Journal: Science Advances

Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

doi: 10.1126/sciadv.1500968

Figure Lengend Snippet: ( A ) Kinetic traces show catalytic reduction of increasing concentrations of HS x − at 100 nM TrxR1 and 250 μM NADPH following the consumption of NADPH (at 340 nm). ( B ) Initial rates of NADPH consumption by TrxR1 using HS x − as substrate show linear dependence on the HS x − concentration up to 1 mM. ( C ) Addition of 50 μM sodium selenite (at the indicated time point by the arrow) to similar reaction mixtures as in (A) resulted in increased NADPH consumption rates (also see fig. S2). ( D ) The GCSG mutant of TrxR1 is inactive in a similar activity assay as in (A), indicating the need for catalysis of the Sec residue (see the activities of further mutants in fig. S3, A to C). ( E ) TrxR1 concentration–corrected initial rates were increased in the presence of 5 μM Trx1 and 2 μM TRP14 compared to TrxR1 alone with linear HS x − concentration dependencies. ( F and G ) Addition of HS x − had no inhibitory potential on TrxR1-coupled ( F ) insulin-reducing Trx1 activities or ( G ) cystine-reducing TRP14 activities. ( H ) Kinetic traces show catalytic reduction of 170 μM BSA-SSH by 50 nM TrxR1 at 250 μM NADPH, which are further accelerated by 5 μM Trx1 or 2 μM TRP14.

Article Snippet: The knockdown was verified in all clones by Western blot using antibodies against TrxR1 (sc-58444) and TRP14 (R&D Systems MAB3504), and GAPDH was used as a loading control (sc-25778).

Techniques: Concentration Assay, Mutagenesis, Activity Assay, Residue

Journal: Science Advances

Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

doi: 10.1126/sciadv.1500968

Figure Lengend Snippet: S3 refers to sampling according to . ( A ) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. ( B ) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. ( C ) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (* P < 0.05 using the paired t test); 100% in the control cells corresponds to 1.52 ± 0.55 μg/mg total protein. Error bars represent SDs of n = 4 experiments. ( D ) Two hours of treatment with 200 μM polysulfide induced higher levels of intracellular protein persulfides in intact TrxR1 and TRP14 knockdown cells compared to control cells. ( E ) A modified SRB cytotoxicity assay revealed that control cells (•) are significantly more viable upon polysulfide exposure than TrxR1 knockdown (○) cells (*** P < 0.0001 at 0.5 and 1 mM polysulfide concentration). Data points and error bars represent the average and SD, respectively, of n = 4 independent experiments of triplicate measurements.

Article Snippet: The knockdown was verified in all clones by Western blot using antibodies against TrxR1 (sc-58444) and TRP14 (R&D Systems MAB3504), and GAPDH was used as a loading control (sc-25778).

Techniques: Sampling, Western Blot, Knockdown, Stable Transfection, Transfection, Control, Staining, Plasmid Preparation, shRNA, Modification, Cytotoxicity Assay, Concentration Assay

Journal: Nucleic Acids Research

Article Title: HCR-Proxy resolves site-specific proximal RNA microenvironments at subcompartmental resolution

doi: 10.1093/nar/gkag086

Figure Lengend Snippet: Spatially resolved rRNA-proximal proteomes within subnucleolar compartments. ( A ) Schematics of probes designed for HCR-Proxy MS to spatially resolve pre-rRNA proteomes within multiphased nucleoli. ( B ) HCR-FISH deconvolved STED micrographs of probes targeting distinct regions of the nascent rRNA transcript (magenta, A′ UP; yellow, A′ DOWN; blue, ITS2). ( C and D ) Dot plots displaying enriched subnucleolar HCR-Proxy components, demonstrating the ability of HCR-Proxy MS to spatially resolve multiphased nucleolar interactomes between (C) A′ UP and A′ DOWN, and (D) A′ UP and the TS2 region, respectively. Error bars present the SDs. ( E ) Volcano plot of enriched interactors specific for A′ UP region (magenta), its newly validated interactor (green) and A′ DOWN region (yellow). The cut-off for identification of bona fide interactors was set on LASSO regularization-based log2 fold-change > 1 and associated −log10( P -value) > 1. For visualization purposes, −log10( P ) values ( y -axis) were thresholded at 5 (dashed line). ( F ) HCR-FISH IF of the newly validated A′ UP interactor. RNA FISH (magenta, A′ UP; yellow, A′ DOWN) and newly validated nucleolar protein localized to FC (grey, TXNRD1). Quantification of the signal co-localization between RNA target (magenta, A′ UP; yellow, A′ DOWN) and candidate protein TXNRD1 with the correlation analysis; direct image correlation at zero displacement (i.e. overlap, left), and displacement-dependent correlation (right; C > 1 indicates above-random co-localization; see the Materials and methods for details). Each dot or thin curve represents one of the ~10 images per condition acquired, the bands represent 80% confidence intervals, and thick curves the mean values (one-way Mann–Whitney test; *** P < 0.001, ** P < 0.01, * P < 0.05).

Article Snippet: To validate co-localization with nuclear condensates and to confirm localization of candidate proteins, we used the following antibodies against: SC-35 (mouse, Santa Cruz Biotechnology, cat #sc-53518, 1:200 dilution), NPM1 (mouse, ThermoFisher Scientific, cat #32-5200, 1:100 dilution), FBL (rabbit, Abcam, cat #ab5821, 1:200 dilution), TXNRD1 (rabbit, Proteintech, cat #11117-1-AP, 1:200 dilution), and GFP (rabbit, ThermoFisher Scientific, cat #A-21311, 1:100 dilution).

Techniques: MANN-WHITNEY

Journal: RNA

Article Title: Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

doi: 10.1261/rna.1905910

Figure Lengend Snippet: Western blot analysis after miRNA transfections helped validate RIP-Chip results. H4 cells were first transfected with full-length cDNA plasmids: pCMV6-LDLR, pCMV6-TXNIP, and pCMV6-DBI for 24 h, and were then transfected with 25 nM of miR-107, miR-124, miR-128, miR-320 precursors, or a negative control miRNA for additional 48 h. Cells were harvested, and Western blot analyses were performed using antibodies against each expressing proteins. Anti-β-actin antibody was used to probe each blot to monitor the total protein loadings. The three miR-128 targeted proteins shown were effectively knocked down by miR-128 transfection. LDLR was also strongly reduced by miR-124 transfection. RIP-Chip did not identify LDLR as miR-124 target, however, microarray analysis of total cell RNA showed that LDLR mRNA was significant decreased upon miR-124 transfection.

Article Snippet: Full-length cDNA (all UTR-containing) cloned in pCMV6-XL5 plasmid vectors were obtained that express human low density lipoprotein receptor (LDLR, {"type":"entrez-nucleotide","attrs":{"text":"NM_000527.2","term_id":"8051613","term_text":"NM_000527.2"}} NM_000527.2 ), thioredoxin interacting protein (TXNIP, {"type":"entrez-nucleotide","attrs":{"text":"NM_006472","term_id":"928192547","term_text":"NM_006472"}} NM_006472 ), and diazepam binding inhibitor (DBI, {"type":"entrez-nucleotide","attrs":{"text":"NM_020548.4","term_id":"54262129","term_text":"NM_020548.4"}} NM_020548.4 ) (OriGene Technologies, Inc.).

Techniques: Western Blot, Transfection, RNA Immunoprecipitation - Chromatin Immunoprecipitation, Negative Control, Expressing, Microarray

Journal: bioRxiv

Article Title: Alterations of redox and iron metabolism accompany development of HIV latency

doi: 10.1101/549014

Figure Lengend Snippet: (A) Subcellular localization of Nrf2 in CD4 + T-cells infected with HIV-1 or mock infected (3 dpi) as analyzed by biochemical fractionation. (B) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA + and HIV-1 RNA - cells (7 dpi) as measured by combining IF and HIV-1 RNA FISH. Scale bar = 2μm. n= number of cells from 3 donors. (C, D) Time course of the relative (infected vs mock infected) mRNA (C) and protein (D) levels of main targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection as measured by qPCR and western blot, respectively. TrxR1 and HMOX-1 were probed upon membrane stripping. (E) Comparison of the effect of different HIV-1 mutations on the mean relative (infected vs mock infected) mRNA level of the Nrf2 targets described in panel D. Values shown in panel B were calculated as nuclear corrected total cell fluorescence (as in) and analyzed by two-tailed unpaired t -test. For panels C, E, raw data were first normalized using GAPDH as housekeeping control and then expressed as Log 2 fold mRNA expression in infected vs mock infected cells (calculated using the 2-ΔΔCTmethod . In panel E the average of the six genes listed in Panel C is shown. For both panels data were analyzed by-two way ANOVA followed by Turkey’s post-test for multiple comparisons. ** P <0.01; *** P <0.001; **** P <0.001. Trx= thioredoxin; NQO1= NAD(P)H Quinone Dehydrogenase 1; HMOX-1= Heme Oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= Glutamate—cysteine ligase; TrxR1= thioredoxin reductase 1.

Article Snippet: Membranes were then blocked for 1 hr at RT with 5% skim milk in 0,1 % PBS-Tween and incubated overnight at 4o with the following primary antibodies diluted in 5% milk: α-beta-actin (1:1000), (Sigma Aldrich, Saint Louis, MI, USA), α-Trx (1:500; sc-58440), α-NQO1 (1:500, sc-32793) (Santa Cruz Biotechnology, Dallas, TX, USA), α-TrxR1 (1:500, MAB7428) (R&D systems, Minneapolis, MN), α-HMOX-1 (1:500, ab13248), FTH-1 (1:100, ab75972), α SLC40A1 (1:500, ab78066) (Abcam, Cambridge, UK) α-PML (1:500) (A301-167A Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Infection, Fractionation, Western Blot, Membrane, Stripping Membranes, Comparison, Fluorescence, Two Tailed Test, Control, Expressing