Journal: Scientific Reports

Article Title: Proliferation of Perivascular Macrophages Contributes to the Development of Encephalitic Lesions in HIV-Infected Humans and in SIV-Infected Macaques

doi: 10.1038/srep32900

Figure Lengend Snippet: Antibodies Used in the Present Study.

Article Snippet: GFAP , 5C10 , Mouse IgG1 , Hu, Mk, Rt, Ms , BioLegend , IF.

Techniques: Immunohistochemistry-IF, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: A synonymous mutation of rs1137070 cause the mice Maoa gene transcription and translation to decrease

doi: 10.3389/fnmol.2024.1406708

Figure Lengend Snippet: Key resources.

Article Snippet: Rabbit (DA1E) mAb IgG XP® isotype control (Alexa Fluor® 488 Conjugate) , Cell Signaling Technology , Cat2975S; RRID:AB_1196589.

Techniques: Control, Plasmid Preparation, Reporter Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Avian pathology : journal of the W.V.P.A

Article Title: Identification of catalytically active domain epitopes in neuraminidase protein of H9N2 subtype of avian influenza virus.

doi: 10.1080/03079457.2023.2239191

Figure Lengend Snippet: Figure 2. Screening and identification of monoclonal antibodies. (A) Antibody levels of the immunized mice. At 1 week after the third immunization, the potency of antibodies from all mice was measured by indirect ELISA, and the results are presented as the absorbance value at OD450 nm. (B) Recognition between mAbs 1B10, 2B6 and 5B10 and AIV-infected cells. A549 cells were infected with AIV, and the protein samples were collected at 24 h to detect recognition between NA protein and mAbs by western blotting. (C) Reactivity of three mAbs detected by IFA. MDCK cells were infected with AIV and incubated with the screened mAbs to detect the reactivity of the mAbs with the NA protein of AIV.

Article Snippet: The type and isotype of the monoclonal antibodies were determined by using a mouse monoclonal antibody isotype identification kit (Proteintech, Wuhan, China).

Techniques: Bioprocessing, Indirect ELISA, Infection, Western Blot, Incubation

Journal: Avian pathology : journal of the W.V.P.A

Article Title: Identification of catalytically active domain epitopes in neuraminidase protein of H9N2 subtype of avian influenza virus.

doi: 10.1080/03079457.2023.2239191

Figure Lengend Snippet: Figure 3. Identification of B cell epitopes of mAbs specific to NA protein. (A) Schematic diagram of NA epitope identification. (B) PCR amplification. Lane 1, PCR product of NA-69; lane 2, PCR product of NA-153; lane 3, PCR product of NA-176; lane 4, PCR pro- duct of NA-183; lane 5-7, negative control. (C) Identification of the recombinant plasmids pET-28a-NA-69, pET-28a-NA-153, pET28a-NA-176 and pET-28a NA-183 with the double enzyme digestion. Lane 1, pET-28a-NA-69; lane 2, pET-28a-NA-153; lane 3, pET28a-NA-176; lane 4, pET-28a NA-183; lane 5, pET-28a vector. (D) PCR amplification. Lane 1 and 2, PCR product of NA- 196; lane 3 and 4, PCR product of NA-220; lane 5 and 6, PCR product of NA-230; lane 7 and 8, PCR product of NA-240; lane 9, negative control. (E) Identification of the recombinant plasmids pET-28a-NA-196, pET-28a-NA-220 pET28a-NA-230 and pET-28a NA-240 with the double enzyme digestion. Lane 1, pET-28a-NA-196; lane 2, pET-28a vector; lane 3 and 4, pET28a-NA-220; lane 5 and 6, pET-28a NA-230; lane 7 and 8, pET-28a-240. (F) Identification of the truncated NA-69 and NA-153 with mAbs following western blot analysis. (G) identification of the truncated NA-176 and NA-183 with mAbs following western blot analysis. (H) identification of the truncated NA-196, NA-220, NA-230 and NA-240 with mAbs following western blot analysis; The reactivities of mAbs with different truncated NA proteins are shown in Table 5.

Article Snippet: The type and isotype of the monoclonal antibodies were determined by using a mouse monoclonal antibody isotype identification kit (Proteintech, Wuhan, China).

Techniques: Negative Control, Recombinant, Plasmid Preparation, Western Blot

Journal: Avian pathology : journal of the W.V.P.A

Article Title: Identification of catalytically active domain epitopes in neuraminidase protein of H9N2 subtype of avian influenza virus.

doi: 10.1080/03079457.2023.2239191

Figure Lengend Snippet: Figure 5. Localization prediction of the identified epitope in NA protein. (A) The epitope recognized by the 5B2 mAb is located in the catalytically active region (marked with an arrow). (B) The epitope recognized by the 2B6 mAb is also located in the cataly- tically active region (marked with an arrow). (C) The epitope recognized by the 1B10 mAb is also located in the catalytically active region (marked with an arrow). (D) Overlapping location prediction for three epitopes of NA protein.

Article Snippet: The type and isotype of the monoclonal antibodies were determined by using a mouse monoclonal antibody isotype identification kit (Proteintech, Wuhan, China).

Techniques:

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) List of 19 test compounds (plus S-2HG) used in screen in described in . (B) Percentage viability of CD8 + T cells following 7 days of treatment with 400μM of test compound, as determined by flow cytometry. Ordinary one-way ANOVA relative to CT; n=4. (C) Percentage of CD62L hi /CD44 hi CD8 + T cells following 7 days of treatment with increasing concentrations of DEG (left) or S-2HG (right). Ordinary one-way ANOVA; n=4. (D) Number of viable CD8 + cells following 7 days of treatment with increasing concentration of DEG (right) or S-2HG (left), as determined by counting beads and flow cytometry. Ordinary one-way ANOVA relative to CT; n=4. All scatter plots show median and 95% CI where each dot represents one murine donor. *p<0.05, **p<0.01, ***p<0.001, #p<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A and B) Percentage of CD62L hi /CD44 hi and number of total murine CD8 + T cells following 7 days of treatment with 400μM of test compound. Ordinary one-way ANOVA relative to control treated cells (CT); n=4. (C) Representative flow cytometry counter plots representing the expression of CD44/CD62L in mouse CD8 + T cells and CD45RO/CCR7 in human CD8 + T cells treated +/− 500μM DEG for 7 days. Pre-gated on live, single, CD8 + events. (D) Heatmap representing proportion of mouse CD8 + T cells expressing CD62L/CD44 (left) and the proportion of human CD8 + T cells expressing CCR7/CD45RO (right) following 7 days of treatment with increasing concentrations of DEG. Two-way ANOVA relative to CT; n=4-5. (E) Cell surface expression of CD25 and CD95 on human CD8 + T cells following 7 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by Median Fluorescence Intensity (MFI). Paired test; n=8. (F) Expression of markers associated with exhaustion on human CD8 + T cells following 7-10 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by MFI. Paired t test; n= 4-8. (G) Expression of cytotoxic granules on human CD8 + T cells following 7 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by MFI. Paired t test; n=4. (H) IFNγ secretion by human CD8 + T cells, 3 days post activation and treatment +/− DEG 500μM, as measured by ELISA. Paired t-test; n=4 All scatter plots show median and 95% confidence interval (CI). Each dot represents one donor (human or murine as indicated). *p<0.05, **p<0.01, ***p<0.001, #P<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Control, Flow Cytometry, Expressing, Fluorescence, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Chromatograms of isotope tracing. Human CD8 + T cells were treated with 13 C5-DEG for varying time length and mass spectrometry for glutarate was performed. (B) Quantified 13 C5-Glutarate from cells treated with 13 C5-DEG for varying time lengths, as described and shown as chromatograms in . (C) Glutarate and 2HG levels in activated human CD8 + T cells. Wilcoxon test: n=5. (D) Glutarate levels in human CD8 + T cells following 7 days of treatment +/− DEG 500μM. Paired t test; n=6. (D) Glutarate levels in human CD8 + T cells following 7 days of treatment +/− 500μM DEG. Paired t test; n=6. (E) Gcdh levels in transcripts HIF1 OE (HIF1AAA) and HIF2 OE (HIF2AAA) – transduced mouse CD8 + T cells, relative to vector control ( Veliça et al ., 2021 ). (F) GCDH levels in T cells transduced with an shGCDH or GCDH overexpressing vector as determined by western blot analysis. Paired t test; n=3. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05, **p<0.01, ***p<0.001, #p<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Mass Spectrometry, Plasmid Preparation, Control, Transduction, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Pathway of lysine and tryptophan catabolism. (B) Glutarate levels in naïve and 72 hr activated mouse CD8 + T cells. Paired t test; n=3. (C) Glutarate levels in naïve or 72 hr activated CD8 + T cells isolated from Hif-1αfl/fl or Hif-1αfl/fl dlckcre mice (HIF ko). Two-way ANOVA; n=3. (D) Glutarate levels in mouse CD8 + T cells cultured at 21% or 1% oxygen for 24 h from day 5 post activation. Paired t test; n=3. (E) GCDH protein copy number in naïve or activated CD8 + T cells from P14 transgenic mice. Unaired t test; n=3. Data from ImmPRes database ( Howden et al ., 2019 ). (F) GCDH counts per million (CPM) in HIF overexpressing (HIF OE) mouse CD8 + T cells. Unpaired t test; n=3. ( Veliça et al ., 2021 ). (G) Expression of markers of T cell differentiation and exhaustion in human CD8 + T cells following 7-10 days treatment +/− DEG 500μM and/or transduction with either shGCDH or GCDH OE vector. Expression as determined by flow cytometry and shown as MFI FC relative to relative untreated control (black dotted line). One sample t test; n=6-12. (J All scatter plot shows median + 95% CI where each dot represents one donor (murine or human as indicated). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Isolation, Cell Culture, Activation Assay, Transgenic Assay, Expressing, Cell Differentiation, Transduction, Plasmid Preparation, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Illustration of alphaketoglutarate (αKG) dependent dioxygenase (αKGDD) reactions investigated in this study. (B) Representative western blot and log2 FC protein expression of H3K9me2 in human CD8 + T cells treated with +/− DEG and/or αKG for 7 days. Graph shows median + 95% CI. Each dot represents one human donor. Kruskal-Wallis test; n=3-9.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Cell free enzymatic inhibition assay for TET2. Inhibition was determined using increasing concentrations of glutarate. Graph shows Michaelis-Menten line of best fit. (B) 5hmC MFI, as determined by flow cytometry, in human CD8 + T cells following 7 days of treatment with increasing concentrations of DEG. Ordinary one-way ANOVA; n=4. (C) Cell free enzymatic inhibition assay for HIF-P4-H1. Values are means of at least 3 independent experiments. Graph shows Michaelis-Menten line of best fit. (D) HIF-PH activity luciferase reporter assay in MEFs treated for 16h with DEG. N=4-6. (E) qPCR analysis of CD8 + T cells treated with DEG for 7 days. Each dot represents one human donor relative to untreated donor control (dotted black line), normalised to HPRT. One sample t test; n=5-8. (F) Representative western blot and log 2 FC in protein expression of specific histone H3 (H3) methylation sites in human CD8 + T cells treated for 7 days +/− DEG 500μM. Each dot represents one human donor normalised to total H3, and relative to untreated control (dotted black line). One-sample Wilcoxon test; n=6-12. (G) Representative western blots and log2 FC protein expression of H3K27me3 and H3K9me3 in human CD8 + T cells treated with +/− DEG and/or αKG for 7 days. Kruskal-Wallis test; n=3-12. (H) Cell free enzymatic inhibition assay for KDM6A. Values are means of at least 3 independent experiments. Graph shows Michaelis-Menten line of best fit. All scatter plots show median + 95% CI, where each dot represents one human donor. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Inhibition, Flow Cytometry, Activity Assay, Luciferase, Reporter Assay, Control, Western Blot, Expressing, Methylation

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Model of protein lysine-glutarylation. (B) Representative western of naïve and activated human CD8 + T cells. N=3. (C) Representative western blot of activated human CD8 + T cells cultured at different oxygen tensions (21%, 5% or 1%). N=4. (D) Representative western of human CD8 + T cells cultured with increasing concentrations of DEG for 7 days. N=4. (E) Representative western of activated human CD8 + T cells cultured with DEG for various time lengths as indicated. All samples were harvested 7 days post activation. N=4. (F) Representative western of CD8 + T cells transduced with shNTC or shGCDH vectors. RQR8 + cells were purified by FACS prior to analysis. N=3. (G) Coomassie staining of proteins immunoprecipitated with a pan-k-glutarylation antibody and separated by SDA-PAGE. 30×10 6 mouse CD8 + T cells, 7 days post activation were used. (H) PDHc isolated from 30×10 6 mouse CD8 + T cells by immunoprecipitation. (I) k-glutarylation WB of immunoprecipitated PDHc as described in . (J) Mode of PDHc. (K) Lipoate and PDHE2 glutarylation levels in HeLa cells treated with DEG. (K) % lipoylation of DLAT (PDHE2) in HEK293 cells treated with DEG for 24h as determined by western blot analysis and relative to βactin. Unpaired t test; n=4. *p<0.05.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Cell Culture, Activation Assay, Transduction, Purification, Staining, Immunoprecipitation, Isolation

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of cells treated with increasing concentrations of DEG for 7 days (as described in ). Ordinary one-way ANOVA; n=3. (B) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of human CD8 + T cells 7 days post activation. Cells were treated with DEG 500μM for different time lengths as indication and harvested at the same time 7 days post activation (as described in ). Ordinary one-way ANOVA; n=3. (C) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of human CD8 + T cells which were cultured at different oxygen tensions for 24hr prior to harvest (as in described in ). (D) Model of lysine-glutarylated protein isolation and identification by mass spectrometry. Lysine-glutarylation immunoprecipitation was performed on 30×10 6 mouse CD8 + T cells, 7 days post activation. Following protein separation by SDS-PAGE, gel was stained with Coomassie blue and stained bands were excised, gel was digested and mass spectrometry was performed. Data was analysed using the Mascot Server v.2.3.1 and the SwissProt protein database. 3 independent experiments and analysis performed. (E) Top hits for each excised band from mass spectrometry described in . (F) Oxoglutarate dehydrogenase (OGDH) complex isolated from 30×10 6 mouse CD8 + T cells by immunoprecipitation. (G) k-glutarylation WB of immunoprecipitated OGDH complex as described in S4I. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Activation Assay, Cell Culture, Isolation, Mass Spectrometry, Immunoprecipitation, SDS Page, Staining

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Schematic of glycolysis and TCA cycle. (B) PDHc activity of human CD8 + T cells treated with DEG for 30 min (acute) or 7 days (chronic), normalised to total protein concentration. Paired t test; n=9-14. (C) PDHc activity of CD8 + T cells transduced with shGCDH relative to NTC control. RQR8 + cells were purified by FACS before assay. Unpaired t test; n= 3. (D) Basal ECAR levels in CD8 + T cells as determined by Seahorse FX analysis following 30 min DEG treatment via seahorse injection. ECAR normalised to ECAR prior to DEG/CT injection. Paired t test; 30 min n=10. (E) Basal ECAR levels in CD8 + T cells following 7 days of treatment +/− DEG 500μM as determined by Seahorse FX analysis Paired t test; 30 min n=5. (F) Basal OCR levels in CD8 + T cells as determined by Seahorse FX analysis following 30 min DEG treatment via seahorse injection. During assay cells were plated in FX media +/− glucose and glutamine as indicated. OCR represented at FC relative to untreated control. One-sample t test; n=6-10. (G) Basal OCR levels in CD8 + T cells following 7 days of treatment +/− DEG 500μM as determined by Seahorse FX analysis. During assay cells were plates in FX media +/− glucose and glutamine as indicated. One sample t test; n=6-10. (H) Seahorse XF Analysis of ECAR levels in CD8 + T cells (7 days post activation). Injections 1-4: DEG/CT, Glucose (Glc), Oligomycin (O), 2-Deoxy-D-Glucose (2-DG), as indicated. Mean and Error with 95% CI; n=10. (I) ECAR measurements during a standard GST of CD8 + T cells treated with +/− DEG 500μM for 7 days. Mean and Error with 95% CI; n=6. (J) Seahorse XF Analysis of OCR levels in CD8 + T cells (7 days post activation). Injections 1-4: DEG/CT, Oligomycin (O), Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (F), Rotenone/Antimycin (R/A). Mean and Error with 95% CI; n=10. (K) ECAR measurements during a standard GST of CD8 + T cells treated with +/− DEG 500μM for 7 days. Mean and Error with 95% CI; n=6. (L) mtDNA copy number in cells treated with +/− DEG 500μM. Acute treatment of CD8 + T cells 7 days post activation for 24h. Chronic treatment of CD8 + T cells for 7 days, from activation. Paired t test; n=4 (M) Mitochondrial fatty acid (FA) and glucose (Glc) oxidative capacity of CD8 + T cells treated +/− DEG 500μM for 7 days, as determined by OCR measurement pre and post addition of BPTES (BP), UK5099 (UK) and etomoxir (ETO) as indicated in . Paired t test; n=6. (N) Protein expression of phosphorylated PDH (ser239) and PDHK1, as determined by western blot analysis and normalised to PPIB. Acute treatment of CD8 + T cells 7 days post activation for 30 min. Chronic treatment of CD8 + T cells for 7 days, from activation. Protein levels normalised to PPIB or complex V expression as indicate. Wilcoxon test; acute n=10, chronic n=5. All scatter plots show median + 95% CI where each dot represents one human. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Activity Assay, Protein Concentration, Transduction, Control, Purification, Injection, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A and B) Quantitative analysis of MST and GST following acute (30 min) (left) and chronic (7 days) (right) DEG exposure, as described in , shown as log2 FC relative to untreated control (black dotted line). One sample t test; n=6-10. (C) Model of mitochondrial fuel flexibility tests. (D) Glutamine oxidative capacity of CD8 + T cells treated +/− DEG 500 μM for 7 days, as determined by OCR measurement pre and post addition of BPTES (BP), UK5099 (UK) and etomoxir (ETO) as indicated. Paired t test; n=5. (E) Gene expression of CD8 + T cells following 7 days of treatment with DEG, as determined by qPCR analysis, and represented as fold change relative to untreated control (dotted black line). One sample t test; n=3-4. (F) Protein expression of PDHc in CD8 + T cells treated +/− DEG 500μM. Acute treatment of CD8 + T cells 7 days post activation for 30 min. Chronic treatment of CD8 + T cells for 7 days, from activation. Protein levels normalised to PPIB or complex V expression as indicated. One-sample t-test; n=4. (G) Representative western blot of CD8 + T cells 7 days post activation as described and shown in . Cells were treated with DEG 500μM for different time lengths as indication and harvested at the same time 7 days post activation. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Control, Gene Expression, Expressing, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Model of in vitro cytotoxicity assays. (B) Cytotoxicity and IFNγ expression (left) of CD19-CAR T cells following co culture with CD19 + Raji cells. Cytotoxicity of OT-1 following co culture with B16F10-OVA cells (right). CAR-T/OT-1 T cells were treated +/− DEG 500μM for 7 days prior to assay. Paired t test; n=9-12. (C) Model of HER2-CAR-T_shRNA cell generation. (D) Percentage cytotoxicity of HER2-CAR T cells with embedded shGCDH or NTC as determined by alamar blue assay following co-culture with HER2 expressing SKOV3 cells. Paired t test; n=4. (E) Adoptive cell therapy (ACT) model with CAR-T cells. 1×10 6 SKOV3 cells were implanted subcutaneously (s.c.) in NSG animals that were injected 21 days later with human shNTC- or shGCDH-expressing CAR-T cells generated as shown in . Tumour growth was monitored every 2-3 days until day 70. (F) Tumor growth data. Thin lines represent tumor growth from individual animals and thick lines represent an exponential (Malthusian) growth curve. No ATC n=5, HER2-CAR-T_shNTC n=11, HER2-CAR-T_shGCDH n=12. (G) Survival curves using 1000mm 3 in tumor volume as the threshold. Log-rank (mantel-Cox) test. (H) Tumor growth model. 1.0×10 6 B16F10 cells were s.c. injected in C57BL/6j mice. From day 4 post tumor inoculation, mice were injected intraperitoneally with 10mg/kg DEG, or control, every 2-3 days. On day 14 after tumor inoculation, peripheral blood, tumor, spleen and tumor draining lymph node from some animals were processed to single cell suspensions and analysed by flow cytometry. Tumor growth was monitored until day 30. (I) Tumor growth data. Thin lines represent tumor growth from individual animals and thick lines represent an exponential (Malthusian) growth curve (n=16). (J) Survival curves using 1000mm 3 in tumor volume as the threshold. Log-rank (mantel-Cox) test; n=14. (K) Frequency of CD8 + T cells in the peripheral blood, 14 days post tumor inoculation. Mann-Whitney test; n=25. (L) Frequency of CD8 + T cells in the tumours. Mann-Whitney test; n=14-16. All scatter plots show median and 95% CI where each dot represents one donor (human or murine as indicated). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: In Vitro, Expressing, Co-Culture Assay, shRNA, Alamar Blue Assay, Injection, Generated, Control, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Cell count of human CAR-T cells treated +/− 500μM for 7 days. Paired t test; n=6. (B) Cell count of murine OT-1 CD8 + T cells treated +/− 500μM for 7 days. Paired t test; n=6. (C) Cell count of HER2-CAR-T_shRNA cell numbers 7 days post transduction. Paired t test; n=4. (D) Cell count of different cancer cell lines treated with increasing concentrations of DEG for 72hr. Cell count relative to appropriate untreated control. One way ANOVA; n=3. (E) In vitro B16F10 cell growth +/− DEG 500μM as determined by cell counting and represented as log2 FC. One way ANOVA; n=3. (F) Gating strategy used for analysis of and . (G) Frequency of cell types in peripheral blood, tumor, spleen and tumour draining lymph nodes, 14 days post tumor inoculation, represented as log2 FC relative to CD45 + count. Graph shows median + 95% CI where each dot represents one mouse. Unpaired t test; n=14-16. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Cell Counting, shRNA, Transduction, Control, In Vitro

Journal: Scientific Reports

Article Title: Biological Activity of Masked Endotoxin

doi: 10.1038/srep44750

Figure Lengend Snippet: ( a ) Monocytes were stimulated with different concentrations of a recombinant protein (Tth) expressed in E. coli BL21 Star™ (DE3) or ClearColi ® (CC) BL21 (DE3), or with different amounts of LPS (0.1 ng/ml ~1 EU/ml). After 24 h of incubation, supernatants were analysed by ELISA. Single values of two independent experiments are shown. Mean values are indicated by bars. ( b ) HEK293 cells were transfected with an NF-κB-luciferase reporter plasmid and a plasmid-mix encoding a functional LPS receptor (TLR4, CD14 and MD2). 24 h after transfection, the medium was replaced and cells were stimulated as indicated (LPS: 0.1 ng/ml ~1 EU/ml). After another 24 h, luciferase activity was measured. Relative luminescence values were calculated relative to the untreated controls. Mean values and SD of at least 3 independent experiments are shown. ( c ) Monocytes were either left untreated (black bars) or treated with 1 μg/ml of a neutralizing IgG monoclonal antibody to human TLR4 for 1 h (gray bars). Thereafter, cells were either not further treated (−) or stimulated with 0.1 ng/ml (~1 EU/ml) LPS or 0.5 μg/ml Tth expressed in E. coli BL21 Star™ (DE3). After 24 h, supernatants were analysed by ELISA. Mean and SD of 7 independent experiments are shown. For statistical analysis, one-way ANOVA with a Tukey post-test was performed. **p < 0.01, ***p < 0.001, n.s. not significant.

Article Snippet: Mouse IgG1-APC isotype control (11711) was purchased from R&D Systems (Biomedica, Vienna, Austria).

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Plasmid Preparation, Functional Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table .

Article Snippet: For assessing maturation state, moDCs were labeled with Mo-DC Differentiation Inspector (Miltenyi, 130-093-567), a commercially available mouse monoclonal conjugated antibody cocktail containing CD14 antibody conjugated to fluorescein-isothiocyanate (FITC; clone Tük4, isotype: mouse IgG2a), CD83 conjugated to allophycocyanin (APC; clone: HB15, isotype: mouse IgG1), and CD209 conjugated toR-phycoerythrin (PE; clone DCN-47.5.4, isotype: mouse IgG1) or isotype control cocktail containing monoclonal mouse IgG2a-FITC, IgG1-PE, and IgG1-APC conjugates in corresponding concentrations (Miltenyi, 130-093-567) following manufacturer's instructions.

Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, SYBR Green Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: DPPA5 Supports Pluripotency and Reprogramming by Regulating NANOG Turnover.

doi: 10.1002/stem.2252

Figure Lengend Snippet: Figure 5. Protein interaction and stabilization between DPPA5 and NANOG in human pluripotent stem cells (hPSCs). (A): Representative confocal microscopy images of immune fluorescent staining of human embryonic stem cells (hESCs) with NANOG and DPPA5 primary antibodies. DAPI shows nuclear staining. The merged images indicate the colocalization of DPPA5 and NANOG in hESC nucleus. Scale bars 5 5 mm. (B): Co-IP of endogenous NANOG and DPPA5 in hESCs. hESCs extracts prepared in CHAPS lysis and IP buffer were immuno- precipitated with anti-NANOG or control IgG antibody. The immune complexes and the input (whole hESC lysate) were analyzed by Western blot with antibodies specific to NANOG or DPPA5. (C): Co-IP of DDK-DPPA5 and NANOG in hESCs. hESCs were transfected with DDK-tagged DPPA5 plasmid. hESC extracts prepared in CHAPS lysis and IP buffer were immunoprecipitated with anti-DDK or control IgG antibody. The immune complexes and the input (whole hESC lysate) were analyzed by Western blot with antibodies specific to DDK and NANOG. (D): The protein levels of NANOG and DPPA5 in control hESCs or hESCs with DPPA5 overexpression were analyzed at the indi- cated time points after CHX (45 mg/ml) treatment. Western blot analysis and relative signal intensity indicated that DPPA5 supports NANOG protein stability in hESCs. b-Actin was used as a loading control. Plot data is presented as mean 6 SD from three independent experiments (*, p < .05). Abbreviations: CHX, cycloheximide; DAPI, 40,6-diamidino-2-phenylindole; DPPA5, developmental pluripotency associated 5; IP, immunoprecipitation.

Article Snippet: Background fluorescence and autofluorescence were determined using cells incubated with Mouse IgG1 PE isotype control (Cat. 1c002p, R&D systems).

Techniques: Confocal Microscopy, Staining, Co-Immunoprecipitation Assay, Lysis, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Over Expression

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Thrombospondin-2 spatiotemporal expression in skeletal fractures

doi: 10.1002/jor.24749

Figure Lengend Snippet: Fracture callus TSP2 positive cells express a MSC immunophenotype. Transverse femoral fractures were induced in TSP2-GFP reporter mice. Calluses from six mice were harvested 7 days post-fracture, pooled, and digested in trypsin, collagenase A, and hyaluronidase. Cells were labeled with anti-mouse directly conjugated MSC antibodies (CD44, CD105, CD45, and Sca-1) and DAPI before flow cytometry. A, Cells were gated by size and granularity using an SSC-A/FSC-A color density plot. Single cells were sub-gated by FSC-W/FSC-A and dead (DAPI+) cells were discriminated by sub-gating on a DAPI-A/SSC-A. B, Included cells were gated by GFP-A as either T2GFP-neg (red) or T2GFP-pos (green). C, Representative histograms of cell surface markers in T2GFP-pos cells. D, Representative histograms of cell surface markers in T2GFP-neg cells. Histograms were gated for positive selection of CD44, CD105, and Sca-1 and negative selection of CD45 to match the known MSC immunophenotype of CD44+/CD105+/CD45−/Sca-1+. Flow cytometry was repeated two times with an n = 2 per experiment. MSC, mesenchymal progenitor cells; TSP2, thrombospondin-2

Article Snippet: Conjugated antibodies for CD105 (562759, preparation: conjugated PE, host: rat, isotype: IgG2a, clone: MJ7/18; BD Biosciences), CD44 (63–0441-80, preparation: conjugated Super Bright 600, host: rat, isotype: IgG2b, clone: IM7; Invitrogen), CD45 (559864, preparation: conjugated APC, host: rat, isotype: IgG2b, clone: 30F-11; BD Biosciences), and Sca-1 (Ly-6A/Ly-6E)-PE-Cy7 (561021, preparation: conjugated PE-Cy7, host: rat, isotype: IgG2a, clone: D7; BD Biosciences) were added to the cells at 4°C for 1 hour.

Techniques: Labeling, Flow Cytometry, Selection