Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: STUbL subunits Slx5 and Slx8 alleviate toxicity of poly-Q expanded Htt. (A) WT, slx5 Δ, and slx8 Δ strains expressing Htt-25Q, Htt-103Q, or empty vector (EV) were grown to mid-logarithmic phase and 5 μl of 10-fold serial dilutions of each culture were spotted on SC-URA medium. Plates were incubated at 30°C for 3 days. (B) Yeast transformants in A and the indicated controls were grown overnight in 5 ml of SC-URA medium. Ten OD 600 readings of cultures were recorded every hour until the OD 600 reached ∼2.0. The average doubling times of four independent experiments were graphed with +/− standard error. EV (empty vector) (C) A shuffle strain, slx5 Δ with SLX5/TRP plasmid (YOK 2990), was transformed with either Htt-25Q or Htt-103Q constructs. Transformants were patched in duplicate on selective medium (SC-TRP URA) and rich medium (YPD). Patches were then replica plated on SC-URA medium with 5FAA to counter-select against the TRP1 marked plasmid.

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Expressing, Plasmid Preparation, Incubation, Transformation Assay, Construct

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Plasmid-borne SLX5 reduces aggregates of poly-Q expanded huntingtin. (A) Representative images of Htt-103Q in WT cells with and without an SLX5/CEN plasmid. Example of aggregates (clearly defined, bright cytoplasmic structures – red arrow-heads) and speckles (multiple small, not clearly defined cytoplasmic granules – blue arrow-heads) (B) Quantitation of phenotypes observed in 2A. WT strain expressing Htt-103Q-GFP alone (YOK 2842) or Htt-103Q-GFP with SLX5 (YOK 2843) were grown to mid-logarithmic phase in selective medium. Images of 103Q diffuse staining, aggregates, and speckles in the indicated strains were recorded and then quantitated. Average counts for three independent experiments were graphed +/− standard deviation. Y-axis: percent of cells ( n = 100/experiment). Y-axis: phenotypes scored (C) Aggregates of poly-Q expanded Htt are localized in the cytoplasm. WT and slx5 Δ strains transformed with GAL-Htt-97Q-DsRed (YOK 3112 and YOK 3114) were grown overnight in SC-TRP medium with 2% raffinose. Cultures were diluted to ∼0.2 OD in a fresh medium with 2% galactose and incubated for an additional 16 h for expression of Htt-97Q-DsRed prior to imaging Htt aggregates using a fluorescence microscope. Nuclei were stained with Hoechst dye. Merged images indicate the absence of Htt-97Q aggregates in nuclei (yellow arrow-heads).

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Plasmid Preparation, Quantitation Assay, Expressing, Staining, Standard Deviation, Transformation Assay, Incubation, Imaging, Fluorescence, Microscopy

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Slx5 modulates the transcriptional activity due to expression of Htt-103Q-NLS. Depiction of 398 SLX5 modulated genes identified by RNA sequencing. (A) RNA-seq analysis shows that Htt103Q-NLS leads to a global effect on the transcriptome as it affects the expression of 3438 genes (25Q [V] vs. 103Q [V]). Plasmid-borne SLX5 affects the expression of 398 genes in Htt103Q-NLS cells (103Q [V] vs. 103Q [ SLX5 ]) as shown in Supplementary Table . The overlap between Htt25Q-NLS and Htt103Q-NLS (25Q [V] vs. 103Q [V]) is 363 genes. The expanded view of A shows that expression of most of the 363 genes (99.4%) inversely correlates between 25Q [V] vs. 103Q [V] and 103Q [V] vs. 103Q [ SLX5 ]. Expression of 247 of the 363 genes (68.0%) is down-regulated by Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Down – Up). Expression of 114 of the 363 genes (31.4%) is up-regulated by the Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Up – Down). (B) Subcellular localization of differentially expressed genes. The localization of proteins encoded by the 398 genes indicated in Supplementary Table was analyzed using cellular components assignment from the PANTHER Classification System and the Saccharomyces Genome Database. Pie chart shows a ratio of the genes placed into cellular component categories. Individual genes are listed in Supplementary Table . (C) Schematic of gene expression in Htt25Q-NLS and Htt103Q-NLS cells and the effect of plasmid-borne SLX5 on the transcriptome of Htt103Q-NLS cells. Expression of genes A and B are downregulated and upregulated in Htt103Q-NLS cells relative to Htt25Q-NLS cells, respectively. Slx5 reduces the association of Htt with chromatin and this contributes to the reversal in gene expression such that gene A is upregulated and gene B is downregulated. (D) RT-PCR validation of gene expression analysis. Total RNAs were purified from strains expressing either Htt25Q-NLS or Htt103Q-NLS from a GAL promoter for 4 h with or without plasmid-borne SLX5 . RT-PCR analyses was performed using the same samples used for the RNA-seq. Relative intensities are reported as the mean ± SD of three biological repeats. Reactions for YDL223C/HBT1 and YPL186C/UIP4 were performed in duplicate. N = 3 for YOR339C/UBC11 and YFL039C/ACT1 , N = 6 for YDL223C/HBT1 and YPL186C/UIP4 .

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Activity Assay, Expressing, RNA Sequencing, Plasmid Preparation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Purification

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: STUbL subunits Slx5 and Slx8 alleviate toxicity of poly-Q expanded Htt. (A) WT, slx5 Δ, and slx8 Δ strains expressing Htt-25Q, Htt-103Q, or empty vector (EV) were grown to mid-logarithmic phase and 5 μl of 10-fold serial dilutions of each culture were spotted on SC-URA medium. Plates were incubated at 30°C for 3 days. (B) Yeast transformants in A and the indicated controls were grown overnight in 5 ml of SC-URA medium. Ten OD 600 readings of cultures were recorded every hour until the OD 600 reached ∼2.0. The average doubling times of four independent experiments were graphed with +/− standard error. EV (empty vector) (C) A shuffle strain, slx5 Δ with SLX5/TRP plasmid (YOK 2990), was transformed with either Htt-25Q or Htt-103Q constructs. Transformants were patched in duplicate on selective medium (SC-TRP URA) and rich medium (YPD). Patches were then replica plated on SC-URA medium with 5FAA to counter-select against the TRP1 marked plasmid.

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Expressing, Plasmid Preparation, Incubation, Transformation Assay, Construct

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Plasmid-borne SLX5 reduces aggregates of poly-Q expanded huntingtin. (A) Representative images of Htt-103Q in WT cells with and without an SLX5/CEN plasmid. Example of aggregates (clearly defined, bright cytoplasmic structures – red arrow-heads) and speckles (multiple small, not clearly defined cytoplasmic granules – blue arrow-heads) (B) Quantitation of phenotypes observed in 2A. WT strain expressing Htt-103Q-GFP alone (YOK 2842) or Htt-103Q-GFP with SLX5 (YOK 2843) were grown to mid-logarithmic phase in selective medium. Images of 103Q diffuse staining, aggregates, and speckles in the indicated strains were recorded and then quantitated. Average counts for three independent experiments were graphed +/− standard deviation. Y-axis: percent of cells ( n = 100/experiment). Y-axis: phenotypes scored (C) Aggregates of poly-Q expanded Htt are localized in the cytoplasm. WT and slx5 Δ strains transformed with GAL-Htt-97Q-DsRed (YOK 3112 and YOK 3114) were grown overnight in SC-TRP medium with 2% raffinose. Cultures were diluted to ∼0.2 OD in a fresh medium with 2% galactose and incubated for an additional 16 h for expression of Htt-97Q-DsRed prior to imaging Htt aggregates using a fluorescence microscope. Nuclei were stained with Hoechst dye. Merged images indicate the absence of Htt-97Q aggregates in nuclei (yellow arrow-heads).

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Plasmid Preparation, Quantitation Assay, Expressing, Staining, Standard Deviation, Transformation Assay, Incubation, Imaging, Fluorescence, Microscopy

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Slx5 modulates the transcriptional activity due to expression of Htt-103Q-NLS. Depiction of 398 SLX5 modulated genes identified by RNA sequencing. (A) RNA-seq analysis shows that Htt103Q-NLS leads to a global effect on the transcriptome as it affects the expression of 3438 genes (25Q [V] vs. 103Q [V]). Plasmid-borne SLX5 affects the expression of 398 genes in Htt103Q-NLS cells (103Q [V] vs. 103Q [ SLX5 ]) as shown in Supplementary Table . The overlap between Htt25Q-NLS and Htt103Q-NLS (25Q [V] vs. 103Q [V]) is 363 genes. The expanded view of A shows that expression of most of the 363 genes (99.4%) inversely correlates between 25Q [V] vs. 103Q [V] and 103Q [V] vs. 103Q [ SLX5 ]. Expression of 247 of the 363 genes (68.0%) is down-regulated by Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Down – Up). Expression of 114 of the 363 genes (31.4%) is up-regulated by the Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Up – Down). (B) Subcellular localization of differentially expressed genes. The localization of proteins encoded by the 398 genes indicated in Supplementary Table was analyzed using cellular components assignment from the PANTHER Classification System and the Saccharomyces Genome Database. Pie chart shows a ratio of the genes placed into cellular component categories. Individual genes are listed in Supplementary Table . (C) Schematic of gene expression in Htt25Q-NLS and Htt103Q-NLS cells and the effect of plasmid-borne SLX5 on the transcriptome of Htt103Q-NLS cells. Expression of genes A and B are downregulated and upregulated in Htt103Q-NLS cells relative to Htt25Q-NLS cells, respectively. Slx5 reduces the association of Htt with chromatin and this contributes to the reversal in gene expression such that gene A is upregulated and gene B is downregulated. (D) RT-PCR validation of gene expression analysis. Total RNAs were purified from strains expressing either Htt25Q-NLS or Htt103Q-NLS from a GAL promoter for 4 h with or without plasmid-borne SLX5 . RT-PCR analyses was performed using the same samples used for the RNA-seq. Relative intensities are reported as the mean ± SD of three biological repeats. Reactions for YDL223C/HBT1 and YPL186C/UIP4 were performed in duplicate. N = 3 for YOR339C/UBC11 and YFL039C/ACT1 , N = 6 for YDL223C/HBT1 and YPL186C/UIP4 .

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Activity Assay, Expressing, RNA Sequencing Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Purification

Journal: Oncotarget

Article Title: C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis

doi: 10.18632/oncotarget.22339

Figure Lengend Snippet: ( A ) Immunoprecipitation of C3G from resting (Rest) and thrombin (TH, at 0.2 U/mL) stimulated platelets for 5 min at 37°C under stirring. Vamp-7 in the immunocomplexes was detected by Western blot with anti-TI-VAMP antibody (Santa Cruz Biotechnologies, sc-67060). Platelet cell lysate from 4 mice of each genotype was used. b: lysate incubated with agarose beads. ( B ) Immunoprecipitation of C3G from stably C3G-overexpressing K562 cells untreated (basal) or treated with 20 nM PMA for 10 min. C3G and VAMP-7 in the immunocomplexes were detected with anti-TI-VAMP and anti-C3G antibodies. L: total cell lysate (50 µg). b: lysate incubated with agarose beads. ( C ) HEK293T cells were transiently transfected with a HA-tagged C3G construct together with the indicated CFP-VAMP-7 fusion proteins or the empty pCDNA3-CFP-C4 plasmid (CFP). Ectopic C3G was immunoprecipitated with anti-HA.11 monoclonal antibody and C3G and VAMP-7 detected with anti-C3G and anti-TI-VAMP (arrows). b: agarose beads incubated with non-transfected lysate. L: total cell lysate (50 µg). IP: immunoprecipitated with anti-HA.11. ( D ) HEK293T cells were transiently transfected with pLTR2C3G∆Cat construct or the empty pLTR2 vector , together with the above CFP-VAMP-7 constructs. C3G was immunoprecipitated with anti-C3G antibodies (IP) and C3G and VAMP-7 detected with anti-C3G and anti-GFP (Santa Cruz Biotechnologies, sc-9996) antibodies. L: total cell lysate. VAMP7-NT: N-terminal (longin) domain of VAMP-7 (aminoacids 1-120); VAMP7-CT: C-terminal (SNARE) domain of VAMP-7 (aminoacids 121-188); VAMP7-cyto: VAMP-7 cytosolic domain (aminoacids 1-188).

Article Snippet: Human VAMP-7 longin (aa 1-120), SNARE (aa 121-188) and full cytosolic (aa 1-188) domains were amplified from pEGFP-VAMP7 (1-220), a gift from Thierry Galli (Addgene plasmid #42316) [ ] and cloned into pcDNA3-CFP-C4 (a gift from Dr. J.M Pereda, IBMCC, Salamanca, Spain) as CFP-fusion proteins, with CFP in the N-terminus.

Techniques: Immunoprecipitation, Western Blot, Incubation, Stable Transfection, Transfection, Construct, Plasmid Preparation