Journal:

Article Title: Gene-expression signature of benign monoclonal gammopathy evident in multiple myeloma is linked to good prognosis

doi: 10.1182/blood-2006-07-037077

Figure Lengend Snippet: Fifty-two SAM-defined genes are differentially expressed in NPC, MGUS, and MM

Article Snippet: 2.05 1.55 3.18 1.36 212038_s_at VDAC1 Ion channel for cytochrome c 2.62 1.56 3.17 1.42 208308_s_at GPI Energy metabolism 1.97 1.48 3.17 1.48 201013_s_at PA1CS DNA synthesis 2.81 1.43 3.17 1.45 202708_s_at HIST2H2BE Chromosome organization and biogenesis 2.35 1.71 3.14 2.11 215071_s_at HIST1H2AC Chromosome organization and biogenesis 3.47 3.62 3.13 1.97 225361_x_at LOC159090 Unknown 3.43 1.68 3.11 1.53 219366_at AVEN Antiapoptosis 2.63 1.76 3.10 1.47 209398_at H1ST1H1C Chromosome organization and biogenesis 5.39 5.53 3.09 2.02 221652_s_at C12orf11 Unknown; sarcoma antigen NY-SAR-95 2.07 1.37 3.06 1.57 225028_at LOC550643 Unknown 3.83 2.01 3.03 1.45 214214_s_at C1QBP Immunity 2.64 1.45 3.03 1.47 201577_at NME1 Nucleotide biosynthesis 3.57 1.62 2.99 1.58 218280_x_at H1ST2H2AA Chromosome organization and biogenesis 4.24 4.76 2.95 1.92 201479_at DKC1 Telomere maintenance 2.24 1.42 2.92 1.43 208864_s_at TXN Redox reactions 2.90 1.68 2.91 1.51 212297_at ATP13A3 Cation transport 2.64 1.42 2.88 1.54 222825_at OTUD6B Unknown 2.66 1.35 2.88 1.40 209267_s_at SLC39A8 Ion transport 3.41 1.86 2.88 1.56 217898_at C15orf24 Unknown 2.35 1.70 2.83 1.33 210275_s_at ZA20D2 Unknown 2.27 1.36 2.81 1.37 213485_s_at ABCC10 ATP-dependent efflux pump; multidrug resistance pump 3.17 1.51 2.76 1.34 200994_at IPO7 Nuclear trafficking 2.08 1.46 2.72 1.37 222428_s_at LARS Protein synthesis 3.64 1.68 2.71 1.32 202591_s_at SSBP1 Mitochondrial DNA replication 2.22 1.42 2.69 1.35 204244_s_at ASK Cell cycle 3.25 1.77 2.67 1.37 225916_at ZNF131 Gene transcription 2.47 1.39 2.63 1.36 202396_at TCERG1 Gene transcription 3.85 1.62 2.63 1.32 213340_s_at K1AA0495 Unknown −2.40 0.78 −2.52 0.75 206150_at TNFRSF7 Immune cell signaling −3.82 0.55 −2.56 0.75 228139_at RIPK3 Cell signaling −2.63 0.74 −2.59 0.77 220066_at CARD15 Antiapoptosis −4.02 0.50 −2.66 0.62 210347_s_at BCL11A Gene transcription 3.48 2.04 −2.67 0.64 232511_at RANBP2L1 Nuclear import 2.07 1.54 −2.71 0.64 214041_x_at RPL37A Ribosomal protein 2.22 1.80 −3.09 0.60 219371_s_at KLF2 Gene transcription 3.81 1.78 −3.11 0.66 202724_s_at FOXO1A Gene transcription 2.49 1.55 −3.27 0.64 215671_at PDE4B Drug metabolism 2.07 1.53 −3.30 0.59 219675_s_at UXS1 Glycosaminoglycan biosynthesis −2.33 0.76 2.97 1.35 Open in a separate window Genes are ordered based on the SAM score in the MM vs MGUS comparison.

Techniques: Binding Assay, DNA Synthesis

Journal: bioRxiv

Article Title: The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

doi: 10.1101/2020.08.27.270223

Figure Lengend Snippet: (A, B) MCF-7 (A) and MCF-10 (B) cells were pretreated with CTL1/2 inhibitor hemicholinium (HC-3, 200μM) or anti-CTL1 antibody (1:500) and then radiolabeled with 0.2 μCi [ 14 C]-Etn or [ 3 H]-Cho for 24h. Both [ 3 H]PC and [ 14 C]PE were significantly reduced in HC-3 and anti-CTL1 treated cells relative to untreated (-) cells. (C) 0.2 μCi [ 14 C]-Etn or [ 3 H]-Cho uptake measured after 20 min in MCF7 cells were significantly reduced with HC-3 (200 μM) added 20 min prior to the labeling). (D) Separately or together, excess of ‘cold’ Cho and Etn (200 μM each, 20 min) inhibited the transport of both radiolabeled substrates. (E-G) Anti-CTL1 and anti-CTL2 antibodies inhibited [ 14 C]-Etn (E, F) and [ 3 H]-Cho (G) transport in primary human fibroblasts in a dose-response manner. Each bar represents the mean ± SD (n = 4); * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Confluent cells were transfected with 5 μg of pCMV3-ORF-C-Myc-SLC44A1 cDNA (Sino Biologics, # RG80408-CM), pCMV-SPORT6-SLC44A2 cDNA (Genomics Online, # ABIN3822596) or empty vector (pcDNA4His-Max B, # V86420) using Lipofectamine 2000 (Invitrogen).

Techniques: Labeling

Journal: bioRxiv

Article Title: The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

doi: 10.1101/2020.08.27.270223

Figure Lengend Snippet: (A ) Saturation curves of Etn transport (V (velocity) vs. S (substrate conc), were produced by measuring the uptake of [ 14 C]-Etn (0-1000 μM, 20 min) in control human fibroblasts (Ctrl), CTL1 deficient human fibroblasts (M1 and M2) and monkey COS-7 cell line (B) The Eadie-Hofstee plots derived from those curves demonstrated the presence of two Etn transport systems, with binding constants K1 (higher affinity) and K2 (lower affinity), in Ctrl and COS-7 cells. Only one transport system with the lower affinity was present in CTL1 deficient M1 (279.6 ± 17.1 μM (K 2 )) and M2 (275.4 ± 13.9 μM (K 2 )) cells. (C) CTL1 protein (72 kDa) was detected in Ctrl and COS-7 cells while a truncated, low abundant 58 kDa protein was detected in M1 cells. No CTL1 protein was detected in M2 cells showing that the Eadie-Hofstee plots K2 Etn transport system in those cells is CTL2 related. An intact 72 kDa CTL2 protein was detected in M1 and M2 cells. (D) CTL2 was not affected while CTL1 mRNA was almost diminished in M2 cells. (E) The [ 14 C]-Etn uptake is down-regulated with low extracellular pH (high [H + ]) and upregulated at high pH (low [H + ]) in both Ctrl (CTL1+CTL2) and M2 (CTL2 only) cells. (F) Saturation curves and Eadie-Hofstee plots of siRNA-CTL2 treated Ctrl cells showed the presence of a single Etn transport of a higher affinity (CTL1-mediated, K1, transport); the low affinity, CTL2-mediated, K2 transport was specifically depleted with the siRNA-CTL2 treatment. (G) Time course (0-30 min) of 10 μM [ 14 C]-Etn uptake in the presence and absence of Na + ions in Ctrl and M2 cells. Each bar or point represents the mean ± SD (n = 4), * p < 0.05, ** p < 0.01.

Article Snippet: Confluent cells were transfected with 5 μg of pCMV3-ORF-C-Myc-SLC44A1 cDNA (Sino Biologics, # RG80408-CM), pCMV-SPORT6-SLC44A2 cDNA (Genomics Online, # ABIN3822596) or empty vector (pcDNA4His-Max B, # V86420) using Lipofectamine 2000 (Invitrogen).

Techniques: Produced, Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

doi: 10.1101/2020.08.27.270223

Figure Lengend Snippet: (A) CTL1 and CTL2 protein levels in Ctrl and M2 cells transfected with CTL1 cDNA, CTL2 cDNA and CTL2 siRNA. (B) In M2 cells transfected with CTL1 cDNA, Etn uptake was increased by 25% after 24h and 75% after 48h. In M2 cells transfected with CTL2 cDNA, Etn uptake increased by 25% after 48h. (C) Cho uptake at 48h was increased by 50% in Ctrl cells transfected with CTL1 cDNA and 25% in Ctrl cells transfected with CTL2 cDNA. (D) Etn uptake was increased by 25% in Ctrl cells transfected with CTL1 cDNA and by 25% in Ctrl cells transfected with CTL2 cDNA. (E) [ 14 C]-Etn radiolabeling of CDP-Etn pathway and PE synthesis in CTL1 overexpressing M2 cells. (F) [ 14 C]-Etn 1-3 h pulse-chase analysis of CDP-Etn pathway and PE degradation in CTL1 overexpressing M2 cells. Each bar or point represents the mean ± SD (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Confluent cells were transfected with 5 μg of pCMV3-ORF-C-Myc-SLC44A1 cDNA (Sino Biologics, # RG80408-CM), pCMV-SPORT6-SLC44A2 cDNA (Genomics Online, # ABIN3822596) or empty vector (pcDNA4His-Max B, # V86420) using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Radioactivity, Pulse Chase

Journal: bioRxiv

Article Title: The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

doi: 10.1101/2020.08.27.270223

Figure Lengend Snippet: (A) Western blots of COXIV mitochondrial marker demonstrating purity of Ctrl and M2 mitochondria; and CTL2 mRNA expression in siRNA-CTL2 treated and untreated M2 cells with complement protein data in . (B) Mitochondrial Etn transport and the effect of CTL2 knockdown in M2 cells; Etn uptake in Ctrl mitochondria was reduced to 25% in M2 (CTL1 deficient) mitochondria and further diminished with siRNA depletion of CTL2. Time course inhibition of Etn uptake in (C) Ctrl mitochondria and (D) whole cells treated with 20 μM HC-3. Time course of Etn uptake in (E) M2 mitochondria and (F) M2 cells treated with 20 μM HC-3. Each point or bar represents the mean ± SD (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001. (G) Schematic representation of ethanolamine transport mechanisms at the cell surface and mitochondria. CTL1 and CTL2 are ethanolamine/proton antiporters of high and low affinity, respectively. They are responsible for extracellular uptake and intracellular balance of [Etn]. Extracellular Etn is immediately phosphorylated by ethanolamine kinase (EK) and further consumed by the CDP-Etn pathway to form PE phospholipid. The intracellular Etn is released by PE degradation by lipolysis and PE exchange to PS by PS synthase 2 (PSS2) and it can enter mitochondria by both transporters.

Article Snippet: Confluent cells were transfected with 5 μg of pCMV3-ORF-C-Myc-SLC44A1 cDNA (Sino Biologics, # RG80408-CM), pCMV-SPORT6-SLC44A2 cDNA (Genomics Online, # ABIN3822596) or empty vector (pcDNA4His-Max B, # V86420) using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Marker, Expressing, Inhibition

Journal: Nature immunology

Article Title: The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

doi: 10.1038/ni.2863

Figure Lengend Snippet: Figure 1 Naive polyclonal Traf5−/− CD4+ T cells display an enhanced TH17 phenotype in vitro. (a) Expression of IFN-γ, IL-4, Foxp3 and IL-17A in activated CD4+ T cells generated from naive wild-type (WT) or Traf5−/− (KO) B6 CD4+ T cells cultured with anti-CD3 and anti-CD28 in various polarizing conditions (left margin; α-, anti-) and restimulated for 5 h with the phorbol ester PMA and ionomycin. Numbers adjacent to outlined areas (left) indicate percent IFN-γ+, IL-4+, Foxp3+ or IL-17A+ (top to bottom) CD4+ T cells. (b) Quantitative RT-PCR analysis of the expression of TH17-associated genes (horizontal axis) in activated CD4+ T cells generated from naive wild-type or Traf5−/− B6 CD4+ T cells cultured for 24 h with anti-CD3 and anti-CD28 in TH17-polarizing conditions (right margin), presented relative to the expression of the gene encoding β-actin. ND, not detected. (c) Enzyme-linked immunosorbent assay of primary IL-17A, IL-21 and IFN-γ in supernatants of cells as in a (polarizing conditions, below graphs), assessed at day 2 (IL-17A and IFN-γ) or at day 3 (IL-21) after initial activation of naive CD4+ T cells. (d) Expression of IFN-γ, IL-4, IL-17A and Foxp3 in activated CD4+ T cells generated from naive wild-type or Traf5−/− B6 CD4+ T cells cultured with anti-CD3 plus wild-type B6 splenic APCs (after depletion of T cells), in various polarizing conditions (left margin), then restimulated for 5 h with PMA and ionomycin. Numbers adjacent to outlined areas indicate percent IFN-γ+IL-4− cells (top left) or IFN-γ−IL-4+ cells (bottom right) (top two rows), or IL-17A+Foxp3− cells (top left) or IL-17A−Foxp3+ cells (bottom right) (bottom two rows). NS, not significant; *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results (average and s.d. of triplicate wells).

Article Snippet: A pCMV6 entry expression vector containing cDNA encoding mouse TRAF5 (TRAF5–c-Myc; MR208836) was from Origene.

Techniques: In Vitro, Expressing, Generated, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Nature immunology

Article Title: The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

doi: 10.1038/ni.2863

Figure Lengend Snippet: Figure 2 Traf5−/− OT-II CD4+ T cells exhibit enhanced TH17 development in vitro and in vivo. (a) Enzyme-linked immunosorbent assay of primary IL-17A and IL-21 in supernatants of activated CD4+ T cells generated from naive wild-type or Traf5−/− OT-II CD4+ T cells stimulated for 3 d with 0.1 µM OVA peptide (amino acids 323–339) and wild-type B6 splenic APCs (after depletion of T cells) in the presence of various concentrations of IL-6 (horizontal axes). (b) Recall IL-17A production in activated CD4+ T cells generated from naive wild-type or Traf5−/− OT-II CD4+ T cells stimulated for 5 d with OVA and APCs as in a in the presence of 10 ng/ml of IL-6, then restimulated for 5 h with PMA and ionomycin. Numbers adjacent to outlined areas (left) indicate percent IL-17A+CD4+ cells. (c) Quantification of IL-17A+ OT-II (CD4+CD45.2+) cells obtained from the draining lymph nodes of CD45.1+ B6.SJL hosts given naive wild-type or Traf5−/− OT-II (CD45.2+) donor cells on day 0, followed by immunization of the host with OVA in CFA on day 1 and node harvest on day 8, then restimulation of the cells in vitro for 5 h with PMA and ionomycin (numbers adjacent to outlined areas (left), as in b). *P < 0.05 (Student’s t-test). Data are from one experiment representative of three independent experiments with similar results (average and s.d. of triplicate wells in a; average and s.e.m. of three replicates in b; average and s.e.m. of four mice per group in c).

Article Snippet: A pCMV6 entry expression vector containing cDNA encoding mouse TRAF5 (TRAF5–c-Myc; MR208836) was from Origene.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Generated

Journal: Nature immunology

Article Title: The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

doi: 10.1038/ni.2863

Figure Lengend Snippet: Figure 4 Constitutive binding of TRAF5 to gp130 inhibits the IL-6-mediated recruitment of STAT3 to gp130. (a) Immunoassay of HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged TRAF5 and c-Myc-tagged gp130, followed by immunoprecipitation (IP) of proteins from lysates with anti-V5, anti-c-Myc or control immunoglobulin G (IgG) and immunoblot analysis with anti-c-Myc and anti-V5. (b) Immunoprecipitation of endogenous gp130 from lysates of splenic wild-type or Traf5−/− B6 CD4+ T cells (2.5 × 108 per sample) with monoclonal antibody to gp130, followed by immunoblot analysis with anti-gp130 or anti-TRAF5. Input (bottom), immunoblot analysis of lysates without immunoprecipitation. *, heavy chain. (c) Immunoprecipitation of gp130 from lysates of HEK cells transiently transfected with plasmid vectors encoding c-Myc-tagged gp130 together with V5-tagged TRAF5(1–558), TRAF5(1–241) or TRAF5(242–558) (above lanes; identified at top), followed by immunoblot analysis with anti-V5 or anti-c-Myc. *, heavy chain. (d) Immunoassay of HEK cells transduced to express c-Myc-tagged gp130 and STAT3 together with V5-tagged TRAF5(242–558) (lanes 2, 4 and 5) or empty vector (lanes 1 and 3), then left unstimulated (US) or stimulated for 15 min with IL-6–IL-6R (500 ng/ml), followed by immunoprecipitation of proteins from lysates with anti-c-Myc or control IgG and immunoblot analysis with anti-c-Myc, anti-STAT3 or anti-V5. (e–g) Immunoassay of HEK cells transduced with plasmids encoding gp130 mutants (above lanes) with various deletions in the cytoplasmic region in positions 774–848 (above blot, e) or with substitution of alanine for other amino acids in TRAF-binding sites 1 and 2 (above blots, f,g) and cotransfected to express V5-TRAF5(242–558), followed by immunoprecipitation of proteins from lysates with control IgG or anti-c-Myc and immunoblot analysis with anti-V5 or anti-c-Myc. Data are from one experiment representative of at least two independent experiments with similar results.

Article Snippet: A pCMV6 entry expression vector containing cDNA encoding mouse TRAF5 (TRAF5–c-Myc; MR208836) was from Origene.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Control, Western Blot, Transduction