Journal: PLoS ONE

Article Title: OsSpo11-4, a Rice Homologue of the Archaeal TopVIA Protein, Mediates Double-Strand DNA Cleavage and Interacts with OsTopVIB

doi: 10.1371/journal.pone.0020327

Figure Lengend Snippet: Seed setting (A, B), pollen viability (C) and endogenous OsSpo11-4 expression (D) were decreased in OsSpo11-4 RNAi lines. A , Panicle morphology of wild-type (left) and OsSpo11-4 RNAi T0 lines with different seed setting (right 3). B , Seed setting of wild-type (WT) and 6 RNAi T1 generation lines (L11, L19, L8, L38, L39 and L45). C , Alexander staining of anthers (a and b) and mature pollen grains (c and d) from wild-type (a and c) and RNAi (b and d) plants showing reduced pollen viability in RNAi lines. Scale bar = 100 µm in (a) and (b), and 50 µm in (c) and (d). D , Semi-quantitative RT-PCR analysis of endogenous OsSpo11-4 mRNA levels in wild-type and RNAi lines. TubA mRNA was amplified as an internal control. All PCR products were separated on 1% agarose gel. E , Quantitative real-time RT-PCR analysis for expression of OsSpo11-4 in wild type and RNAi lines. The expression levels of SPO11-4 in different RNAi lines were firstly normalized by computing to the internal standard gene, tubA , and then compared to the wild type by the ΔΔC T method.

Article Snippet: The relative expression of OsSPO11-4 in RNAi lines versus wild type was computed by the ΔΔCT method (Applied Biosystems, USA).

Techniques: Expressing, Staining, Quantitative RT-PCR, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

doi: 10.1093/nar/gkl181

Figure Lengend Snippet: RT–PCR analysis on mitochondrial transcripts spanning the cyt b/ND1-binding site in DmTTF-depleted cells. ( A ) Schematic illustration of the cyt b/ND1-binding site. Black arrows represent the forward (11 494–11 520 nt) and reverse (11 855–11 834 nt) primers used for RT–PCR. Dashed regions indicate non-coding nucleotides. ( B ) Total RNA (600 ng) extracted from untreated (control) and treated (RNAi) D.Mel-2 cells was used as template in RT–PCR; 10 µl-samples were collected at the indicated cycles, run on a 1.5% agarose gel and stained with ethidium bromide. Nuclear encoded 28S rRNA was used as endogenous control.

Article Snippet: Drosophila cell culture and conditions for RNAi Drosophila embryonic cell line D.Mel-2 (GIBCO-Invitrogen) was maintained in Drosophila SFM (GIBCO-Invitrogen) supplemented with 16 mM l -glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin, at 28°C in 75 cm2 flasks.

Techniques: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis, Staining

Journal: Nucleic Acids Research

Article Title: The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

doi: 10.1093/nar/gkl181

Figure Lengend Snippet: Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.

Article Snippet: Drosophila cell culture and conditions for RNAi Drosophila embryonic cell line D.Mel-2 (GIBCO-Invitrogen) was maintained in Drosophila SFM (GIBCO-Invitrogen) supplemented with 16 mM l -glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin, at 28°C in 75 cm2 flasks.

Techniques: Western Blot, Incubation, Recombinant

Journal: Nucleic Acids Research

Article Title: The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

doi: 10.1093/nar/gkl181

Figure Lengend Snippet: RNase protection assay on mitochondrial (−)strand transcripts mapping around the cyt b/ND1-binding site in DmTTF-depleted cells. ( A ) Schematic representation of digestion products using probes Ribo-1 (295 nt) and Ribo-2 (218 nt). Riboprobes (grey bold arrows), mature transcripts (continuous arrows) and read-through transcripts (dotted arrows) are indicated above the cyt b/ND1 region map. Dashed regions indicate non-coding sequences. ( B ) Total cellular RNA (50 µg), extracted from untreated (control) and DmTTF-dsRNA treated (RNAi) D.Mel-2 cells, was hybridized with about 1.5 × 10 5 c.p.m. of Ribo-1 and Ribo-2 probes and digested with RNase A and T1. Digestion products were denatured and run on a 10% polyacrylamide/7 M urea gel. Y, sample containing 50 µg of yeast total RNA. M, Decade RNA marker (Ambion).

Article Snippet: Drosophila cell culture and conditions for RNAi Drosophila embryonic cell line D.Mel-2 (GIBCO-Invitrogen) was maintained in Drosophila SFM (GIBCO-Invitrogen) supplemented with 16 mM l -glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin, at 28°C in 75 cm2 flasks.

Techniques: Rnase Protection Assay, Binding Assay, Marker

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: SAV1 and MST2 are present in the cytosol. FLAG-MST2 and Myc-SAV1 were co-transfected into 293FT cells. Cells were collected after 24 hr and subjected to cytosol-membrane fractionation using the membrane protein extraction kit (Thermo Scientific). The cytosol and membrane fractions were blotted with the indicated antibodies. Tubulin, NF2 and Pan Cadherin were used as markers for cytosolic, peripheral membrane, and integral membrane proteins, respectively.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Transfection, Fractionation, Protein Extraction

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: The N-terminal 90 residues of SAV1 associates with PP2A_A subunit in human cells. Control and SLMAP KO 293FT cells were transfected with the indicated FLAG-SAV1 plasmids. Anti-FLAG IP and cell lysates (input) were blotted with the indicated antibodies. Asterisk designates non-specific bands.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Transfection

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: CRISPR/Cas9-induced indel mutations in MCF10A and 293FT cells. ( A and B ) Intact genomic sequences of SLMAP and SAV1 are shown in the top panels. The indel mutations in SLMAP KO or SAV1 KO cells are shown in the bottom panels.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: CRISPR, Genomic Sequencing

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: Feedback inhibition of MST2 activation by SLMAP binding to autophosphorylated MST2 linker. ( A and B ) Anti-FLAG and anti-pMST2 (T180) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. ( C ) Anti-FLAG, anti-pMST2 (T336), and anti-pMST2 (T378) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. ( D ) In vitro kinase assay of MST2 kinase domain using GST-MST2 D146N as the substrate. Immunoblots of the kinase reaction mixtures were shown. Anti-GST blot was used to evaluate protein levels. ( E ) The in vitro kinase assays of MST2-FL and MST2-∆L using myelin basic protein (MBP) as substrate. The reaction mixtures were separated on SDS-PAGE and analyzed by a phosphoimager (top) and Coomassie blue staining (middle). FL, full-length; ΔL, linker deletion. The kinetic profiles of MBP phosphorylation by activated MST2 FL and ∆L as monitored by 32 P incorporation (bottom). The relative 32 P-MBP signal intensities, normalized to those of MST2 FL and ∆L at 60 min (100%), respectively, are plotted against time. Means ± range for two independent experiments are plotted. ( F ) Coomassie-stained gel of the indicated MST2 proteins bound to GST-SLMAP FHA (residues 1–140) beads. FL, full-length; KD, kinase domain; ΔL, linker deletion.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Inhibition, Activation Assay, Binding Assay, Transfection, In Vitro, Kinase Assay, Western Blot, SDS Page, Staining

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: STRIPAK SLMAP inhibits the Hippo pathway in human cells. ( A ) 293FT cells were transfected with siSTRIP1 with or without FLAG-STRIP1. The total cell lysates were blotted with the indicated antibodies. ( B ) Immunoblots with the indicated antibodies of lysates of control MCF10A cells, SLMAP KO cells, and SLMAP KO cells stably expressing GFP-SLMAP WT or ΔFHA. ( C ) Relative expression of YAP target genes CTGF and CYR61 in control MCF10A cells, SLMAP KO cells, and SLMAP KO cells stably expressing GFP-SLMAP WT or ΔFHA. YAP target gene expression was analyzed by quantitative real-time RT-PCR and normalized to GAPDH. Data are plotted as mean ± SEM of three independent experiments (**p

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Transfection, Western Blot, Stable Transfection, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: The N-terminal region of SAV1 is required for MST2 activation. Immunoblots of cell lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated N-terminal truncation of Myc-SAV1 constructs. FBM, FERM-binding motif.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Activation Assay, Western Blot, Transfection, Construct, Binding Assay

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: SARAH and WW domains of SAV1 mediate the formation of the MST2-SAV1 heterotetramer. ( A ) Binding between GST-MST2-SARAH and in vitro translated SAV1 SARAH proteins. SAV1 mutants that are defective in MST2-binding are labeled red. ( B ) UV traces of molecular weight standards (dashed line), MST2-FL (black line), MST2-FL/SAV1-ΔN320 (purple line), MST2-FL/SAV1-ΔN290 (blue line), and MST2-FL/SAV1-ΔN198 (red line) fractionated on a Superdex 200 gel filtration column. ( C ) Cartoon drawing of the solution structure of the mouse SAV1 WW2 homodimer (PDB ID: 2DWV) ( Ohnishi et al., 2007 ). Monomer A is colored cyan and monomer B is colored blue. ( D ) UV traces of human SAV1 WW1 (blue line), and WW2 (red line) fractionated on a Superdex 75 gel filtration column. The molecular weight standard is indicated. ( E ) Anti-HA and anti-FLAG blots of cell lysates (input) and anti-FLAG IP of 293FT cells co-transfected with HA-SAV1 ∆SARAH and the indicated FLAG-SAV1 plasmids.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Binding Assay, In Vitro, Labeling, Molecular Weight, Filtration, Transfection

Journal: eLife

Article Title: SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

doi: 10.7554/eLife.30278

Figure Lengend Snippet: Crystal structures of SLMAP FHA and SLMAP FHA bound to pMST2. ( A ) Cartoon drawing of the crystal structure of human SLMAP FHA. SLMAP FHA is colored cyan. ( B ) Cartoon drawing of the crystal structure of human SLMAP FHA in complex with the pMST2 peptide. SLMAP FHA is colored wheat. pMST2 is colored yellow and shown as sticks. The pMST2 residues are labeled. ( C ) Zoomed-in view of the pT-binding site in the SLMAP FHA-pMST2 complex. The backbones and side chains of SLMAP FHA interface residues are shown as sticks and are labeled. ( D ) Anti-Myc and anti-SLMAP blots of lysates (input) and anti-FLAG IP of 293FT cells transfected with the indicated plasmids.

Article Snippet: Mammalian cell culture, transfection, and RNA interference 293FT cells (Thermo Scientific, catalogue #R70007; not independently authenticated) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

Techniques: Labeling, Binding Assay, Transfection

Journal: PLoS ONE

Article Title: HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells

doi: 10.1371/journal.pone.0062729

Figure Lengend Snippet: Depletion of hnRNP A1/A2 or SF2/ASF increases exclusion of exons 2 and 3 of IRF-3. (A) Schematic diagram showing the three splicing variants FL-IRF-3 (full-length IRF-3), Type I (only E2 exclusion) and Type II (both E2 and E3 exclusion) isoforms. E, exon. IRF-3 exons from 1 to 4 are numbered. The black solid line represents introns. The arrows above the transcripts show the location of specific primer sets designed for RT-PCR analysis of IRF-3 splicing variants. (B) Splicing factors hnRNP A1/A2 or SF2/ASF were depleted by specific siRNA transfection in human NSCLC cells A549 and Calu-6, respectively. A1, hnRNP A1; A2, hnRNP A2. The mismatched control siRNA was used for mock-transfected control. After siRNA transfection and subsequent Poly(I:C) stimulation, cells were harvested and semi-quantitative RT-PCR was performed to determine the impact of RNA interference on expression of target genes and IRF-3 splicing variants. Products corresponding to FL-IRF-3 and its two kinds of splicing variant (Type I and Type II) are indicated with arrows. For all reactions, total RNA extracted from A549 cells without reverse transcription was used as negative control. PCR products of FL-IRF-3, Type I and Type II isoforms were quantified by TotalLab Quant scanning. The graph indicates the ratio isoform FL/(FL+I+II) and the values are mean ± SD for n = 3 experiments. (C) Western blot analysis was performed with antibodies directed against the proteins indicated on the right. Protein bands of IRF-3 were also quantified and normalized to internal control Actin. The graph indicates the ratio IRF-3/Actin and the values are mean ± SD for n = 3 experiments. # P and ## P

Article Snippet: Cell Culture and RNA Interference Human NSCLC cell lines A549 and Calu-6 were obtained from ATCC (Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, Expressing, Variant Assay, Negative Control, Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells

doi: 10.1371/journal.pone.0062729

Figure Lengend Snippet: Depletion of PTB shows no effect on IRF-3 splicing pattern. Splicing factor PTB was depleted by specific siRNA transfection in human NSCLC cells A549 and Calu-6. The mismatched control siRNA was used as mock-transfected control. After siRNA transfection and subsequent Poly(I:C) stimulation, total cellular RNA and protein were collected and tested by semi-quantitative RT-PCR (A) and Western blot analysis (B) to examine the expression levels of target genes and IRF-3 splicing variants indicated on the right. For RT-PCR reactions, total RNA extracted from A549 cells without reverse transcription was used as negative control. For all RT-PCR and Western blot analysis, Actin was used as internal control.

Article Snippet: Cell Culture and RNA Interference Human NSCLC cell lines A549 and Calu-6 were obtained from ATCC (Manassas, VA).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: PLoS ONE

Article Title: HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells

doi: 10.1371/journal.pone.0062729

Figure Lengend Snippet: Depletion of hnRNP A1/A2 or SF2/ASF reduces IFNβ and IP-10 expression in human NSCLC cells. A549 and Calu-6 cells were performed specific siRNA-mediated knockdown of hnRNP A1/A2 or SF2/ASF. Mock, cells transfected with the mismatched control siRNA. Control, cells without specific or mock siRNA transfection and Poly(I:C) stimulation. (A) After siRNA transfection and subsequent Poly(I:C) stimulation, the mRNA expression of IFNβ and IP-10 genes was analyzed by semi-quantitative RT-PCR. Total RNA isolated from A549 cells without reverse transcription was used as negative control. (B) Secretion of IFNβ (top) and IP-10 (bottom) proteins was examined by ELISA assay. The value for each assay is presented as mean ± S.D. for three independent experiments. # P and ## P

Article Snippet: Cell Culture and RNA Interference Human NSCLC cell lines A549 and Calu-6 were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Isolation, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: eNeuro

Article Title: The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

doi: 10.1523/ENEURO.0134-20.2020

Figure Lengend Snippet: Ubc9 expression protects N27 cells from MPP+ or PFF-induced toxicity, enhancing cell viability and reducing cytotoxicity. A , MPP+ exposure reduces the number of viable cells in EGFP cells in a dose-dependent manner, whereas Ubc9 overexpression protects N27 cells from MPP+ toxicity in MTT assay. B , In LDH assay, Ubc9 overexpression reduces the toxic effect derived from MPP+ exposure. C , In MTT assay, Ubc9 knock-down by RNAi exacerbates the cell viability induced by MPP+ exposure, compared with NC1 random cocktail control. D , In LDH assay, Ubc9-RNAi significantly increases the cytotoxicity derived from MPP+ treatment. E , In MTT assay, PFF treatment reduces cell viability in EGFP cells compared with WGA/GluNAc-treated control, while Ubc9 overexpression ameliorates the toxic effect from PFF. F , In LDH assay, PFF-induced cytotoxicity was suppressed by Ubc9 overexpression. G , In MTT assay, Ubc9-RNAi further exacerbates the cell viability induced by PFF treatment, compared with NC1 random cocktail control with WGA/GluNAc. H , In LDH assay, Ubc9 knock-down substantially enhances the cytotoxicity derived from PFF treatment compared with NC1 control. All the treatments were exposed for 24 h, and each dot represents the number of experiments and each experiment was performed in triplicate. Statistical analysis was applied using two-way ANOVA, Tukey’s post hoc test. Scattered dot plots represent mean ± SEM; * p

Article Snippet: Cell lines with plasmids or RNAi treatment N27 rat dopaminergic parental cell line (SCC048, EMD Millipore) was used to generate enhanced green fluorescent protein (EGFP) or Ubc9-EGFP-overexpressing (Ubc9-OE) stable cell lines by transfection.

Techniques: Expressing, Over Expression, MTT Assay, Lactate Dehydrogenase Assay, Derivative Assay, Whole Genome Amplification

Journal: eNeuro

Article Title: The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

doi: 10.1523/ENEURO.0134-20.2020

Figure Lengend Snippet: Ubc9 overexpression increases the endogenous level of α-syn protein in dopaminergic N27 cells. A , Immunofluorescent signals for α-syn and EGFP in N27 cell lines stably expressing either Ubc9-EGFP or EGFP, are shown as indicated. Image scale bar: 20 μm. B , In the quantification of fluorescence intensity of endogenous α-syn in immunocytochemistry, higher level of α-syn was detected in Ubc9 cells than in EGFP cells ( n = 15). Statistical analysis was applied to Student’s unpaired t test. C , In the qRT-PCR, there is no difference in mRNA level of α-syn between Ubc9 and EGFP cells ( n = 3 × 3). D , In WBs, the levels of α-syn and Ubc9 are displayed with the treatment of Ubc9-OE or -RNAi, in comparison to EGFP and NC1 controls. E , Ubc9 expression was upregulated by Ubc9-OE and downregulated by Ubc9-RNAi in the quantification of WBs ( n = 5 per group). F , In the analysis of 24-h chase assays, the mixed Ubc9-RNAi constructs significantly reduces the level of α-syn, compared with the control (NC1) in WBs, whereas Ubc9-OE increases the level of α-syn, compared with EGFP control ( n = 6). Bars represent mean ± SEM, and each dot represents the mean of each experiment, which was performed in triplicate. Statistical analysis was applied to one-way ANOVA, Tukey’s post hoc test; * p

Article Snippet: Cell lines with plasmids or RNAi treatment N27 rat dopaminergic parental cell line (SCC048, EMD Millipore) was used to generate enhanced green fluorescent protein (EGFP) or Ubc9-EGFP-overexpressing (Ubc9-OE) stable cell lines by transfection.

Techniques: Over Expression, Stable Transfection, Expressing, Fluorescence, Immunocytochemistry, Quantitative RT-PCR, Construct

Journal: eNeuro

Article Title: The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

doi: 10.1523/ENEURO.0134-20.2020

Figure Lengend Snippet: Ubc9 knock-down by RNAi exacerbates PFF-induced protein aggregation in thioflavin T staining. A , Bright field images show the location of N27 cells (top row). Immunocytochemical images of thioflavin T staining in dark field show PFF-induced protein aggregation in green fluorescent label (second row). α-Syn staining (red) in immunocytochemistry was detected in thioflavin T -labeled protein aggregates (third row). The merged images (yellow, the bottom row) demonstrate that α-syn is co-localized with thioflavin T-stained protein aggregates. B , PFF exposure to N27 cells for 24-h results in the accumulation of protein aggregation labeled in thioflavin T. Ubc9-RNAi further enhances PFF-induced protein aggregation, compared with NC1 random cocktail control with WGA/GluNAc. C , PFF increases the level of α-syn in thioflavin T-positive aggregates with Ubc9-RNAi, and Ubc9 knock-down aggravates α-syn accumulation in the protein aggregates compared with NC1 control after PFF treatment, although PFF did not significantly increase the level of α-syn in the aggregates in NC1 control-treated cells. Integrated density of images was measured by ImageJ and presented in scattered dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. All dot plots were displayed as individual values and mean differences are depicted as horizontal lines; SEM is indicated by the end of the vertical error bars; *** p

Article Snippet: Cell lines with plasmids or RNAi treatment N27 rat dopaminergic parental cell line (SCC048, EMD Millipore) was used to generate enhanced green fluorescent protein (EGFP) or Ubc9-EGFP-overexpressing (Ubc9-OE) stable cell lines by transfection.

Techniques: Staining, Immunocytochemistry, Labeling, Whole Genome Amplification

Journal: eNeuro

Article Title: The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

doi: 10.1523/ENEURO.0134-20.2020

Figure Lengend Snippet: Ubc9 expression suppresses ROS generation triggered by MPP+ or PFF treatment. A , Examples of ROS-labeled cells show that MPP+ (640 μ m ) for 24 h stimulates the generation of ROS in N27 EGFP cells, which was labeled with CellROX red fluorescent dye in dark field microscopy. B , MPP+ triggers a striking increase in ROS generation, which was prevented by Ubc9 overexpression. C , Examples of cell images showing that Ubc9-RNAi exacerbates the ROS production induced by MPP+ exposure. D , The treatment of Ubc9-RNAi (100 p m ) overnight enhances MPP+-induced ROS generation compared with NC1 random construct control. E , Cell images show that PFF (1 μg/ml) exposure for 24 h stimulates ROS production (red label) in EGFP cells, which was not clearly detected with Ubc9 overexpression. F , The ROS generation induced by PFF was almost completely suppressed by Ubc9 overexpression, compared with WGA/GluNAc control. G , Cell images show that Ubc9-RNAi exacerbates the PFF-induced ROS production, compared with no treatment or WGA/GluNAc control. H , Ubc9-RNAi significantly enhances ROS production induced by PFF treatment, compared with NC1 control. Integrated density of images was measured by ImageJ and presented in scattered dot plots. Statistical analysis was applied using two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. All dot plots were displayed as individual values and mean differences are depicted as horizontal line ( n = 7–10); SEM is indicated by the end of the vertical error bars; ** p

Article Snippet: Cell lines with plasmids or RNAi treatment N27 rat dopaminergic parental cell line (SCC048, EMD Millipore) was used to generate enhanced green fluorescent protein (EGFP) or Ubc9-EGFP-overexpressing (Ubc9-OE) stable cell lines by transfection.

Techniques: Expressing, Labeling, Microscopy, Over Expression, Construct, Whole Genome Amplification

Journal: Genetics and Molecular Biology

Article Title: Regulation of adipogenesis by nuclear receptor PPAR? is modulated by the histone demethylase JMJD2C

doi: 10.1590/S1415-47572010005000105

Figure Lengend Snippet: JMJD2C expression in the 3T3-L1 cell line, capacity of binding to histone deacetylases and – to PPARγ (A) RNA extractions and RT-PCR assays for JMJD2c were performed on 3T3-L1 cell line at days 0, 4 and 8 of differentiation; (B) Western blot showing JMJD2C expression at all days of differentiation; (C) Co-immunoprecipitation of JMJD2C and HDAC type I.

Article Snippet: RNA interference 3T3-L1 cell lines were obtained from American Tissue Culture Collection (ATCC) and transfected with double-stranded oligonucleotides targeting JMJD2C (Invitrogen, USA) in order to select the oligos and the concentration that most efficiently silenced JMJD2C expression.

Techniques: Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Cohesin alters the transcriptional bursting frequency of boundary-proximal genes. a , Representative images of intronic RNA FISH to the HS3ST1 transcript before or after auxin treatment. Dashed lines represent nuclear edges. Scale bar equals 5 μm. b and distance to nearest boundary for genes assayed by RNA FISH. The mean domain size denoted by a hashed line is 343.9 kb. c , Hi-C contact matrices of the loci surrounding GALNT5 and HS3ST1 . Hi-C maps shown for HCT-116 cells before or after auxin treatment to degrade RAD21. d , Change in bursting frequency of each gene following auxin treatment by intronic RNA FISH. n > 227 chromosomes. Average of 3 biological replicates per gene. e versus change in bursting frequency detected by intronic RNA FISH (R 2 = 0.9047, two-sided Pearson correlation). P

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Fluorescence In Situ Hybridization, Hi-C

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Additional information related to . a , Distribution of domain sizes that harbor either a significantly differentially expressed (median = 183734, n = 4196) or non-differentially expressed genes (median = 194447, n = 8026) in the HCT-116 cell line following auxin treatment. P = 0.102, two-tailed Mann-Whitney test. b , Scatterplot of log 2 (fold change)] of HCT-116 differentially expressed genes (DEGs) versus the distance between their TSS and the center of the nearest domain boundary. c in HCT-116 cells for DEGs with > 30% fold change in expression following auxin treatment. Genes were categorized by their proximity to a domain boundary (

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Confirmation and quantification of RAD21 degradation. a , Immunofluorescence for RAD21 (cyan). DNA (Hoescht stain) is shown in grey. Dashed lines represent nuclear edges. Scale bar equals 5 μm. b , Western blot to RAD21 protein in the chromatin-bound fraction of HCT-116-RAD21-AID cells with no auxin treatment (−) or following 6 hours of auxin treatment (+). Histone H3 as loading control. Protein was labeled using fluorescent secondary antibodies. c , Fluorescence quantification of RAD21 and H3 isolated from the chromatin-bound fraction of protein corresponding to blot in b using Image Lab v5.2.1. Protein intensity normalized to total protein per well (via stain-free technology) and presented as fraction of protein observed in untreated (− auxin) conditions; we observe a 96% reduction in chromatin-bound RAD21 following auxin treatment.

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Immunofluorescence, Staining, Western Blot, Labeling, Fluorescence, Isolation

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Additional information related to . a , HCT-116-RAD21-AID cells were synchronized at the G1/S transition. Immunofluorescence for CENPF (green) to indicate cells in G2 and PCNA (grey) to mark cells in S phase. DNA (Hoescht stain) is shown in grey in first column. Dashed lines represent nuclear edges. Scale bar equals 5 μm. b , Cumulative frequency distribution of spatial overlap between neighboring domains on chr12:11.6Mb-13.6Mb (n = 716 chromosomes) and chr22:33.2Mb-36.8Mb (n = 1410 in asynchronous HCT-116 cells. Overlap normalized to the volume of the upstream domain. n > 716 chromosomes. c , Hi-C contact matrix and Oligopaint designs corresponding to ( c-e ). d , Representative FISH images of each subdomain and the downstream D2. Scale bar equals 1 μm. Corresponding 3D segmentation of FISH signals below each image. e , Cumulative distribution plot of spatial overlap between the subdomains [S1 (n = 1932); S2 (n = 2283); S3 (n =1977)] and D2, normalized to the volume of D2. *** P

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Immunofluorescence, Staining, Hi-C, Fluorescence In Situ Hybridization

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Cohesin promotes interactions within and across domain boundaries. a , Hi-C contact matrices and Oligopaint probe design of chr12:11.6Mb-13.6Mb and chr22:33.2–36.8Mb in HCT-116-RAD21-AID cells prior to or after 6 hours of auxin treatment. b , Representative FISH images of neighboring domains across chr12:11.6Mb-13.6Mb prior to and after auxin treatment. Dashed lines represent nuclear edges. Scale bar equals 5 μm (left) or 1 μm (zoomed images, below). c , Locus-specific differences in the percentage of domain pairs in contact following auxin treatment. Each bar represents an average of two biological replicates. d , Fold-change in contact frequency between neighboring domains following auxin treatment versus the insulation score of their intervening boundary. Each point represents an average of two biological replicates. n = 17 boundaries. e , Cumulative distribution of overlap between the neighboring domains at chr12:11.6–13.6Mb prior to (n = 1,625) and after auxin treatment (n = 1,607). Overlap normalized to the volume of the upstream domain. P

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Hi-C, Fluorescence In Situ Hybridization

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: WAPL and CTCF restrict cohesin-dependent interactions across domain boundaries. a , Representative FISH images of neighboring domains on chr22:33.2–36.8Mb in RNAi control, NIPBL-, WAPL-, or CTCF-depleted HCT-116 cells. Dashed lines represent nuclear edges. Scale bar equals 5 μm (left) or 1 μm (zoomed images, below). b , Difference in the percentage of domain pairs in contact following depletion of NIPBL or WAPL. Each bar represents an average of three biological replicates. P

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Fluorescence In Situ Hybridization

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Additional information related to . a , Western blot of NIPBL, WAPL, and CTCF protein of HCT-116 cells following RNAi. H3 as loading control. b , Representative FISH images of neighboring domains on chr12:11.6Mb-13.6Mb in RNAi control, NIPBL-, WAPL-, or CTCF-depleted cells. Dashed lines represent nuclear edges. Scale bar equals 5 μm (left) or 1 μm (zoomed images, below). c , Spatial overlap between neighboring domains on chr12:11.6Mb-13.6Mb in control (n = 643), NIPBL- (n = 636), WAPL- (n = 819) or both NIPBL and WAPL- (n = 922) depleted cells. *** P

Article Snippet: RNAi HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 .

Techniques: Western Blot, Fluorescence In Situ Hybridization