Journal: PLoS ONE

Article Title: The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

doi: 10.1371/journal.pone.0008229

Figure Lengend Snippet: ( A ) Kaplan–Meyer distribution graph showing the percentage of survival rates of mice of the indicated genotypes. NS, non–statistically significant values; *, P <0.01; **, P <0.05. ( B ) Examples of non–tumorigenic (NT) and tumorigenic (T) tissues obtained from two Vav1 –/– ; Rasgrf2 –/– mice. Scale bar, 1 cm. ( C ) Hematoxylin/eosin stained sections of healthy and tumoral tissues obtained from a healthy wild type (upper panel) and a tumor–bearing Vav1 –/– ; Rasgrf2 –/– (lower panel) mouse, respectively. Scale bar, 100 µm. Asterisks, tumoral cells infiltrates in non–hematopoietic tissues.

Article Snippet: Number LYRT101, Origene Technologies) was used to analyze the expression of VAV1 and RASGRF2 transcripts in different subsets of lymphomas.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

doi: 10.1371/journal.pone.0008229

Figure Lengend Snippet: ( A ) Cell suspensions from healthy and tumoral tissues obtained from mice of the indicated genotypes (right) were stained with anti–CD3 antibodies and subjected to flow cytometry analysis. Red squares highlight CD3 + positive cell populations present in the animals under study. The percentage of the CD3 + population in each case is indicated inside each panel. SSC, side scatter. ( B ) Tissues from a tumor–bearing Vav1 –/– ; Rasgrf2 –/– mouse (panels 3–7 counting from the left) were stained with either anti–CD3 (top panels) or anti–PCNA (bottom panels) using immunohistochemistry techniques. As control, we stained thymus and spleen sections obtained from a wild type mouse (first and second panel from left, respectively). Scale bar, 100 µm. ( C ) Cell suspensions were isolated from a healthy and three tumor–bearing Vav1 –/– ; Rasgrf2 –/– animals, stained with anti–CD4 and anti–CD8 antibodies, and analyzed by flow cytometry. ( D ) Cell suspensions derived from the indicated tissues of a healthy and two tumoral Vav1 –/– ; Rasgrf2 –/– mice were collected and blasts visualized by flow cytometry. In the y and x axes, the SSC and FCS values range from 0 to 1,024, respectively. The vertical lines indicate the FCS point from where gated cells were considered as blasts. FCS, forward scatter. The percentage of blasts in each case is indicated inside each panel. n = 5 and 10 for healthy and tumor–bearing animals, respectively (panels A–D).

Article Snippet: Number LYRT101, Origene Technologies) was used to analyze the expression of VAV1 and RASGRF2 transcripts in different subsets of lymphomas.

Techniques: Staining, Flow Cytometry, Immunohistochemistry, Isolation, Derivative Assay

Journal: PLoS ONE

Article Title: The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

doi: 10.1371/journal.pone.0008229

Figure Lengend Snippet: ( A ) Hematoxylin/eosin, anti–CD3 and anti–PCNA staining of tissue sections obtained from a Vav1 –/– ; Rasgrf2 –/– mouse with a thymus–localized lymphoma (panels 3–7 counting from left). As control, we stained thymus and spleen sections obtained from a wild type mouse (first and second panel from left, respectively). Scale bar, 100 µm. ( B ) Cell suspensions were obtained from a healthy Vav1 –/– ; Rasgrf2 –/– (top) and a Vav1 –/– ; Rasgrf2 –/– mouse with a thymus–localized tumor (bottom), stained with anti–CD3 antibodies and analyzed by flow cytometry. The red square highlights the abnormal, CD3 + positive cell population found in the thymus–localized tumor. The percentage of CD3 + cells in each case is indicated inside each panel. ( C ) Cell suspensions were obtained from a Vav1 –/– ; Rasgrf2 +/+ (top) and a Vav1 –/– ; Rasgrf2 –/– (bottom) mouse containing thymus–localized tumors, stained with anti–CD4 and anti–CD8 antibodies and analyzed by flow cytometry. Results similar to those shown in panels A–C were observed in 5 independent determinations. ( D ) Graph showing the percentage of one–year old mice of the indicated genotypes with thymus–localized tumors.

Article Snippet: Number LYRT101, Origene Technologies) was used to analyze the expression of VAV1 and RASGRF2 transcripts in different subsets of lymphomas.

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

doi: 10.1371/journal.pone.0008229

Figure Lengend Snippet: Outlier analysis of the expression of the RASGRF2 mRNA in hematopoietic tumors.

Article Snippet: Number LYRT101, Origene Technologies) was used to analyze the expression of VAV1 and RASGRF2 transcripts in different subsets of lymphomas.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Impaired AJC formation in Albatross knockdown cells. (A) Double staining for Albatross (red) and the undercoat proteins (green) for each AJC component: TJ, ZO-1; AJ, afadin; DS, desmoplakin. Top and bottom columns show projections of x-y planes and z sections, respectively. Albatross knockdown A549 (Albatross KD) cells lack accumulation of these proteins at the cell–cell borders except in regions where residual Albatross is present. (B) Cell–cell adhesive properties evaluated by a cell aggregation assay. In the differential interference contrast images, control cells show cell aggregation. With Albatross knockdown A549 (A1050 and A1160) cells, the aggregated cell population is reduced and free cells are increased. The percentages of single cells in total cells (mean ± SD) are: control, 36.1 ± 3.9; A1050, 52.4 ± 2.8; A1160 cells, 59.4 ± 10.2. n = 4 and P < 0.01. (C) Immunoelectron microscopy of A549 cells with anti-Albatross antibodies. Note that the cytoplasm in the vicinity of AJCs is labeled. TJ, AJ, and DS are indicated. Arrows indicate cell–cell contacts. (D) Quantitative data from C. (E) BC fraction and AJ fraction were immunostained for Albatross with the indicated AJC proteins, PKCζ or Par3. Note that Albatross is well colocalized with them. (F) Immunoblotting of fractions derived from mouse liver: homogenates (left), BC (middle), and AJ (right). Not only Albatross but also Par3 is enriched in line with the concentrations of the indicated AJC components. (G) Immunoprecipitation of A549 cells with anti-Albatross antibodies. Start and IP indicate starting lysates and immunoprecipitates with preimmune (Pre.) and anti-Albatross (αAlb.) antibodies, respectively. Note the Par3 precipitation with Albatross. Among AJC components, ZO-1 also coprecipitated. (H) Immunoprecipitation analysis with tagged Albatross and Par3. Start and IP indicate starting lysates and immunoprecipitates with anti-GFP antibodies, respectively. Left lanes show results for negative controls expressing GFP alone. Par3 was the most precipitated with GFP-Albatross among coexpressed myc-Par3, -Par6, and -PKCλ. Bars: (A) 10 μm; (B) 100 μm; (C) 0.1 μm; (E, BC) 13 μm; (E, AJ) 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Double Staining, Immuno-Electron Microscopy, Labeling, Western Blot, Derivative Assay, Immunoprecipitation, Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Functions of keratins and Albatross–Par3 complexes. (A–C) The amounts of Albatross protein and mRNA were analyzed in both keratin 8 and keratin 18 (K8/18)-introduced SW13 cells. As a control, an empty vector was transfected. As loading controls, α-tubulin and GAPDH were used. Two independent experiments were performed. (A) Immunoblotting. In transiently K8/18-introduced SW13 cells, the amount of Albatross protein is elevated, along with the amount of keratin 18. (B) With stable lines, the same results were obtained. (C) RT-PCR. In K8/18-introduced SW13 cells, the mRNA level of K18 is elevated, but not that of Albatross. β-actin is included as an internal control. (D) Double staining for K8/18 and the indicated proteins: Albatross, AJC components of ZO-1 and afadin, and Par3. (top) In control cells, K8/18 is absent and only limited amounts of Albatross are apparent at cell–cell junctions. In stably K8/18-introduced SW13 cells, Albatross is well localized in cell–cell junctions compared with control cells. (middle and bottom) ZO-1, afadin, and Par3 similarly accumulated at the cell–cell borders in stably K8/18-introduced SW13 cells. (E) Immunostaining of stably K8/18-introduced SW13 cells transfected with control or Albatross siRNA. Note that ZO-1, afadin, and Par3 are reduced at cell–cell borders with knockdown of Albatross. (F) A model for the regulation of AJC and lateral domains with the Albatross–Par3 complex and keratins. Albatross–Par3 complexes regulate the formation of AJC and maintain lateral membrane identity. However, Par3 without Albatross regulates apical structures. Keratins stabilize Albatross, promoting the formation of AJC. Knockdown effects are also indicated. Bars, 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Staining, Stable Transfection, Immunostaining

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 reverses the ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) by tripartite motif-containing protein 21 (TRIM21) and ameliorates the phenotype of polycystic ovary syndrome (PCOS) mice. (A) Western blot (WB) analysis of neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) and CPT1A treated with MLN4924, with or without TRIM21 knockdown in KGN cell. (B and C) Coimmunoprecipitation (Co-IP) and WB analysis of TRIM21 neddylation in KGN cells treated with or without MLN4924. (D) Co-IP and WB analysis of exogenous K48 ubiquitination of CPT1A in KGN cells overexpressing UBE2M, with or without NEDD8 knockdown in the presence of MG132 (10 μM, 4 h). (E and F) Evaluation of the mitochondrial oxidative phosphorylation (OXPHOS) function by oxygen consumption rate (OCR) in KGN cells, including basal respiration, maximum respiration, ATP generation, and coupling efficiency ( n = 6). (G and H) Evaluation of fatty acid oxidation (FAO)-dependent mitochondrial function by OCR in KGN cells, including basal respiration and maximum respiration ( n = 6). (I) Activity of mitochondrial complex V in KGN cells ( n = 6). (J and K) Testosterone and luteinizing hormone (LH) levels of serum ( n = 15). (L) Anogenital distance in adult female mice ( n = 15). (M) Insulin tolerance test (ITT) test in adult female mice after 4 h of fasting ( n = 5). (N) Number of pups per birth ( n = 10). (O and P) WB and reverse transcription polymerase chain reaction (RT-PCR) analysis of TRIM21 and CPT1A in ovarian granulosa cells (GCs). (Q) Immunohistochemical staining of TRIM21 in mouse ovarian tissues (scale bars, 100 μm). Data are expressed as means ± standard error of the mean (SEM), and each symbol represents a biologically independent mouse. Significance was calculated by 1-way analysis of variance (ANOVA) multiple comparison test. Blood glucose analysis between groups (M) was determined by 2-way ANOVA and multiple comparison test. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IB, immunoblot.

Article Snippet: Cells were exposed to 100 nM DHT for 72 h. To investigate specific cellular processes, cells were also subjected to various inhibitors: 100 nM MLN4924 (Selleck, cat# S7109) for 24 h to inhibit neddylation; 5 μM MG132 (Selleck, cat# S2619) for 24 h to inhibit proteasomal degradation; 500 nM CHX (MCE, cat# HY-12320) for 4 h to inhibit protein synthesis; and 50 μM etomoxir (MCE, cat# HY-50202) for 24 h to inhibit FAO.

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Co-Immunoprecipitation Assay, Phospho-proteomics, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Comparison

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 improves the quality of oocytes of polycystic ovary syndrome (PCOS) mice. (A) Hematoxylin and eosin (H&E) staining of ovaries and the number of follicles in ovary including primordial follicles (*), growing follicles (#), and atretic follicles (arrows; scale bars, 100 μm). (B) Number of oocytes after superovulation ( n = 5 mice; scale bars, 100 μm). (C) Representative images and percentage of first polar body extrusion in mouse oocytes, fertilization rate, and blastocyst maturation rate ( n = 5 mice; scale bars, 100 μm). (D) Transmission electron microscopy (TEM) analysis of mitochondrial morphology of mouse ovarian granulosa cells. Green arrows indicate normal mitochondria, red arrows indicate abnormal mitochondria, and # indicates lipid droplets ( n = 5 mice; scale bar, 500 nm). (E) Representative images of spindle morphology and chromosome alignment in oocytes at the metaphase II stage by confocal microscopy ( n = 5 mice; scale bar, 10 μm). The percentage of aberrant spindles and misaligned chromosomes were quantified in oocytes at metaphase II from control ( n = 23), PCOS ( n = 19), and MLN4924 ( n = 21) mice. (F) Representative images for JC-1 staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected (G) Representative images for tetramethylrhodamine ethyl ester perchlorate (TMRE) staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected. (H) Representative images for lipid content in oocytes ( n = 15; scale bar, 50 μm) and ratio of the fluorescence intensities. Three fields of view of each mouse were selected. (I) Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA levels of follicle development-related genes in mouse cumulus–oocyte complexes (COCs) ( n = 5). Data are expressed as means ± SD, and each symbol represents a biologically independent mouse. (J) Schematic diagram of the pathway by which tripartite motif-containing protein 21 (TRIM21) regulates ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) leading to abnormal fatty acid oxidation in ovarian granulosa cells. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were exposed to 100 nM DHT for 72 h. To investigate specific cellular processes, cells were also subjected to various inhibitors: 100 nM MLN4924 (Selleck, cat# S7109) for 24 h to inhibit neddylation; 5 μM MG132 (Selleck, cat# S2619) for 24 h to inhibit proteasomal degradation; 500 nM CHX (MCE, cat# HY-12320) for 4 h to inhibit protein synthesis; and 50 μM etomoxir (MCE, cat# HY-50202) for 24 h to inhibit FAO.

Techniques: Staining, Transmission Assay, Electron Microscopy, Confocal Microscopy, Control, Fluorescence, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Pyruvate metabolism controls chromatin remodeling during CD4 + T cell activation.

doi: 10.1016/j.celrep.2023.112583

Figure Lengend Snippet: Figure 2. ACSS2 inhibition or ACLY deficiency does not affect epigenome remodeling during T cell activation (A) Immunoblots of ACLY, ACSS2, PDCE1, and actin from human T cells activated for 8, 16, 24, or 48 h with anti-CD3/CD28 stimulation. (B and C) CD25 protein expression was measured by flow cytometry (FACS) and IL2ra gene expression was determined by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) in human CD4+ T cells activated with anti-CD3/CD28 in the presence and absence of ACSS2 inhibitor (15.6 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FCCP, mitochondrial oxidative phosphorylation uncoupler Abcam # ab120081 Rotenone Merck Millipore # R8875 UK-5099 (Synonyms: PF-1005023) Med Chem Express # HY-15475 3PO R98% (HPLC) Sigma-Aldrich # SML1343 Sodium acetate Sigma-Aldrich # S1429 DCA Tocris Bioscience # 2755 Etomoxir sodium salt hydrate Sigma-Aldrich # E1905 ACSS2 inhibitor Selleck # S8588 6,8-Bis(benzylthio)-octanoic acid Sigma-Aldrich # SML0404 Sodium Pyruvate Thermo Fisher # 11360070 SB204990 Med Chem Express # HY-16450 BMS303141 Sigma-Aldrich # SML0784 HEPES Sigma-Aldrich #H4034-100G EDTA Sigma-Aldrich #EDS-100G DL-Dithiothreitol solution (DTT) Sigma-Aldrich #43816-10ML Bovine serum albumin (BSA) Sigma-Aldrich #A9647-500G D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML 13C6-glucose Cambridge Isotope Lab #CLM-1396 DMSO Sigma-Aldrich # D2650 Ficoll Paque Plus Sigma-Aldrich # GE17-1440-02 13C6-glucose Cambridge Isotope Lab #CLM-1396 Cell Trace Violet Invitrogen #C34557 Molecular ProbesTM 2-NBDG Invitrogen #N13195 SYBR Thermo fisher #S33102 iScript cDNA kit biorad #170-8891 Formaldehyde Sigma-Aldrich #252549-1L DAPI (1:2000) Invitrogen #D1306 Zombie NIRTM Fixable Viability Kit Biolegend #423106 Zombie GreenTM Fixable Viability Kit Biolegend #423112 7-AAD Invitrogen #A1310 D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML GolgiPlugTM (Protein Transport Inhibitor) BD Biosciences #555029 Tween 20 Sigma-Aldrich #P7949-500ML HALT protease inhibitor Thermo scientific #78439 Powdered milk Carl Roth #T145.2 PierceTM Protein A/G Magnetic Beads thermo fisher #88802 Proteinase K, recombinant, PCR Grade Sigma #3115828001 RNeasy Mini Kit Qiagen #74106 Seahorse XF DMEM medium Agilent Technologies #103575-100 Critical commercial assays MagniSortTM Human CD4+ T cell Enrichment Kit eBioscience #8804-6811-74 CD4 (L3T4) MicroBeads, mouse Miltenyi # 130-117-043 Duolink In Situ Detection Reagents Red Sigma DUO92008 Duolink Green PLA Sigma # DUO92014 Foxp3/Transcription Factor Staining Buffer Set eBioscience #00-5523-00 BD Cytofix/Cytoperm Fixation/Permeabilization kit BD Biosciences #554714 seahorse XFe24 Flux Packs Agilent #1023-40-100 Acetyl-CoA Assay Kit Biovision, Milpitas CA #K317-100 (Continued on next page) Cell Reports 42, 112583, June 27, 2023 19

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Cytometry, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR