Journal:

Article Title: Higher expression of Bax in regulatory T cells increases vascular inflammation

doi:

Figure Lengend Snippet: Generation of TCTP transgenic mouse model. A. Bax transgenic construct. A c-myc expression tag-fused Bax cDNA was placed under the direction of mouse CD25 promoter. The locations of primers used for PCR detection of transgenic Bax DNA, and RT-PCR detection of transgenic Bax transcript and endogenous Bax transcript are presented with arrows. In the lower panel, the primer sequences for detection of β-actin by RT-PCR are presented. B. The sequences of six primers used in this study are presented. C. Detection of Bax transgenic mice by PCR using mouse tail genomic DNAs as templates. No DNA template control and non-transgenic mouse (wild-type) tail DNA control are included. D. Detection of transgenic Bax transcripts by RT-PCR in lymphoid tissues of Bax transgenic mice (upper panel). Equal amounts of RNAs prepared from non-lymphoid tissues (kidney and liver) from Bax transgenic mice and counterpart tissues from non-transgenic wild-type control mice were used in the experiment as shown by RT-PCR detection of β-actin transcripts (lower panel). E. Detection of Bax upregulation in CD4+CD25high Tregs by flow cytometry using anti-Bax monoclonal antibody. Bax expression in splenic CD4+CD25− T cells in six splenocyte preparations from wild-type control mice and six Bax Tg mice, which generated the background ratios of Bax expression in Bax Tg samples over Bax expression in wild-type controls. The statistical analysis of the background ratios generated a mean ± 2 × standard deviations (0.93 ± 2 × 0.11), as shown by the two dash lines. In comparison, the ratio of Bax expression in CD4+CD25+ Tregs from Bax Tg mice over the Bax expression in CD4+CD25+ Tregs from wild-type control mice was significantly higher than the upper limit of the Bax expression ratios in CD4+CD25− T cells, suggesting significant upregulation of Bax expression in CD4+CD25high Tregs in Bax transgenic mice.

Article Snippet: The 3.818 kb transgenic DNA fragment “Nru I-CD25 promoter-C-Myc-Bax-Sex AI” was prepared by digesting the pCD25-Bax-Tg vector with three restriction enzymes, Nru I, Sex AI, and Pvu I (New England Biolabs, Ipswich, MA), gel-purified, and microinjected into zygotes from C57BL/6 mice in the Baylor College of Medicine Genetically Engineered Mouse Core.

Techniques: Transgenic Assay, Construct, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Generated

Journal:

Article Title: Higher expression of Bax in regulatory T cells increases vascular inflammation

doi:

Figure Lengend Snippet: Association of IL-2 withdrawal-induced Bax upregulation and Treg apoptosis. A. Quantitation of wild-type splenic CD4+CD25− T cells, CD4+CD25low and CD4+CD25high Tregs by flow cytometry with PE-conjugated CD25 and PE-Cy7-conjugated CD4 antibodies. In the left upper gate are shown the percentages of gated cell fractions. B. IL-2 withdrawal induced T cell apoptosis. The apoptotic rates of three CD4+ fractions, such as CD25−, CD25low and CD25high in each of four indicated conditions (IL-2 withdrawal for 0 hours, IL-2 withdrawal for 12 hours, cultured with IL-2 for 12 hours, and IL-2 withdrawal plus incubation with Bax channel inhibitor for 12 hours) are presented as percentages of FITC-conjugated annexin V staining positive cells. The experiments were repeated four times and the statistical data with the mean and standard deviations are presented. C1. Bax expression in three gated CD4+ cell fractions was measured in three conditions including IL-2 withdrawal for 0 hours, and IL-2 withdrawal for 12 hours by flow cytometry using FITC-conjugated anti-Bax monoclonal antibody. The upper gate in each FACS plot indicates the total Bax+ cells whereas the lower gate in each plot shows the cell fractions with higher fluorescence and elevated expression of Bax. C2. The experiments were repeated three times and the statistical data with the mean and standard deviations are presented. D1. Bax expression in three gated CD4+ cell fractions was measured in two conditions including IL-2 withdrawal for 12 hours in the presence of vehicle-control, and cultured without IL-2 but with p53 transcription factor inhibitor Pifithrin for 12 hours by flow cytometry using FITC-conjugated anti-Bax monoclonal antibody. D2. The experiments were repeated three times and the statistical data with the mean and standard deviations are presented. E. A working hypothesis of Bax upregulation in Tregs induced by IL-2 withdrawal-triggered signals.

Article Snippet: The 3.818 kb transgenic DNA fragment “Nru I-CD25 promoter-C-Myc-Bax-Sex AI” was prepared by digesting the pCD25-Bax-Tg vector with three restriction enzymes, Nru I, Sex AI, and Pvu I (New England Biolabs, Ipswich, MA), gel-purified, and microinjected into zygotes from C57BL/6 mice in the Baylor College of Medicine Genetically Engineered Mouse Core.

Techniques: Quantitation Assay, Flow Cytometry, Cell Culture, Incubation, Staining, Expressing, Fluorescence

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: Upregulation of lipid metabolic genes by MYC. (A) Diagram of MYC’s regulation of the fatty acid synthesis pathway. (B) qPCR shows fatty acid synthesis mRNA expression in high MYC (MYC ON, N=3 per cell line) compared to 24 hours of MYC inactivation (MYC OFF, N=3 per cell line) in BCL (P493–6) and RCC (E28). Expression of lipogenesis proteins for BCL (P493–6) and RCC (E28) cells with MYC ON vs. MYC OFF, by Western blot analysis. Statistical significance by t-test, ***P<0.001. (C) RNA-seq analysis of lipid metabolic pathways in fold-change of RPKM comparing MYC ON/MYC OFF in BCL (P493–6).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Expressing, Western Blot, RNA Sequencing Assay

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: MYC regulates SREBP and together they regulate lipogenesis. (A) mRNA expression of fatty acid synthesis genes: Acly, Acaca, Fasn, and Scd1 upon siRNA knockdown of SREBP1 or scramble RNA in HCC(EC4) under MYC ON and OFF conditions (N=3 per condition). Statistical significance by two-way ANOVA using Tukey’s multiple comparisons post tests, ***P<0.001. Overall, ***P<0.001 for SREBP1 effect, ***P<0.001 for MYC effect and ***P<0.001 for interaction between the two. (B) SREBP1 ChIP-qPCR on BCL P493–6 line shows decreased binding of SREBP1 upon MYC inactivation (top panel). MYC ChIP-qPCR on HCC EC4 line shows no change of MYC binding upon SREBP1 knockdown (bottom panel). (C) Glucose and glutamine incorporation into lipids by NMR in MYC ON (upper panel) and OFF (bottom panel) in BCL(P493–6). For close-up look see Figure S3B. (D) Incorporation of glucose and glutamine carbons to de novo synthesized fatty acids by mass spectrometry based on the fold change of 2C13 peak in BCL(P493–6) as measured upon 24 hours of MYC inactivation (N=5). Fold change in the de novo fatty acid synthesis when MYC is activated or inactivated (left panel); and glucose and glutamine incorporation into fatty acids when MYC is activated (middle panel), or inactivated (right panel). Statistical significance by t-test, **P<0.01, ***P<0.001.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Expressing, Binding Assay, Synthesized, Mass Spectrometry

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Recombinant, Chromatin Immunoprecipitation, RNA Extraction, Transgenic Assay, Software

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, Representative profile of peaks obtained for the Pgk1 gene by ChIP-Seq. ChIP-seq was performed using sPLA 2 -V KD cells transfected with empty vector, sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V, and sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V-H48Q using an anti-Myc-tag mouse monoclonal antibody. Input of sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V was used as a ChIP-seq control. ChIP-seq analysis showed a Myc-tagged sPLA 2 -V-binding peak at the upstream region of Pgk1 gene locus. B, ChIP-qPCR validation of Myc-tagged sPLA 2 -V binding to the Pgk1 gene. Data are shown as a percentage expression of input control. The amplification sites (binding site and unrelated site) for PCR are indicated in panel A (ChIP-seq). Each bar represents the mean±SEM of 2–3 independent experiments. C, Promoter assay for Pgk1 gene transcriptional activity. sPLA 2 -V WT cells, sPLA 2 -V KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q were transfected with Cypridina and Renilla luciferase expression vectors. The promoter activity is expressed as the relative luciferase activity normalized to Renilla activity. Values in each bar were normalized to that of WT (=1). Each bar represents the mean±SEM of 6–9 independent experiments. ＊＊ , P ＜0.01 vs. WT. †† , P ＜0.01 vs KD. Upper panel shows a schematic illustration of the promoter construct used in this Cypridina luciferase reporter assay.

Article Snippet: Rabbit monoclonal antibody against c-Src (clone no. 32G6; catalog. no. 2123), rabbit monoclonal antibody against phospho-c-Src at the active site (Tyr416; clone no. D49G4; cat. no. 6943), mouse and rabbit monoclonal antibody against Myc-tag (clone no. 9B11 and 71D10, cat. no. 2276 and 2278, respectively), rabbit monoclonal antibody against Beclin1 (clone no. D40C5; cat. no. 3495), rabbit polyclonal antibody against phospho-Beclin1 at the active site (Ser30) (cat. no. 54101), rabbit monoclonal antibodies against Rab5 (clone no. C8B1; cat. no. 3547) and Rab7 (clone no. D95F2; cat. no. 9367), rabbit polyclonal antibody against Lamin (cat. no. 2032), and rabbit monoclonal antibody against β-tubulin (clone no. 9F3; cat. no. 2128) were purchased from Cell Signaling Technology Japan (Tokyo, Japan).

Techniques: ChIP-sequencing, ChIP-qPCR, Promoter Assay, Transfection, Plasmid Preparation, Expressing, Control, Binding Assay, Biomarker Discovery, Amplification, Activity Assay, Luciferase, Construct, Reporter Assay

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, Representative profile of peaks obtained for the Pgk1 gene by ChIP-Seq. ChIP-seq was performed using sPLA 2 -V KD cells transfected with empty vector, sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V, and sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V-H48Q using an anti-Myc-tag mouse monoclonal antibody. Input of sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V was used as a ChIP-seq control. ChIP-seq analysis showed a Myc-tagged sPLA 2 -V-binding peak at the upstream region of Pgk1 gene locus. B, ChIP-qPCR validation of Myc-tagged sPLA 2 -V binding to the Pgk1 gene. Data are shown as a percentage expression of input control. The amplification sites (binding site and unrelated site) for PCR are indicated in panel A (ChIP-seq). Each bar represents the mean±SEM of 2–3 independent experiments. C, Promoter assay for Pgk1 gene transcriptional activity. sPLA 2 -V WT cells, sPLA 2 -V KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q were transfected with Cypridina and Renilla luciferase expression vectors. The promoter activity is expressed as the relative luciferase activity normalized to Renilla activity. Values in each bar were normalized to that of WT (=1). Each bar represents the mean±SEM of 6–9 independent experiments. ＊＊ , P ＜0.01 vs. WT. †† , P ＜0.01 vs KD. Upper panel shows a schematic illustration of the promoter construct used in this Cypridina luciferase reporter assay.

Article Snippet: The remainder of the chromatin lysate was used for the ChIP experiment using mouse monoclonal antibody against Myc-tag (1:100 dilution; clone no. 9B11, catalog no. 2276, Cell Signaling Technology Japan, Tokyo, Japan).

Techniques: ChIP-sequencing, ChIP-qPCR, Promoter Assay, Transfection, Plasmid Preparation, Expressing, Control, Binding Assay, Biomarker Discovery, Amplification, Activity Assay, Luciferase, Construct, Reporter Assay

Journal: Nature metabolism

Article Title: A large-scale genome-lipid association map guides lipid identification

doi: 10.1038/s42255-020-00278-3

Figure Lengend Snippet: a, We quantified 2,558 features in B6 plasma (n=4 for each sex). 254 features were sex-specific (FC > 1.0, p < 0.05, non-paired, two-sided Student’s t-test). Precursor m/z (±10 ppm) matching to our DO database provided genetic information for ⅓ of the otherwise unidentified features. b , Six male-specific unidentified features (red) share a QTL on Chr 6:91 Mbp with a common A/J down effect . c , The features further clustered in m/z-RT space. d-g , Targeted fragmentation spectra (exemplary spectra for two species ([M+H]+ m/z 1156 and 1158) in positive (MS2) and negative (MS2 and MS3) mode) exhibited signals consistent with a lipid class built of a PC headgroup and three FAs. h , The DO database further confirmed LysoPC 14:0 mapping to Abhd1 60 with j , an enrichment of FA 14:0 containing lipids k , We compare Hepa1-6 overexpressing ABHD1 and ABHD3 versus a control overexpressing GFP (n=12 for each, 4 biological x 3 technical replicates, boxplots are defined with first and third quartiles for lower and upper hinges, 1.5x interquartile range for the length of the whiskers, center line at median). The boxplots show absolute FC of each mutant over GFP by lipid class; the dashed line is at FC=0.4 l , The lowest (negative) FC for both is observed for LysoPC 14:0; isomers are summed. m , All 14:0 containing PCs exhibit a negative FC for ABHD1 and ABHD3 mutants consistently, while 18:0 containing species are showing opposing positive FC. Plotted are sum-normalized, log2-transformed FC means with error bars representing 95% confidence interval, significance indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001) of non-paired two-sided Student’s t-test, equal variance, n=12 for each, details in source data. Abbreviations: DO (diversity outbred), FC (fold change), m/z (mass-to-charge), QTL (quantitative trait loci), Chr (chromosome), Mbp (megabase pair), SNP (single nucleotide polymorphism), PC (phosphatidylcholine), F/M (female-to-male), RT (retention time), FA (fatty acid), PE (phosphatidylethanolamine), GFP (green fluorescent protein)

Article Snippet: Mouse Abhd1 (CMV6 promoter, Myc-DDK-tagged, MR206471) and mouse Abhd3 (CMV6 promoter, Myc-DDK-tagged, MR206458) plasmids were obtained from Origene.

Techniques: Clinical Proteomics, Control, Mutagenesis, Transformation Assay

Journal: Nature metabolism

Article Title: A large-scale genome-lipid association map guides lipid identification

doi: 10.1038/s42255-020-00278-3

Figure Lengend Snippet: a, Experimental design of the validation experiment featuring three technical and four biological replicates of Hepa1-6 cells either untransfected (CTL), transfected with a His-tag GFP control (GFP), or transfected with MYC-tagged ABHD1 or ABHD3. b, Western blot of Hepa1-6 overexpression of ABHD1 and ABHD3. Shown is an overlay of membrane and ECL blot for MYC-tag. c, Heatmap of top 49 features from discovery lipidomics experiment with p < 0.05 (ANOVA, Fisher’s LSD post-hoc). Features were sum-normalized and log2-transformed. Hierarchical clustering (Ward clustering, Euclidean distance) shows two clusters with opposite fold changes distinguishing between ABHD1 and ABHD3 and the GFP control.

Article Snippet: Mouse Abhd1 (CMV6 promoter, Myc-DDK-tagged, MR206471) and mouse Abhd3 (CMV6 promoter, Myc-DDK-tagged, MR206458) plasmids were obtained from Origene.

Techniques: Biomarker Discovery, Transfection, Control, Western Blot, Over Expression, Membrane, Transformation Assay