Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Blimp1 expression in CD4 + effector and Foxp3 + Treg cells under homeostatic conditions. ( A ) Quantitative Real time-PCR (qRT-PCR) analysis Prdm1 ( Blimp1 ) mRNA (relative to β2-microglobulin) (left) and Western blotting (right) of CD4 + Foxp3 GFP + Treg (nTreg) sorted from Ctrl Foxp3 GFP mice or in vitro differentiated Treg (iTreg,), Th1, Th17 or pathogenic (p) Th17 cells differentiated from naïve cells from the same mice (C57BL/6). (N = 3 mice/group, qPCR and N = 2 mice/sample, Western blotting). ( B ) FACS plot shows Prdm1 mRNA expression (as reported by YFP, Blimp1 YFP ) among peripheral Treg (Foxp3 + as determined by intracellular staining of Foxp3 protein), effector (Foxp3 − CD44 high ) and naïve (Foxp3 − CD44 low ) TCRβ + CD4 + cells in mesenteric lymph nodes (MLN, left) and spleen (SP, right). Bar graph shows the average percentage of Blimp1 YFP + cells among TCRβ + CD4 + Foxp3 + , TCRβ + CD4 + Foxp3 − CD44 high or TCRβ + CD4 + Foxp3 − CD44 low cells. ( C ) FACS plots and histograms overlay show percent of Blimp1 YFP + cells in gated TCRβ + CD4 + Foxp3 + cells from thymus (THY), spleen (SP), mesenteric lymph nodes (MLN) and large (LI) intestines lamina propria (LP) from control (open histogram) and Blimp1 YFP (filled histogram) mice. Gating of Foxp3 + cells (as determined by intracellular staining of Foxp3 protein) is shown in FACS plots on the left. Cumulative data from several mice is shown on graph (right). ( D ) FACS histograms show analysis of Blimp1 YFP expression in gated TCRβ + CD4 + Foxp3 + Neuropilin-1 (Nrp-1) + (full line, empty histograms) and TCRβ + CD4 + Foxp3 + Nrp-1 − (dashed line, filled histograms) cells in THY, SP, MLN and LI-LP from Blimp1 YFP mice. Lower panel shows percent of Blimp1 YFP + cells in CD4 + Foxp3 + Nrp-1 + (filled circles) and CD4 + Foxp3 + Nrp-1 − (open circles) cells iin different organs. For all graphs, each symbol represents one mouse. (N = 2–11 mice/group). * P < 0.05 and ** P < 0.01, one-way ANOVA ( B ) and paired t - test ( C ). Error bars indicate SEM.

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, In Vitro, Staining, Control

Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Increased Treg numbers in Blimp1 CKO mice is a cell-intrinsic effect associated with increased proliferation and decreased cell death. ( A ) Expression of Prdm1 (Blimp) mRNA, (qRT-PCR, relative to β2Microglobulin) in sorted Treg (CD4 + Foxp3 GFP+ ) and Teff (CD4 + Foxp3 − CD44 high ) cells from control ( Prdm1 F/F ), Foxp3 CRE CKO ( Prdm1 F/F Foxp3 cre+ ), and CD4 CRE CKO ( Prdm1 F/F CD4 CRE+ ) mice ( B ) Left panel; representative FACS plots of TCRβ + CD4 + Foxp3 + cells (as determined by intracellular staining of Foxp3 protein) in large intestine lamina propria (LI-LP), mesenteric lymph nodes (MLN), spleen (SP) and thymus (THY) from control (Ctrl) or Blimp1 CKO (CD4 cre CKO or Foxp3 cre CKO) mice. Right graphs; percentage of Foxp3 + cells among TCRβ + CD4 + cells (top) and absolute numbers of Foxp3 + cells (bottom) in LI-LP, MLN, SP and THY. Each symbol represents one mouse (N ≥ 4mice/group). Bars indicate average ± SEM. * P < 0.05, ** P < 0.01, one-way ANOVA. ( C ) Top: graphs showing the average percentage ± SEM of Ki-67 + or Zombie (fixable viability dye) + cells among CD4 + Foxp3 + (as determined by intracellular staining of Foxp3 protein) Foxp3 YFP-cre+ or CD4 + Foxp3 + Foxp3 YFP-cre- cells from Foxp3 cre CKO female mice. Each circle represents one mouse. Bottom: representative FACS plots showing analysis of Ki-67 + or Zombie + cells (N = 5 mice). ** P < 0.01, paired t -test. In ( A ) results representative of two different experiments; In ( B ) cell numbers were calculated based on the total number of cells obtained from each organ and population frequency determined by FACS analysis.

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: Expressing, Quantitative RT-PCR, Control, Staining

Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Blimp1 regulates unique and commonly shared genetic programs in CD4 + Treg and Teff cells. ( A ) Schematic representation of experimental approach used to obtain allotype-marked Blimp1-sufficient (Ctrl, Thy1.1 + ) and deficient (CKO, Thy1.2 + ) Teff (CD4 + Foxp3 − ) and Treg (CD4 + Foxp 3+ ) cells differentiated in the same environment in vivo . ( B ) Venn diagrams representing the number of deferentially expressed (DE) genes upregulated (top) and downregulated (bottom) in pTreg and Teff cells (CD4 cre CKO versus Ctrl) generated from Ctrl and CD4 cre CKO CD4 + naive T cells co-injected into RAG1 −/− mice and used for mRNA microarray analysis. Three biological replicates were used for each sample. Bar graphs under Venn diagram show fold change for selected genes in each group. ( C ) IPA analysis of DE genes (1.5-fold at P < 0.05): shown are comparison analysis of diseases and bio functions between common DE genes in both Foxp3 GFP + pTreg (pTreg ) and Foxp3 GFP − CD44 high (CD4 + Teff) cells, DE genes in CD4 + Teff cells only and in pTreg cells only (−log 10 p value cut-off; 1.3).

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: In Vivo, Generated, Injection, Microarray, Comparison

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Schematic of copper transport between the blood and brain in controls (A) and schizophrenia (B). Stars show copper. CTR1, white arrows; ATP7A, black arrows; ATP7B, striped arrows; BBB, blood brain barrier; BCB, blood cerebrospinal barrier. Thinner arrows indicate decreased protein levels.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques:

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Protein levels of ATP7A (A-B), CTR1 (C-D), dysbindin 1A, 1B/C (E-F) and ATP7B (G-H) for analysis of diagnostic group and treatment status. Error bars represent standard deviation. Significant omnibus ANOVA results are: N-terminal ATP7A: p=0.02; C-terminal ATP7A: p=0.01; transmembrane CTR1: p=0.001. Significant ANOVAs were followed by post hoc tests illustrated by the following: *: p<0.05; **: p<0.01; ***: p<0.001.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Diagnostic Assay, Standard Deviation

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Numerical labeling of amino acid ranges are shown for each protein segment. Dotted boxes indicate antibody-specific epitopes. A. ATP7A protein structure. N-terminal antibody specifically binds to 225-273aa; C-terminal antibody binds to 1403-1452aa. B. CTR1 protein structure. Extracellular CTR1 antibody binds to 19-68aa; transmembrane CTR1 antibody specifically binds to 140-190aa. C. Intracellular diagram of copper chaperones and enzymes and schizophrenia-related alterations. Copper enters neurons after transport from astrocytes via CTR1 and is immediately bound by MT/ GSH. MT/GSH then delivers copper to chaperone ATOX1 or the TGN where it is delivered to other metalloenzymes (e.g., SCO1 → COX) via ATP7A. The SN exhibits more ATOX1 than other brain regions. SCO1 is a copper-requiring enzyme involved in the last step of the electron transport chain of ATP synthesis. SCO1, MT/GSH, COX, ATP, ATP7A and CTR1 are all downregulated or altered in schizophrenia. Abbreviations: MT, metallothionein; GSH, glutathione; APD, antipsychotic drug; SN, substantia nigra; COX, cytochrome c oxidase. References: 1) The present paper; 2) ; 3) ; 4) ; 5) ; 6) ; ; 7) ; 8) ; 9) ; 10) ; and 11) .

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Labeling