (F–J). (A) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different cytokines in the presence of 5-OP-RU. (B–E) Frequency of MAIT cells expressing IFN-γ (B), TNF-α (C), GrB (D), or CD69 (E) upon stimulation with the indicated cytokines. (F) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different cytokines in the presence of 5-OP-RU. (G–J) Frequency of MAIT cells expressing IFN-γ (G), TNF-α (H), GrB (I), or CD69 (J) upon stimulation with the indicated cytokines. Data were acquired from seven donors in two experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001. See also Figure S2 .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: FACS, Cell Culture, Expressing, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: Gut-Derived MAIT Cells Show a Broadly Similar Response Pattern toward Innate and Adaptive Stimuli Compared with Their Blood-Derived Counterparts Representative plots showing the percentage of cells positive for the indicated effector molecules as a proportion of CD8 + MAIT cells. (A–C) Proportions of blood-derived (n = 32) CD8 + MAIT cells producing IFN-γ (A), TNF-α (B), or GrB (C) following overnight stimulation with combinations of suboptimal concentrations of IL-12 and IL-18, TL1A, and αCD3/CD28 beads as indicated. (D–F) Proportions of gut-derived (n = 13) CD8 + MAIT cells producing IFN-γ (D), TNF-α (E), or GrB (F) stimulated in the same way as in (A)–(C). (G and H) Expression of IFN-γ, TNF-α, and GrB by blood-derived (G, n = 7) or gut-derived (H, n = 6) CD8 + MAIT cells 20 h after coculture with THP1 cells alone or THP1 cells incubated with 25 fixed E. coli bacteria per cell. Data were acquired from multiple donors as indicated in 3–5 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests (A–F), two-way ANOVA (G), or Wilcoxon tests (H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001. See also Figure S3 .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Derivative Assay, Expressing, Incubation, Bacteria, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR- and Cytokine-Activated MAIT Cells Possess Distinct Transcriptional Profiles (A–C) Venn diagrams showing genes that are significantly differentially modulated (p < 0.05, fold change > 4) in TCR (T)-, cytokine (C)-, or TCR and cytokine (TC)-treated CD8 + MAIT cells compared with untreated (UT) MAIT cells of three healthy individuals. The cytokine (C) stimulation consisted of a cocktail of 4 cytokines: IL-12 (2 ng/mL), IL-18 (50 ng/mL), IL-15 (25 ng/mL), and TL1A (100 ng/mL). Genes with significantly altered expression levels (A) are divided into two sets: those are that are upregulated upon stimulation (B) and those that are downregulated upon stimulation (C). (D) Heatmap showing 1,594 significantly differentially expressed transcripts (p < 0.05, fold change > 4) between TCR/C/TC-stimulated and UT CD8 + MAIT cells among the same three healthy individuals. (E) Visualization of the CD8 + MAIT cell transcripts elicited by differential stimulations in the subspace of the first principle components (PCs). Each colored circle represents a sample and is color coded in accordance with the conditions with which cells were stimulated, as illustrated on the right-hand side of the graph. (F–K) Volcano plots to visualize differentially expressed transcriptional profiles of activated CD8 + MAIT cells stimulated in different ways. Each point represents a single gene, and genes expressed at significantly higher or lower levels between the compared conditions are depicted, respectively, in the upper-right or upper-left corner of each plot. Genes discussed in the text are highlighted in blue (tissue repair associated) or in red (inflammation associated). The gene expression of untreated MAIT cells was compared to (F) T-, (G) C-, or (H) TC-stimulated MAIT cells. Further, gene expression in those cells was also compared directly between the different stimulation conditions: (I) T- to C- stimulation, (J) T- to TC-stimulation, and finally (K) C- to TC-stimulation. Data were acquired from three donors in one experiment. See also Figure S4 and , , and .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Expressing, Gene Expression

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR-Mediated Activation of MAIT Cells Leads to the Expression of Tissue-Repair-Associated Molecules and Accelerates Wound Healing (A–C) Gene set enrichment summary plots for stimulated sorted MAIT cell-versus-unstimulated cell-ranked genes. Depicted are the individual plots for TCR-stimulated versus UT in (A), TC-stimulated versus UT in (B), and C versus unstimulated in (C). Non-significant for C versus UT, normalized enrichment score (NES) = 1.63; p < 0.0002 for TCR versus UT, NES = 1.57; and p < 0.0002 for TC versus UT. Data were acquired from three donors in one experiment. (D) Flow cytometry analysis of the expression of TNF-α, furin, and CCL3 by CD161 ++ /MAIT CD8 + T cells in response to fixed E. coli presented by THP1 cells in the presence or absence of an anti-MR1 (αMR1) blocking antibody at the 72-h time point. (E) Statistical analysis of the expression of the effector molecules shown in (D). (F) Caco2 cells were grown to confluency and scratched with a WoundMaker device to perform in vitro wound-healing assays. Cells were supplemented with different supernatants collected from 72-h cocultures of enriched CD8 T cells with E. coli -loaded THP1 cells in the presence or absence of αMR1, as indicated. The open wound areas were quantified as percentages of the initial wound size in the Caco2 cultures. Data points are mean ± SEM and were acquired from five biological replicates in two experiments. (G) Representative pictures of the closure of the wounds in Caco2 cultures treated as in (F) were assessed with time-lapse imaging over a time course of 36 h. Data were acquired from seven donors in three experiments. Differences among conditions were analyzed by two-way ANOVA. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001. Scale bars, 250 μm. See also Figure S5 and .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Activation Assay, Expressing, Flow Cytometry, Blocking Assay, In Vitro, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: Integrated Transcriptional Analyses Reveal the Relationship between In Vitro -Activated Human MAIT Cells and In Vivo -Activated Murine MAIT and Tc17 Cells Hierarchical clustering analysis of the transcriptomic profiles of the indicated cell populations is shown. Similarity between the expression profiles is measured using a Euclidean distance (height). Datasets were derived from ImmGen ( Heng et al., 2008 ), Linehan et al. (2018) , and Hinks et al. (2019) and were integrated as described in the section. The relevant datasets are colored. UT, TCR, C, and TC refer to the conditions used in this paper on in vitro -activated human CD8 + MAIT cells (blue). Topical Tc17 and H2M3_CD8_SLO refer to the H2M3-restricted populations identified in Linehan et al. (2018) in the skin and secondary lymphoid organs of mice, respectively (red). The cells described in Hinks et al. (2019) are marked in yellow (stimulated human MAIT cells) or green (chronic or acute, derived during a late or an early time point after L. longbeachae infection in mice, respectively). See also Figure S6 .

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: In Vitro, In Vivo, Expressing, Derivative Assay, Infection

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: MAIT Cells Can Be Found Close to and within the Colonic Epithelium (A–G) Representative images showing the expression of Va7.2, CD161, CD8, PLZF, CD3, and CD103 in the lamina propria and the epithelium of fixed samples of colonic polyp tissue. Samples were mounted on cytometer chips and iteratively stained with sets of three directly fluorochrome-conjugated antibodies as described in the methods section. Depicted are a merged picture (A) and all the individual stains for Va7.2 (B), CD161(C), CD8 (D), PLZF (E), CD3 (F), and CD103 (G). White arrows mark cells showing co-expression of Va7.2, CD161, PLZF, and CD3 that were defined as MAIT cells here. Note that while CD8 was co-expressed in most of them, CD8− MAITs (arrow + asterisk) could also be found. In contrast, CD103 was rarely co-expressed on MAITs (arrow + diamond). During the iterative staining process dust particles and other detritus can be picked up by the solution flowing over the tissue creating autofluorescent artifacts (1–4). While some of these get washed away after completion of the staining cycle (1, 4), others present during multiple imaging rounds (2, 3). Scale bars, 50μm.

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Expressing, Cytometry, Staining, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet:

Article Snippet: CD8 MicroBeads, human , Miltenyi , Cat# 130-045-201.

Techniques: Virus, Recombinant, Reverse Transcription, Activation Assay, Staining, Software