Journal: Journal of Lipid Research

Article Title: Cholesterol trafficking to the ER leads to the activation of CaMKII/JNK/NLRP3 and promotes atherosclerosis

doi: 10.1016/j.jlr.2024.100534

Figure Lengend Snippet: Cholesterol trafficking from plasma membrane to ER via StARD protein complex increases NLRP3 inflammasome activation. A: IL-1β secretion from control and Myl-Abc dko BMDMs that were treated with LPS for 3 h, vehicle or SMase for 1 h, and with vehicle or ATP for an additional 1 h to induce inflammasome activation. B: Immunoblot of intracellular Caspase-1 and GSDMD cleavage from (A). C: IL-1β secretion from vehicle and acLDL loaded wild-type BMDMs that were primed with LPS for 3 h and treated with vehicle or SMase for 1 h and treated with vehicle or Nigericin for an additional 1 h to induce inflammasome activation. D: IL-1β secretion from control and Myl-Abc dko BMDMs transfected with control (Scrambled) or siRNA against Gramd1b for 48 h. After 48 h of transfection, cells were primed with LPS for 3 h and treated with SMase for 1 h and treated with vehicle or ATP for an additional 1 h. E: Immunoblot of intracellular Caspase-1 and GSDMD cleavage from (D). F: IL-1β secretion from wild-type BMDMs transfected with control (Scrambled) or siRNA against Gramd1b for 48 h. After 48 h of transfection, cells were primed with LPS for 3 h and treated with vehicle or cholesterol microcrystals for additional 6 h to induce inflammasome activation. G: IL-1β secretion from vehicle and acLDL loaded wild-type BMDMs that were pre-treated macrophages with vehicle or ACAT inhibitor (Sandoz 58-035) for 30 min and primed with LPS for 3 h and treated with ATP for an additional 1 h to induce inflammasome activation. H: Immunoblot of intracellular Caspase-1 cleavage from (G). ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05 by t test (A) and two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: For mechanistical inflammasome assays, treatments of various compounds were done as listed below; 5 μg/ml U18666A compound (Millipore, 662015-10MG) for 48 h prior to LPS, 100 milliunits/ml of Sphingomyelinase (Sigma, S8633-50UN) for 1 h prior to ATP or Nigericin, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor Sandoz 58-035 (Sigma, S9318-5MG) for 30 min prior to LPS, 0.1 mM C2 ceramide (Avanti polar lipids, 860502P-5 mg) for 1 h prior to ATP or Nigericin, 1 μM Xestospongin C (Sigma, 682160-10UG) for 30 min prior to LPS, 1 μM KN-93 (ApexBio, B1306) for 30 min prior to LPS, 1 μM SP600125 (SelleckChem, S1460) for 30 min prior to LPS, 1 μM G5 (Ubiquitin isopeptidase inhibitor I, Sigma, 662125-10MG) for 30 prior to ATP or Nigericin, 25 nM THL (Thiolutin, Tocris, 1567) for 30 prior to ATP or Nigericin, 25 nM HL (Holomycin, Santa Cruz, sc-490291) for 30 prior to ATP or Nigericin, 100 μg/ml Cyclodextrin (CD)-cholesterol (Sigma, C4951) for 1 h prior to ATP or Nigericin.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Control, Western Blot, Transfection, Comparison

Journal: Journal of Lipid Research

Article Title: Cholesterol trafficking to the ER leads to the activation of CaMKII/JNK/NLRP3 and promotes atherosclerosis

doi: 10.1016/j.jlr.2024.100534

Figure Lengend Snippet: BRCC3-mediated NLRP3 deubiquitylation promotes inflammasome activation by cholesterol accumulation. A: Control and Myl-Abc dko BMDMs were primed with LPS for 3 h and pre-treated with vehicle or G5 (Deubiquitinase inhibitor) for 30 min and treated with vehicle or Nigericin for an additional 1 h to induce inflammasome activation. LDH release and IL-1β secretion in media were assessed. B: Vehicle and acLDL loaded wild-type BMDMs were primed with LPS for 3 h and treated with vehicle or Holomycin for 30 min and treated with vehicle or ATP for an additional 1 h to induce inflammasome activation. LDH release and IL-1β secretion in media were assessed. C: IL-1β secretion from wild-type BMDMs were primed with LPS for 3 h and treated with vehicle or Holomycin or Thiolutin for 30 min and vehicle or cholesterol microcrystals for additional 6 h to induce inflammasome activation. D: IL-1β secretion from control and Myl-Abc dko BMDMs transfected with control (Scrambled) or siRNA against Brcc3 for 48 h. After 48 h of transfection, cells were primed with LPS for 3 h and treated with vehicle or SMase for 1 h and treated with ATP for an additional 1 h. E: Immunoblot of BRCC3 and NLRP3 in cell lysates immunoprecipitated with anti-BRCC3 in vehicle and acLDL loaded wild-type BMDMs primed with LPS for 3 h. F: Immunoblot of NLRP3 ubiquitylation in cell lysates immunoprecipitated with anti-NLRP3 in control and Myl-Abc dko BMDMs that were treated with LPS for 3 h and vehicle or SMase for 1 h. G: Immunoblot of NLRP3 ubiquitylation in cell lysates immunoprecipitated with anti-NLRP3 in Ly6G − CD11b + monocytes/macrophages isolated from Ldlr −/− mice that were transplanted with BM from Myl-Abc dko mice and fed WTD for 8 weeks. ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05 by t test (C, G) and two-way ANOVA with Sidak’s multiple comparison test (A, B, D, and G).

Article Snippet: For mechanistical inflammasome assays, treatments of various compounds were done as listed below; 5 μg/ml U18666A compound (Millipore, 662015-10MG) for 48 h prior to LPS, 100 milliunits/ml of Sphingomyelinase (Sigma, S8633-50UN) for 1 h prior to ATP or Nigericin, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor Sandoz 58-035 (Sigma, S9318-5MG) for 30 min prior to LPS, 0.1 mM C2 ceramide (Avanti polar lipids, 860502P-5 mg) for 1 h prior to ATP or Nigericin, 1 μM Xestospongin C (Sigma, 682160-10UG) for 30 min prior to LPS, 1 μM KN-93 (ApexBio, B1306) for 30 min prior to LPS, 1 μM SP600125 (SelleckChem, S1460) for 30 min prior to LPS, 1 μM G5 (Ubiquitin isopeptidase inhibitor I, Sigma, 662125-10MG) for 30 prior to ATP or Nigericin, 25 nM THL (Thiolutin, Tocris, 1567) for 30 prior to ATP or Nigericin, 25 nM HL (Holomycin, Santa Cruz, sc-490291) for 30 prior to ATP or Nigericin, 100 μg/ml Cyclodextrin (CD)-cholesterol (Sigma, C4951) for 1 h prior to ATP or Nigericin.

Techniques: Activation Assay, Control, Transfection, Western Blot, Immunoprecipitation, Isolation, Comparison

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: CD147 is expressed on brain infiltrating monocytes. ( A ) Representative dot plot of sham and stroke (72hr) brain shows microglia and infiltrating myeloid cells. Representative dot plot of 72hr stroke brain, gated on myeloid cells, shows Ly6C hi monocytes, Ly6C lo monocytes, and Ly6G neutrophils. ( B ) Absolute cell counts at 72hr in stroke brain (and sham microglia). ( C ) Quantified % CD147 positive cells in brain. Representative histogram showing expression of CD147 on microglia (sham and stroke; gray= microglia-specific FMO). Representative histogram showing expression of CD147 on brain-infiltrating myeloid cells after stroke. ( D ) Quantified CD147 + cell counts from brain, n=6. ( E ) Quantified %CD147 positive blood cells. Representative histogram showing expression of CD147 on blood cells after stroke. ( F ) Quantified expression level of MMP-9 on brain-infiltrating myeloid cells (and sham microglia). Representative histogram showing expression of MMP-9 on brain-infiltrating myeloid cells (and sham microglia).

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: Matrix metalloproteinase-9 (MMP-9) protein concentrations and infarct volumes decrease following anti-CD147 antibody administration. ( A ) Timeline depicting the dosing strategy of CD147. ( B ) Representative western blots of MMP-9 protein concentrations in 72-hour post stroke mice following IgG control or anti-CD147 antibody administration, quantified in ( C ) p<0.01, n=4, * indicates statistical significance. ( D ) Representative TTC images of 72 hour post stroke brain slices of IgG control or ant-CD147 antibody. Red color details healthy brain tissue while white is indicative of infarcted tissue. ( E ) Quantification of percent infarct volume of cortex, striatum or total hemisphere relative to contralateral regions, p<0.05 for each region, n=4.

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: CD147 expression in the aged brain. ( A ) Representative dot plot of aged sham and stroke (72hr) brain; shows microglia and infiltrating myeloid cells. Representative dot plot of 72hr stroke brain; gated on myeloid cells, shows Ly6C hi monocytes, Ly6C lo monocytes, and Ly6G neutrophils. ( B ) Quantified counts at 72hr in stroke brain and sham microglia. ( C ) Quantified % CD147 positive cells in brain. ( D ) Absolute counts of CD147 + cells in brain, n=6. ( E ) Quantified %CD147 postive cells from blood. ( F ) Quantified expression level of MMP-9 on brain-infiltrating myeloid cells (and sham microglia).

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: CD147 blocking antibody reduced infarct volume and improved cognitive outcomes in aged male mice. ( A ) CD147 blocking antibody administration reduced the amount of atrophy witnessed in the aged brain following stroke compared to IgG control at day 14. ( B ) Representative cresyl violet stained brain slices. ( C ) Neurological deficit scores showed improvement at day 3 and day 14 following stroke. ( D ) Cognitive impairment, as measured by escape time on the Barnes Maze, was prevented in stroke mice that received CD147 blocking antibody. ( E ) Levels of pro-MMP-9 were reduced in stroke mice receiving blocking antibody. ( F ) Brain hemoglobin, a reflection of blood brain barrier breakdown and hemorrhagic transformation was decreased in blocking antibody treated mice.

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques: Blocking Assay, Staining, Transformation Assay

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: Demographic and characteristics of human post mortem cases.

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques:

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: Correlation between different variables in all human post mortem cases.

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques:

Journal: Aging (Albany NY)

Article Title: EMMPRIN/CD147 plays a detrimental role in clinical and experimental ischemic stroke

doi: 10.18632/aging.102935

Figure Lengend Snippet: Correlation between different variables in the human post mortem cases of acute and sub-acute infarct ages.

Article Snippet: Leukocytes were then washed twice in 300μl permeabilization/wash buffer (BD Biosciences) and resuspended in an intracellular antibody cocktail containing MMP-9-PE (S51-82; StressMarq Biosciences, Inc.) and subsequently fixed (N=4 SH and 6 ST) [ ].

Techniques:

Journal: The Journal of Cell Biology

Article Title: The DExD/H box ATPase Dhh1 functions in translational repression, mRNA decay, and processing body dynamics

doi: 10.1083/jcb.201007151

Figure Lengend Snippet: Dhh1 DQAD has differential effects on tethered and nontethered mRNAs. (A, left) Northern blot analysis of FBA1 -PP7 loop mRNA in dhh1Δ cells expressing Dhh1 DQAD -GFP, GFP-PP7CP, or Dhh1 DQAD -PP7CP normalized to SCR1 . (right) Western blot analysis of FBA1 protein levels compared with Xpo1. Mean values ± SD from three independent experiments are shown. (B) Wild-type Dhh1 or Dhh1 DQAD was expressed in a dhh1Δ cells and 10-fold serially diluted. Growth at 30 and 37° was monitored for >3 d. (C) ACT1 , CRH1 , and RPL25 mRNA levels were measured after transcriptional shutoff with thiolutin. mRNA decay was observed in dhh1Δ cells alone or expressing wild-type Dhh1 or Dhh1 DQAD . Graphs depict mean mRNA levels ± SEM from three independent experiments.

Article Snippet: Time points were collected after the addition of 3 µg/ml thiolutin (Enzo Life Sciences).

Techniques: Northern Blot, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: Thiolutin or reduced thiolutin alone fails to inhibit purified yeast Pol II in vitro . ( A ) Thiolutin and holomycin structures. ( B ) Thiolutin, or thiolutin treated with equimolar DTT, show no inhibition of Pol II transcription in vitro . Transcription activity assay was performed with ssDNA as the template and NTPs, including 32 P labeled UTP. The transcribed 32 P containing RNA was separated from free 32 P UTPs on 10% polyacrylamide gels for visualization. Experiments were performed with at least three times, and a representative experiment is shown.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: Purification, In Vitro, Inhibition, Activity Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: Mutations in diverse cellular pathways confer resistance/sensitivity to thiolutin. ( A ) Four libraries (Variomics libraries for non-essential or essential genes, Deletion libraries for non-essential or essential genes) were used for screening thiolutin resistant or sensitive mutants. Changes in abundance in the library was detected by deep sequencing of the PCR amplicon of the barcode region (Bar-seq). Variomics libraries consist of random gene variants and can be used to screen for both gain-of-function (GOF) and loss-of-function (LOF) alleles, whereas ‘Deletion’ libraries consist of single-gene-deleted mutants that can be used to screen for complete LOF mutants. ( B ) Different thiolutin resistant or sensitive mutant classes are revealed in the Bar-seq based screenings of pooled Variomics and Deletion libraries. ( C ) Thiolutin induced phenotypic profile in the pooled deletion libraries co-clusters with drugs with signature of ‘mitochondrial response to ROS’ and bathophenanthroline, a metal chelator. 4683 deletion mutants’ responses (in columns) to 3356 compounds (in rows) were used for hierarchical clustering, but only the thiolutin closely correlated compounds are shown in this figure for clarity. Resistant strains: yellow; sensitive strains: blue.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: Sequencing, Amplification, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: Divalent metals cations appear to alter thiolutin sensitivity in vivo and interact with reduced thiolutin in vitro . (A–D) 10-fold serial dilutions of yeast strains spotted onto different types of media to examine thiolutin sensitivity. ( A ) zap1Δ is hypersensitive to thiolutin. ( B ) pmr1Δ is hypersensitive to thiolutin. ( C ) Mn 2+ supplementation exacerbates WT yeast sensitivity to thiolutin. ( D ) Distinct sensitivities of thiolutin and 1,10-phenanthroline to Co 2+ , Cu 2+ and Mn 2+ and differential interactions with gene deletions. ( E ) Reduced thiolutin was incubated with a selection of divalent metal cations and UV absorbance was analyzed. Zn 2+ , Mn 2+ and Cu 2+ , but not Mg 2+ , interact with reduced thiolutin, as evident by alteration of the UV spectra of reduced thiolutin.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: In Vivo, In Vitro, Incubation, Selection

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: Apparent thiolutin/Mn 2+ complex inhibits Pol II transcription in vitro and in vivo . ( A ) Thiolutin requires both DTT (reductant) and Mn 2+ to inhibit Pol II transcription in vitro . A representative of multiple experiments is shown. Quantifications of this and a wide range of experimental conditions for thiolutin inhibition are shown in (C,D). ( B ) Pre-binding to DNA renders Pol II resistant to Thiolutin/Mn 2+ complex. This gel is representative of multiple independent experiments. ( C ) Quantification of thiolutin inhibition under a number of experimental conditions. Thiolutin (60 μg/ml) inhibition of Pol II requires DTT and Mn2 + and is blocked by pre-incubation of Pol II with DNA or with excess (12 mM) DTT added after Pol II treatment with thiolutin. All reactions are compared to baseline for individual Pol II in particular experiment, which is activity of Pol II plus vehicle (15% DMSO final). Different symbols indicate three independent Pol II purifications used in experiments. Note that variability in fraction of Pol II inhibited by thiolutin appears to track with Pol II purification (▴• Pol II consistently more inhibited than ⬥). Error bars are mean +/– standard deviation. Statistical analysis for selected pairs is one-way ANOVA with multiple comparisons controlling for false discovery rate (0.01) (method of Benjamini, Krieger, Yekutieli ). **** P < 0.0001, *** P < 0.0002, * P < 0.0332. ( D ) Quantification of thiolutin inhibition at lower thiolutin/DMSO concentrations and additional controls. All reactions are compared to baseline for individual Pol II in particular experiment, which is activity of Pol II in absence of drug or vehicle. Different symbols indicate two separate Pol II purifications (symbols here match Pol II purifications in (C). Statistical analysis for selected pairs is one-way ANOVA with multiple comparisons controlling for false discovery rate (0.01) (method of Benjamini, Krieger, Yekutieli ). **** P < 0.0001. ( E ) In vitro elongation assay for thiolutin-treated Pol II. (Top) Schematic indicates three assembly pathways for making elongation complexes on nucleic acid scaffolds in the absence of thiolutin, incubation with thiolutin after elongation complex assembly, or before elongation complex assembly. (Bottom) Thio/Mn 2+ inhibited Pol II can be directly assembled into a distinct and slow elongating complex, but only if Pol II is treated with thiolutin prior to complex assembly. Two experimental replicates were performed, and a representative replicate is shown. Duration times for incubation with NTPs were 0, 10, 30, 120, 300 and 600 s.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: In Vitro, In Vivo, Inhibition, Binding Assay, Incubation, Activity Assay, Purification, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: Thiolutin reduces Pol II occupancy genome wide. ( A ) Thiolutin inhibits Pol II occupancy at a TEF1 promoter-driven YLR454W reporter in vivo . Schematic indicates positions of PCR primers for chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) for a FLAG-tagged Rpb3 subunit of Pol II at a reporter gene representing a long transcription unit. Thiolutin (10 μg/ml) was added to cells for indicated times prior to crosslinking for ChIP. Three experimental replicates were performed, and error bars represent standard deviation of the mean. ( B ) Schematic of Pol II ChIP-seq experiment thiolutin treatment for the indicated times (control sample with addition of vehicle DMSO was for 8 min treatment). ( C ) Genome-wide decreases of Pol II occupancy at genes separated into quintile based on normalized Pol II occupancy in untreated cells from highest (Q1) to lowest (Q5). Quintiles are ∼846 genes each. Genes are rank ordered from shortest to longest with annotated gene 3′-ends indicated with the dashed line. ( D ) The majority of genes show decreases in Pol II occupancy as determined by fold change in normalized Pol II occupancy per base within each gene. Genes separated by class indicate that ribosomal protein (RP) genes show greater Pol II decreases than do ribosomal biogenesis (RiBi) than do other highly expressed genes in Q1. ( E ) Decrease in Pol II occupancy (y axis) shows some correlation with initial occupancy levels (x-axis) with RP genes showing special sensitivity to thiolutin treatment.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: Genome Wide, In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Standard Deviation, ChIP-sequencing, Control

Journal: Nucleic Acids Research

Article Title: Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

doi: 10.1093/nar/gkad1258

Figure Lengend Snippet: The apparent thiolutin/Mn 2+ complex is unstable in solution but Pol II can be stably altered. ( A ) Time course of changing UV spectra of reduced thiolutin treated with Mn 2+ . Reduced thiolutin (blue, 0 min) was treated with equivalent molar Mn 2+ , and the UV spectra were acquired at different time points. Spectra over time were colored in a color gradient from blue to red. Three experimental replicates were performed, and a representative replicate is shown. ( B ) Thio/Mn 2+ lost inhibitory activity after 20 min in solution though immediately treated Pol II remains inhibited. Two experimental replicates were performed and were consistent. One replicate is shown. ( C ) Thio/Mn 2+ can be reversed by excess DTT. Excess of DTT was added after 20 min of Pol II inhibition by Thio/Mn 2+ . DTT was at 12 mM during thiolutin treatment. Two replicates under these exact conditions were performed, and a representative replicate is shown. Results are representative of additional experiments performed under slightly different conditions.

Article Snippet: Chemicals were commercially obtained from the following: Cayman Chemical (Thiolutin), Gold Biotechnology (DTT, TCEP, 5FOA), Sigma (MnCl 2 ), JT Baker (ZnCl 2 ), BDH Chemicals (MgCl 2 ).

Techniques: Stable Transfection, Activity Assay, Inhibition