Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: HDAC2 in NSCs is a mediator for the role of neuronal nNOS–PSD-95 association in regulating the fate of NSCs. A, B, Immunoblots showing HDAC2 levels (n = 4; A) and bar graph showing the percentage of newborn neurons (n = 3; B) in the NSCs infected by LV-HDAC2-shRNA, LV-control-shRNA, AD-HDAC2-Flag, or AD-Flag. C, Bar graph showing the percentage of newborn neurons in the DG of rats infected by AD-HDAC2-Flag or AD-Flag (n = 9). These rats were subjected to MCAO at 24 h after virus infection and were killed for BrdU/DCX staining at day 14 after MCAO administration. D–F, Bar graphs showing the percentage of newborn neurons (D), the percentage of neurons with multineurites (E), and total neurite length per newborn neuron with multineurites (F) in the NSCs treated with DETA/NONOate (50 μm) with or without NO scavenger C-PTIO (10 μm) for 24 h at day 2 after differentiation. The cultures were stained 4–5 d after treatment. (n = 3). G, Nitrosylation of total proteins or HDAC2 in NSCs. NSCs were treated with 50 μm DETA/NONOate for the indicated times (G1), with 50 μm GSNO for 4 h (G2), or with 50 μm DETA/NONOate for 4 h (G3) at day 2 after differentiation (n = 4). PC, Positive control, in which the lysate of NSCs was treated with GSNO (200 μm) for 30 min; BSA, biotin-switch assay. H, Immunoblots showing HDAC2 levels in the NSCs treated with 50 μm DETA/NONOate for the indicated times at day 2 after differentiation. (n = 3–5). I, Bar graphs showing HDAC2 activity in the NSCs treated with 50 μm DETA/NONOate for the indicated times (left) or by 50 μm DETA/NONOate with or without 10 μm NO scavenger C-PTIO for 4 h (right) at day 2 after differentiation. n = 3–5. J, K, HDAC2 levels (J; n = 5) and enzymatic activity (K; n = 4) in the solo-cultured or neuron-cocultured NSCs with or without C-PTIO. All cultures were exposed to OGD for 2 h at day 2 after NSC differentiation, and the NSCs were collected 6 h after OGD finish. To measure HDAC2 in NSCs selectively, coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells. C-PTIO (10 μm) or vehicle was added to the medium of NSCs in wells at the start of OGD exposure. L, M, The percentage of newborn neurons (L) and the percentage of newborn neurons with multineurites (M) in the cocultures of LV-control-shRNA- or LV-HDAC2-shRNA-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h at day 2 after differentiation and stained at days 4∼5 after OGD (n = 4). N, O, Bar graphs showing the percentage of newborn neurons (N) and the percentage of newborn neurons with multineurites (O) in the cocultures of AD-HDAC2-flag- or AD-flag-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h and treated with 10 μm ZL006 for 26 h beginning at OGD administration, and were stained at days 4∼5 after OGD administration. n = 5. Data are the mean ± SEM. *p < 0.05, **p < 0.01, two-tailed t test for C, ANOVA for others.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, Infection, shRNA, Control, Virus, Staining, Positive Control, Biotin Switch Assay, Activity Assay, Cell Culture, Membrane, Pore Size, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: Association of nNOS–PSD-95 in neurons and consequent NO production may negatively regulate NSC fate via HDAC2-mediated histone deacetylation and NeuroD downregulation. A, B, Immunoblots showing acetyl-H4 (A) and NeuroD (B) levels in the NSCs treated by 50 μm DETA/NONOate with or without 10 μm C-PTIO for 8 h at day 2 after differentiation (n = 4). C, D, Immunoblots showing acetyl-H4 (C) and NeuroD (D) levels in the NSCs treated by 50 μm DETA/NONOate with or without 2 nm TSA (an HDAC inhibitor that was added 30 min before DETA/NONOate) for 8 h at day 2 after differentiation (n = 4). E, F, Immunoblots showing acetyl-H4 (E) and NeuroD (F) levels in the NSCs treated with 50 μm DETA/NONOate with or without LV-HDAC2-shRNA infection for 8 h at day 2 after differentiation (n = 4). DETA/NONOate was added at day 6 after LV-HDAC2-shRNA or LV-control-shRNA infection. G, H, Immunoblots (G) and immunofluorescence (H) showing acetylation of histone H4 in the NSCs cocultured with neurons after OGD. Coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells (n = 4). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ANOVA.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, shRNA, Infection, Control, Immunofluorescence, Membrane, Pore Size

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1 and Gαi3 DKO blocks BDNF-induced Akt–mTORC1 and Erk activation in MEFs. (A–F) WT or Gαi1/3-DKO MEFs were treated with BDNF (applied at the indicated concentrations for the indicated times) or with PDGF-BB (25 ng/mL) and were analyzed by Western blotting for the listed proteins. For Western blot assays, equal amounts of quantified protein lysates were loaded (30 μg per treatment) or the same set of lysate samples was run on identical gels. The indicated proteins were quantified using ImageJ software (A, C–E, and F; n = 5). *P < 0.001 by Student’s t test. Data are reported as mean ± SD. (G and H) Immunofluorescence analysis was performed to test p-Akt (G) and p-Erk1/2 (H). (Scale bars: 10 μm.)

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: Activation Assay, Western Blot, Software, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1 and Gαi3 are required for BDNF-induced Akt–mTORC1 and Erk activation in MEFs. (A and B) WT MEFs, Gαi1- or Gαi3-SKO MEFs, Gαi2-SKO MEFs, and Gαi1/3-DKO MEFs were treated with BDNF (25 ng/mL) for the indicated time and were analyzed by Western blot for the listed proteins. (C) DKO MEFs were transiently transfected with plasmids encoding EE-Gαi1-cDNA, EE-Gαi3-cDNA, or empty vector. MEFs were treated with BDNF (25 ng/mL) for the indicated time and were tested by Western blot for the listed proteins. (D) WT MEFs were transfected with 100 nM of scr-siRNA, Gαi1, or Gαi3 siRNA, and MEFs stably expressing CRISPR/Cas9-Gαi1 and CRISPR/Cas9-Gαi3 were treated with BDNF (25 ng/mL) and were tested by Western blot for the listed proteins. *P < 0.01 vs. WT MEFs (A) and vs. scr-siRNA (D). #P < 0.001 (A and D). #P < 0.001 vs. vector (C).

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, CRISPR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1/3 are required for the TrkB adaptor complex formation. (A and B) WT and Gαi1/3-DKO MEFs were treated with BDNF (25 ng/mL) for 5 min, and the association among TrkB, Gαi3, and adaptor proteins was tested by the immunoprecipitation assay. Input control shows the expression of listed proteins in the total cell lysates. (C–E) WT MEFs, Gαi1- or Gαi3-SKO MEFs, and Gαi1/3-DKO MEFs (C), WT MEFs or Gαi1/3 MEFs transfected with shRNA or scr-shRNA (D), and WT and Gαi2-KO MEFs were treated with BDNF and p-Gab1 and were tested by Western blot analysis. β-Actin served as the loading control. (F) DKO MEFs were transiently transfected with vector encoding EE-Gαi1-cDNA, EE-Gαi3-cDNA, or empty vector, and BDNF (25 ng/mL)-induced Gab1 phosphorylation was analyzed by Western blot. (G) WT or Gab1-KO MEFs were treated with BDNF (25 ng/mL) for the indicated time and were analyzed by Western blotting for the listed proteins. *P < 0.001 vs. WT MEFs.

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: Immunoprecipitation, Control, Expressing, Transfection, shRNA, Western Blot, Plasmid Preparation, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1/3 are required for BDNF-induced signaling and TrkB adaptor complex formation in cerebellar granule neurons. (A and C) Lentiviral-mediated shRNA knockdown of Gαi1/3 disrupts downstream signaling. Primary cultured cerebellar granule neurons transduced with lentiviral Gαi1 shRNA plus Gαi3 shRNA or scr-shRNA for 24 h were treated with BDNF (25 ng/mL), and Akt, S6, MEK1/2–Erk1/2, SHP2, and Gab1 phosphorylation was analyzed by Western blotting. (B and D) Lentiviral-mediated shRNA knockdown of Gαi1/3 decreases the association between TrkB and Gαi1/3, SHP2, APPL1, and Gab1. Primary cerebellar granule neurons transduced with Gαi1 shRNA plus Gαi3 shRNA or scr-shRNA were stimulated with BDNF (25 ng/mL), and the association was tested by immunoprecipitation. *P < 0.001 vs. scr-shRNA.

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: shRNA, Knockdown, Cell Culture, Transduction, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1/3 are required for BDNF signaling, endocytosis, and dendrite outgrowth in the hippocampal neurons. (A) Lentiviral-mediated shRNA knockdown of Gαi1/3 disrupts downstream TrkB signaling and TrkB endocytosis in primary hippocampal neurons. Hippocampal neurons were infected with lentiviral Gαi1 shRNA plus lentiviral Gαi3 shRNA (shGαi1/3) or a lentiviral scr-shRNA for 24 h. Neurons were stimulated with BDNF (25 ng/mL) and analyzed for the listed proteins in total and cell-surface lysates. (B and C) Lentiviral-mediated shRNA Gαi1/3 knockdown decreases dendrite morphology in primary hippocampal neurons. On day 5 after infection, neurons were stained for GFP expression (encoded by the lentiviral shRNA vectors), and the morphology of hippocampal neurons was quantified. (B) Representative images of anti-GFP staining are presented. Arrowheads indicate spines. (Scale bars: 25 μm; magnification: Inset, 6.8×.) (C) Dendrite length, branching, soma area, and the number of dendritic spines were quantified. For spine analysis, 30-μm-long dendritic segments (50–80 μm from soma) were selected, and spines from 40 neurons were counted. (D) A representative image of GFP expression 7 d after hippocampal injection of a lentiviral GFP control virus shows a high efficiency of infection. (E) Intrahippocampal lentiviral shRNA-mediated knockdown of Gαi1/3 decreases Akt and Erk1/2 activity, as analyzed by Western blotting with phospho-specific antibodies. Hippocampi were isolated and tested by Western blotting of listed proteins (n = 5). (F–I) Intrahippocampal lentiviral shRNA-mediated knockdown of Gαi1/3 decreases the formation of secondary dendrites and spines. (F and H) Representative images showing hippocampal neuron morphology are presented for control and shRNA Gαi1/3-infected neurons stained for GFP. Arrowheads in H indicate spines. (Scale bars: 25 μm in F and 5 μm in H.) (G) The number of neurons and dendrites per neuron were counted. (I) Spines were counted from 30-μm-long dendritic segments (50–80 μm from soma) of 40 randomly selected neurons. *P < 0.001 vs. GFP. #P < 0.001.

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: shRNA, Knockdown, Infection, Staining, Expressing, Injection, Control, Virus, Activity Assay, Western Blot, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Gαi1/3 knockdown in the hippocampus produces depressive behaviors. (A–C) CMS exposure decreases Gαi1 and Gαi3 expression in the hippocampus. Western blot analysis of Gαi1 and Gαi3 expression in the hippocampus (A) and cortex (B) of mice exposed to the CMS model for 14–56 d compared with control hippocampus (C). (D–F) Gαi1 shRNA or Gαi3 shRNA injection into the hippocampus elicited depressive behaviors. Immobility times in the FST (D) and TST (E) and sucrose water preference (F) were examined on day 7 after intrahippocampal injection of GFP or lentiviral Gαi1 shRNA and/or Gαi3 shRNA. (G–J) Exogenous Gαi3 expression in the hippocampus induces antidepressive behavior. On day 7 after intrahippocampal injection of Ad-GFP or Ad-Gαi3, immobility times in the FST (I) and TST (J) were tested, and then hippocampi were isolated and analyzed by Western blotting of the listed proteins (G and H). (K–M) Exogenous expression of Gαi3 reversed CMS-induced depressive behavior. The immobility times in the FST (K) and TST (L) and sucrose water preference (M) were tested in control and CMS mice with or without intrahippocampal injection of Ad-GFP or Ad-Gαi3. #P < 0.001 vs. GFP (D–F, I, and J). *P < 0.001 vs. control mice (K–M). #P < 0.001 (D–F and K–M).

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: Knockdown, Expressing, Western Blot, Control, shRNA, Injection, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus

doi: 10.1073/pnas.1722493115

Figure Lengend Snippet: Severe depressive-like behaviors in Gαi1/3-DKO mice. (A) Depletion of Gαi1 and Gαi3 in the DKO mice disrupts signaling. The expression of the listed proteins in the CA1 hippocampus of WT and Gαi1/3-DKO mice was examined by Western blot analysis. (B–D) DKO mice display depressive behaviors. For both WT and Gαi1/3-DKO mice, the FST (B), TST (C), and sucrose water preference test (D) were performed. (E–J) Analysis of DKO hippocampal CA1 neuronal morphology. (E and H) Representative images of CA1 pyramidal hippocampal neuronal morphology. Arrowheads indicate spines. (Scale bars: 25 μm in E and 5 μm in H.) (F) The number of neurons in randomly selected 200 × 200 μm fields was counted. (G and I) The number of secondary dendrites (G) and spines (I) in 40 random neurons were counted. Spines were analyzed from 30-μm-long apical dendritic segments (50–80 μm from soma). (J) The maximum spine width of 200 spines from 10 randomly selected neurons was measured by Image J software. *P < 0.001 vs. WT mice.

Article Snippet: Lentivirus with shRNA targeting murine Gαi1 (sc-41751-V; Santa Cruz Biotechnology) or murine Gαi3 (sc-37255-V; Santa Cruz Biotechnology) were added for 18 h. For stable cell lines, MEFs infected with the lentiviral Gαi1/3 shRNA were selected with puromycin (1.0 μg/mL).

Techniques: Expressing, Western Blot, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bone Marrow CD11c + Cell-Derived Amphiregulin Promotes Pulmonary Fibrosis

doi: 10.4049/jimmunol.1502479

Figure Lengend Snippet: Flow sorted BM-derived CD11c+ cells from SAL or BLM treated donor mice as indicated were transferred endotracheally into recipient mice which had been treated with low dose (1U/kg body weight) BLM or SAL 2 days prior to cell transfer. The effects of this cell transfer on, A) lung hydroxyproline content (n=3), and B) lung Col I mRNA levels are shown (n=3). C) BM-derived CD11c+ cells were transfected with control (‘C’) or AREG shRNA (‘C-sh’), and then transferred endotracheally into recipient mice, which had been treated with SAL or BLM as in (A). The effect of these cell transfers on lung hydroxyproline content of recipient mice are shown (n=3). D) Conditioned media (CM) were prepared from cultures of BM CD11c+ cells isolated from SAL or BLM treated mice as indicated, and their effects on MLF proliferation were measured using the WST-1 cell proliferation assay. The results were expressed as a percentage of the untreated controls (n=5). E) The effects on αSMA and procollagen I (Col I) mRNA levels were evaluated by qRT-PCR in left and right panels, respectively (n=3). F) BM CD11c+ cells from BLM-treated WT or Areg KO (‘KO-CD11c+’) mice were cocultured with MLFs for 48 hours. The MLFs were then harvested for analysis of αSMA mRNA by qRT-PCR (n=3). Data are shown as the mean ± SD. *P<0.05 between the indicated 2 groups.

Article Snippet: Small hairpin RNA (shRNA) transfection BMDCs were transfected with AREG shRNA lentiviral particles or its control (sc-39413-V, Santa Cruz) in accordance with the manufacturer’s instructions.

Techniques: Derivative Assay, Transfection, Control, shRNA, Isolation, Proliferation Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

doi: 10.1371/journal.pone.0092608

Figure Lengend Snippet: ( A ) Transcript knockdown validation of commitment phase candidate genes using Q-PCR (mean ± SD), * p<0.05. ( B ) Q-PCR validation for Ucp-1 transcript levels in cells transduced with or without short hairpin constructs against indicated genes post two days BMP6 pretreatment followed by adipogenic differentiation and 10 uM forskolin stimulation. The expression in parental cells in the absence of forskolin stimulation was set to 1. A reduction in Ucp-1 transcript levels by more than 40% of levels observed in forskolin stimulated parental cells was set as the lower threshold to choose the gene(s) of interest for subsequent investigations. Results represent triplicate analyses of two to three independent biological replicates (mean ± SD). ( C ) Bar graph representation of Optn and Cox2 transcript levels at indicated time points measured using Affymetrix array. ( D ) Verification of Optn and Cox2 induction at the protein level by immunoblot analyses at indicated time points post BMP6 stimulation. Gapdh served as the loading control. ( E ) Knockdown of Optn and Cox2 was validated using immunoblot analyses in basal state and after two days of BMP6 stimulation. ( F ) Q-PCR validation for BAT markers ( Cidea, Cox7a1 ) showing reduced induction in the context of attenuated Optn and Cox2 expression in indicated cells. Expression in Optn and Cox2 proficient cells was set to 1 and Gapdh served as the endogenous control. Results represent triplicate analyses of three independent biological replicates and similar results were obtained in at least two independent analyses (mean ± SD), * p<0.05 . ( G ) Immunoblot analyses for pan-adipogenic markers (PPARγ, Fabp4 and Adiponectin). Quantification of band intensity is provided above the respective blot. Note that the quantification data provided for PPARγ blot corresponds to the intensity of PPARγ2 (top band, panel G). ( H ) Two representative images for Oil-Red-O staining in Optn and Cox2 proficient and knockdown cells are shown. Analyses for panels F, G and H was performed at the end of the differentiation cascade illustrated in following two days of BMP6 (250 ng/mL) stimulation. Also see and .

Article Snippet: The following shRNA lentiviral particles against relevant genes were purchased from Santa Cruz Biotechnology: Optn (sc-39055-V), Sdc3 (sc-41048-V), Smoc2 (sc-63047-V), Lgr6 (sc-60933-V), Prg4 (sc-60973-V), Cox2 (sc-29278-V), Igf-1 (sc-37194-V) and Igf-1Rα/β (sc-35638-V).

Techniques: Knockdown, Biomarker Discovery, Transduction, Construct, Expressing, Western Blot, Control, Staining

Journal: PLoS ONE

Article Title: Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

doi: 10.1371/journal.pone.0092608

Figure Lengend Snippet: (A) A schematic highlighting relevant time points in the differentiation protocol utilized to generate data shown in this figure. (B) Heat map depiction and unsupervised clustering analyses of 4255 significantly changing qualifiers ( p≤0.01, fold change≥1.5 ) in BMP6 stimulated cells from “48 hours post BMP6 stimulation” to “post 3 days in induction media” time points. IGF-1 levels are highlighted in the heat map. Each gene is represented by a single row and each sample in three independent biological replicates for each time point by a column. Two distinct clusters indicate genes induced (red) and repressed (green). (C) Independent Q-PCR validation of IGF-1 and IGF-1R transcripts at indicated time points in the BMP6 stimulated cells. The expression at the 48 hour time point was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), * p<0.05 . (D) Schematic representation of the differentiation protocol for cells with or without IGF-1 or IGF-1R knockdown is illustrated. (E) Knockdown validation of IGF-1 and IGF-1R transcripts as indicated in panel D. The expression in the IGF-1 or IGF-1R proficient cells was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), * p<0.05. (F) Q-PCR validation for Ucp-1 transcript levels in cells transduced with or without short hairpin constructs against indicated genes (IGF-1 or IGF-1R) post two days of BMP6 pretreatment followed by adipogenic differentiation and 4 hours forskolin stimulation. The expression in IGF-1 and IGF-1R proficient cells in absence of forskolin stimulation was set to 1. (G) Q-PCR validation for BAT markers ( Cidea, Cox7a1, Ntrk3 ) in indicated cells pretreated with BMP6 followed by adipogenic differentiation. Expression in IGF-1 or IGF-1R proficient cells was set to 1 and Gapdh served as the endogenous control. Results represent triplicate analyses of three independent biological replicates (mean ± SD), * p<0.05 . (H) Representative bright field images (magnification: 40X) and Oil-Red-O staining (magnification: 10X) in cells with attenuated IGF-1 or IGF-1R expression. (I) Q-PCR assessment of IGF-1 transcript levels in Optn and Cox2 proficient and knockdown cells. Results represent triplicate analyses of three independent biological replicates (mean ± SD) and similar results were obtained in at least two independent analyses. Analyses for panels G and H was performed at the end of differentiation cascade illustrated in following two days of BMP6 (250 ng/mL) stimulation.

Article Snippet: The following shRNA lentiviral particles against relevant genes were purchased from Santa Cruz Biotechnology: Optn (sc-39055-V), Sdc3 (sc-41048-V), Smoc2 (sc-63047-V), Lgr6 (sc-60933-V), Prg4 (sc-60973-V), Cox2 (sc-29278-V), Igf-1 (sc-37194-V) and Igf-1Rα/β (sc-35638-V).

Techniques: Biomarker Discovery, Expressing, Knockdown, Transduction, Construct, Control, Staining

Journal: Nature Communications

Article Title: SUMOylation of ROR-γt inhibits IL-17 expression and inflammation via HDAC2

doi: 10.1038/s41467-018-06924-5

Figure Lengend Snippet: Ubc9 interacts with and SUMOylates ROR-γt. a Lysate was prepared from cLPLs of WT mice and subjected to immunoprecipitation with anti–ROR-γt antibody or control IgG antibody. The precipitated proteins were subjected to SDS-PAGE and in-gel digestion. The resulting peptides were analyzed by high-resolution MS/MS. Ubc9 (SwissProt #P63280) was identified as a specific interactor of ROR-γt protein. An MS/MS spectrum of the peptide 50 GTPWEGGLFK 59 ([M + H] +2 = 546.27 m/z ) belonging to Ubc9 is shown. Observed b - and y- ions are indicated. b Lysates from cLPLs of WT mice were immunoprecipitated with anti-ROR-γt, anti-Ubc9 antibody, or control IgG antibody, and immunoblot analysis with antibody against ROR-γt or Ubc9 was performed. c Sequence alignment was conducted for ROR-γt. The box indicates the conserved SUMOylation (Ψ-K-X-D/E) motif. d Total cell lysates from 293 T cells transfected with Myc-Ubc9, HA-SUMO1, and either Flag-ROR-γt or Flag-K187R-ROR-γt were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were analyzed by immunoblot assay with anti-HA antibody to detect the SUMOylated form of ROR-γt. e Total cell lysates from 293 T cells transfected with Flag-ROR-γt, HA-SUMO1, and either Myc-Ubc9 or Myc-Ubc9-C93A were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibody to detect the SUMOylated form of ROR-γt. f Lysates were prepared from cLPLs of WT mice and immunoprecipitated with anti–ROR-γt antibody or control IgG antibody. The immunoprecipitates were immunoblotted with anti-SUMO1 antibody. The data are representative of three or more independent experiments

Article Snippet: Ubc9 (sc-36774-V), ROR-γt (sc-38881-V), and HDAC2 (sc-29346-V) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Control, SDS Page, Tandem Mass Spectroscopy, Western Blot, Sequencing, Transfection

Journal: Nature Communications

Article Title: SUMOylation of ROR-γt inhibits IL-17 expression and inflammation via HDAC2

doi: 10.1038/s41467-018-06924-5

Figure Lengend Snippet: SUMOylation of ROR-γt inhibits IL-17 expression. a Data show heatmap visualization of upregulated genes determined by RNA seq analysis in ROR-γt –/– CD4 + T cells transduced with either WT ROR-γt or K187R-ROR-γt and differentiated under Th17-inducing conditions. b Real-time PCR analysis was conducted for IL-17a mRNA expression in ROR-γt –/– CD4 + T cells transduced with either WT ROR-γt or K187R-ROR-γt. The relative fold change in the mRNA levels of the genes was normalized against ROR-γt –/– cells as compared with lentivirus-transduced ROR-γt –/– cells. c ELISA was conducted for IL-17A secretion in the culture supernatant of ROR-γt –/– cells transduced with lentivirus encoding V5-tagged WT-ROR-γt or K187R-ROR-γt. d Immunoblot analysis of the expression of lentiviral constructs of V5-ROR-γt in ROR-γt –/– cells. e Luciferase assay was conducted of lysates from Jurkat T cells transfected with various combinations (below plot) of the IL-17-promoter-driven luciferase plasmid (pGL4-mIL17p) and plasmid-encoded HA-SUMO1, Myc-Ubc9, Flag-ROR-γt, and/or Flag-K187R-ROR-γt. f Luciferase assay was conducted of lysates from Jurkat T cells transfected with various combinations (below plot) of plasmid pGL4-mIL17p along with Flag-ROR-γt, HA-SUMO1, and either Myc-Ubc9 or Myc-Ubc9-C93A. Results are presented in relative luciferase units (RLU). g Data show Ubc9 knockdown by Ubc9-specific shRNA (shUbc9) or control shRNA (shCtrl) in WT cLPLs that were stimulated with PMA and ionomycin. ELISA was performed to measure IL-17A secretion in culture supernatant of Ubc9 knockdown cLPLs as well as immunoblotting showing knockdown of Ubc9. h ELISA was performed to measure the IL-17A secretion in the culture supernatant of Ubc9 knockdown cells expressing either WT-ROR-γt or K187R-ROR-γt. i Real-time PCR analysis for IL-17a mRNA expression, in which the relative fold change in the mRNA levels of the genes has been normalized against ROR-γt-expressing cells treated with control shRNA compared to Ubc9-specific shRNA. j Immunoblot analysis shows Ubc9 knockdown in CD4 + T cells expressing either WT-ROR-γt or K187R-ROR-γt. Data are from one experiment representative of three independent experiments with similar results. * p < 0.05, ** p < 0.01, ns: non-significant (two-tail t test) error bars are S.D.

Article Snippet: Ubc9 (sc-36774-V), ROR-γt (sc-38881-V), and HDAC2 (sc-29346-V) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, RNA Sequencing, Transduction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Construct, Luciferase, Transfection, Plasmid Preparation, Knockdown, shRNA, Control