Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: ATAD5-RLC is a robust PCNA unloader. a Deletion mutants of ATAD5 examined for PCNA unloading activity. Top diagram shows the locations of the reported motifs in human ATAD5. Lines between boxes represent predicted coiled-coil structures. Boundaries of motifs are denoted as amino-acid numbers. N-terminal deletion mutants of ATAD5 were fused to its putative NLS sequence located at its N-terminus (residues 1–32 of ATAD5). b The PCNA unloading activity is conferred by the C-terminal domain of ATAD5. The indicated ATAD5 deletion mutants were transiently expressed in ATAD5-depleted 293T cells. Chromatin-bound PCNA was analyzed by immunoblotting after chromatin fractionation. ATAD5 (ΔN692) exhibited PCNA unloading activity. c Quantification of chromatin-bound PCNA in ( b ). Values indicate relative chromatin-bound PCNA amount compared with the control chromatin ( n = 3). Error bars indicate standard deviations in all quantification results. d Purification of ATAD5-RLC. Baculovirus encoding HIS-ATAD5 (ΔN692)-3XFLAG were co-infected to insect cells with a virus encoding RFC2–5. Pentameric ATAD5-RLC was purified from cell extracts through sequential application onto HIS, FLAG, and Heparin affinity columns. Coomassie-stained purified ATAD5-RLC has a stoichiometric amount of each subunit. e Schematic diagram of PCNA unloading assay. DNA substrates were prepared by annealing oligonucleotides to the 5′-biotinylated DNA. Substrate DNA was attached to the streptavidin-coated magnetic beads. PCNA was loaded to the substrate DNA by purified RFC. After washing, ATAD5-RLC was treated to the DNA-loaded PCNA. PCNA that remained on DNA was analyzed by immunoblotting. Error bars indicate standard deviations in all PCNA loading/unloading results. f ATAD5-RLC unloads PCNA. Indicated amounts of purified ATAD5-RLC, RFC, CTF18-RLC, and RAD17-RLC were treated to the DNA-loaded PCNA. 10-nucleotide-gap DNA (130-mer) was used for this assay. Unloading reaction contained 1 mM ATP. g Quantification of ( f ). Relative DNA-loaded PCNA amounts are indicated compared with the control reaction. Error bars indicate standard deviation ( n = 3). See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: Activity Assay, Sequencing, Western Blot, Fractionation, Control, Purification, Infection, Virus, Staining, Magnetic Beads, Standard Deviation

Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: Active ATAD5-RLC formation requires functional RFC2–5 binding motif. a Constructs to identify the RFC2–5 binding motif (RBM). The positions of the deletions and point mutations (CM1–4) are indicated. Four conserved five-amino-acid stretches in RBM were mutated and denoted as CM1–4 (see Supplementary Fig. ). b – d ATAD5 (1602–1844) is a RBM. The indicated ATAD5 fragments were transiently expressed in 293T cells and immunoprecipitated using anti-FLAG beads. Bindings of RFC2–5 were monitored by co-immunoprecipitation of RFC4 or RFC5. b ATAD5 (1602–1719) is important for RFC2–5 binding. ATAD5 (1602–1844) bound to RFC2–5, but ATAD5 (1720–1844) did not. c ATAD5 (1720–1844) is also necessary for RFC2–5 binding. d E 1713 ALSF 1717 of ATAD5 is important for RFC2–5 binding. The CM1 mutant, E 1713 ALSF 1717 to AAGGG, was severely defective in RFC2–5 binding. e – g Proper RFC2–5 binding to ATAD5 is crucial for PCNA unloading. PCNA unloading activity of the indicated ATAD5 variants were examined as described in Fig. . e RBM-deleted ATAD5 (693–1719) is defective in PCNA unloading. Graph shows relative amount of PCNA on the chromatin ( n = 3). f ATAD5 (CM1), which is defective in RFC2–5 binding, is severely defective in PCNA unloading. Graph shows relative amount of PCNA on the chromatin ( n = 3). g RBM-deleted ATAD5 (693–1719) fails to unload PCNA in vitro. In total, 1.4 kbps DNA was used for this assay. Graph shows relative PCNA amounts remained on DNA after unloading reaction ( n = 2). See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: Functional Assay, Binding Assay, Construct, Immunoprecipitation, Mutagenesis, Activity Assay, In Vitro

Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: ATPase interface of ATAD5 is crucial for PCNA unloading. a ATPase-motif mutants examined in this study. Conserved lysine (K1138) in the Walker A motif or negatively charged amino acids in the putative Walker B motif (E1305, E1306, and D1308) were mutated as indicated (denoted as KA and NQA, respectively). E1173K mutation (EK) is located in the conserved region between Walker A and Walker B motifs. b ATPase-motif mutants are defective in PCNA unloading. ATAD5 (ΔN400) harboring mutations in the ATPase-motif were expressed in ATAD5-depleted 293T cells. Graph shows relative amount of PCNA on the chromatin ( n = 3). The amount of PCNA on the chromatin shows that all ATPase-motif mutants are defective in PCNA unloading, similar to the RBM-deleted mutant (ATAD5 (ΔC1720)). c Functional Walker A and Walker B motifs are required for stable interaction between ATAD5 and RFC2–5. The indicated ATAD5 variants were affinity-purified and co-immunoprecipitated RFC3–5 were examined. The KA and NQA mutants show reduced RFC2–5 binding compared with wild-type and EK mutant. d ATPase-motif mutants of ATAD5-RLC fail to unload PCNA in vitro. Unloading activity of purified wild-type or mutant ATAD5-RLC was examined. In total, 1.4 kbps DNA was used for this assay. Graph shows relative PCNA amounts remained on DNA after unloading reaction ( n = 3 for KA and EK experiments and n = 2 for NQA experiments. NQA experiments share control reaction with ATAD5 (693–1719) experiments (Fig. ), because two experiments were performed together). e ATP-γ-S partially reduces the efficiency of PCNA unloading by ATAD5-RLC. The PCNA unloading reaction was performed in the presence of ATP or ATP-γ-S. In total, 1.4 kbps DNA was used for this assay. Graph shows relative PCNA amounts remained on DNA after unloading reaction ( n = 2). See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: Mutagenesis, Functional Assay, Affinity Purification, Immunoprecipitation, Binding Assay, In Vitro, Activity Assay, Purification, Control

Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: Loading and unloading of PCNA occur through distinct steps. a Schematic diagram of smFRET measurements by total internal reflection microscopy to monitor PCNA loading and unloading. Cy3-labeled PCNA was pre-assembled with RFC and loaded to Cy5-labeled primed-template DNA, which was tethered to the surface via biotin (B) to neutravidin (N) binding, and the fluorescence signals were recorded. When loading was complete, ATAD5-RLC was added to observe the PCNA unloading process. b A representative smFRET trace from the PCNA loading experiment, showing Cy3 (green)/Cy5 (magenta) intensities, FRET efficiency (navy), and state sequence found from hidden Markov modeling (light blue). The association moment is defined as 0 s. c A representative smFRET trace from the PCNA unloading experiment. The dissociation moment is defined as 0 s. d Heat map of FRET population dynamics generated from 393 loading traces synchronized at PCNA-DNA association. Three distinct stages, LI1, LI2, and LS, are marked. e Heat map generated from 513 unloading traces synchronized at PCNA-DNA dissociation. Two distinct stages, LS and UI, are marked. f Transition density plot (TDP) generated from hidden Markov modeling on the first 6 s of the loading traces since PCNA-DNA association. LI1→LI2 and LI2→LS transitions are marked in red circles. g TDP generated from hidden Markov modeling on the last 6 s of the unloading traces prior to PCNA-DNA dissociation. LS→UI and UI→LS transitions are marked in red and green circles, respectively. h – j Dwell time distributions of LI1 (H), LI2 (I), and UI (J) states during PCNA loading and unloading processes. Dwell times of LI1 and LI2 were fitted to single exponential decay curve, Ae –t/τ , and τ values are shown. k Heat map generated from 230 loading traces with ATP-γ-S, showing the complex stalled at the initial FRET level. l Heat map generated from 102 unloading traces with ATP-γ-S showing similar transition to that with ATP. m Dwell time distribution of UI state measured with ATP-γ-S. See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: Microscopy, Labeling, Binding Assay, Fluorescence, Sequencing, Generated

Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: ATAD5-RLC unloads ubiquitinated PCNA. a Schematic diagram of PCNA mono- and poly-ubiquitination. After loading onto bead-coupled DNA, PCNA was mono-ubiquitinated with UBA1, RAD6B-RAD18, and ubiquitin. Mono-ubiquitinated PCNA was poly-ubiquitinated with UBA1, HLTF, UBC13-MMS2, and ubiquitin. DNA beads were washed after each reaction to remove ubiquitination enzymes and free ubiquitin. Unloading of mono-ubiquitinated PCNA (Ub-PCNA) or poly-ubiquitinated PCNA (Ub N -PCNA) was monitored by immunoblot using anti-PCNA antibody unless indicated. b Mono-ubiquitination of PCNA. Same amount of free PCNA or DNA-loaded PCNA were ubiquitinated with mono-ubiquitination enzymes (+: 16.7 nM UBA1, 66.7 nM RAD6B-RAD18, ++: 33.4 nM UBA1, 133.4 nM RAD6B-RAD18). +DNA is a reaction in which free PCNA was mixed with bead-coupled DNA without loading reaction. DNA-loaded PCNA was preferentially mono-ubiquitinated. c – d ATAD5 (ΔN692)-RLC unloads mono-ubiquitinated PCNA. The indicated amount of ATAD5 (ΔN692)-RLC was used to treat DNA-loaded unmodified or mono-ubiquitinated PCNA. Graph shows relative PCNA or Ub-PCNA amounts remained on DNA after unloading reaction. In total, 1.4 kbps DNA was used for this assay. c ATAD5 (ΔN692)-RLC were titrated in the unloading reaction of PCNA or mono-ubiquitinated PCNA ( n = 3). d Time-course unloading of PCNA or mono-ubiquitinated PCNA by 1.2 nM ATAD5 (ΔN692)-RLC ( n = 2). e In vitro PCNA poly-ubiquitination. PCNA was loaded to 130-mer substrate DNA, then poly-ubiquitinated with the indicated enzymes. Poly-ubiquitination reaction was performed through a single step (1-step) or two steps (2-step). In two-step reactions, the poly-ubiquitination reaction was separately performed after the mono-ubiquitination reaction. The two-step reaction was more efficient compared with the single-step reaction. f ATAD5 (ΔN692)-RLC unloads mono- and poly-ubiquitinated PCNA with similar efficiency. After the mono- or poly-ubiquitination reaction, ATAD5 (ΔN692)-RLC was treated to DNA-loaded ubiquitinated PCNA. Unloading of ubiquitinated PCNA was monitored with anti-Ub-PCNA antibody. Graph shows relative mono-Ub-PCNA or poly-Ub-PCNA amounts remained on DNA after unloading reaction ( n = 3). See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: Ubiquitin Proteomics, Western Blot, In Vitro

Journal: Nature Communications

Article Title: Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes

doi: 10.1038/s41467-019-10376-w

Figure Lengend Snippet: Okazaki fragment-processing enzymes inhibit PCNA unloading by Elg1-RLC. a Elg1-RLC unloads PCNA in vitro. DNA-loaded yeast PCNA (Pol30p) was treated with indicated amount of purified Elg1-RLC in the presence of ATP or ATP-γ-S. In total, 1.4 kbps DNA was used for PCNA unloading assays shown in this figure. Graphs show relative PCNA amounts remained on DNA after unloading reaction ( n = 3 unless indicated). Error bar indicates standard deviation. b Elg1-RLC unloads PCNA. RFC, Ctf18-RLC, and Rad24-RLC did not unload PCNA from DNA. c The ATPase motif of Elg1-RLC is crucial for PCNA unloading activity. Mutation of the conserved lysine in Walker A motif or mutations of two aspartates in Walker B motif abolished the unloading activity of Elg1-RLC. d Purification of Elg1 N-terminal or C-terminal deletion mutants. Elg1 (ΔC536) failed to interact with Rfc2–5. e Elg1 (216-683) is crucial for PCNA unloading. PCNA unloading activity of indicated deletion mutants of Elg1-RLC were examined ( n = 2). f Okazaki fragment-processing enzymes inhibit Elg1-RLC mediated PCNA unloading. Indicated proteins were added to the PCNA unloading reaction with Elg1-RLC. g , h PCNA-Fen1 interaction is required for the inhibition of Elg1-RLC mediated PCNA unloading. g PIP box mutant of Fen1 (PIP) did not inhibit PCNA unloading compared with the wild-type or nuclease-motif mutant (CAT). h PIP box mutant of Fen1 failed to interact with PCNA in vitro. Purified wild-type or mutant Fen1 were mixed with PCNA, and Fen1 was affinity-purified from the mixture using anti-FLAG beads. Co-isolated PCNA was analyzed by immunoblotting. See also Supplementary Fig.

Article Snippet: The following antibodies were used: anti-PCNA (PC10) antibody (Santa Cruz Biotechnology, sc-56, 1:2000); anti-Ubiquityl-PCNA (Lys164) antibody (Cell Signaling, #13439, 1:1000); anti-FLAG antibody (Sigma, F3165, 1:1000); anti-V5 antibody (Sigma, V8137, 1:2000); anti-RFC3 antibody (Bethyl, A300-186A, 1:500); anti-RFC4 antibody (Abcam, ab182145, 1:1000); anti-RFC5 antibody (Abcam, ab79871, 1:500); anti-Lamin B1 antibody (Abcam, ab16048, 1:1000); anti-Histone H3 antibody (Merck-millipore, 07-690, 1:5000); anti-Strep-Tactin HRP Conjugate antibody (IBA-life sciences, 2-1502-001, 1:1000).

Techniques: In Vitro, Purification, Standard Deviation, Activity Assay, Mutagenesis, Inhibition, Affinity Purification, Isolation, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Trafficking of AFF 1-induced antibodies into the CNS of MBP-α-syn tg mice. (A) Monoclonal AFF 1-induced antibodies were tagged with Alexa-488 and administered to non-tg or MBP-α-syn mice. Alexa-488 tagged mAb-AFF 1 bounded α-syn within cell bodies (arrow-head) and blood vessels (bv). As negative control, a non-immune Alexa-488-tagged IgG1 was used. Scale bar = 5 μm (B) mAb-AFF 1 or non-immune IgG1 were tagged with Alexa-488 and administered to non-tg or MBP-α-syn mice. Time course analysis was performed every 24 h for 3 days, and fluorescence was only increased in brain sections of MBP-α-syn tg animals injected with Alexa-488-tagged mAb-AFF 1. Results are shown as corrected intensity values and expressed as average ± SEM. n = 3 animals per group and time point (C) AFF 1-induced antibodies were detected with and FITC-tagged anti-mouse antibody in brain sections of immunized MBP-α-syn tg mice (green), together with an antibody against Iba1 (microglia) or S100 (astrocytes) (red). Cell nuclei were stained with DAPI (blue). Colocalization was observed in microglial cell bodies and projections, but not in astroglial cells (arrows) (D) AFF 1-induced antibodies detected with and FITC-tagged anti-mouse antibody in brain sections of immunized MBP-α-syn tg mice (green), together with an antibody against p25 (oligodendrocytes) or NeuN (neurons) (red). Cell nuclei were stained with DAPI (blue). Colocalization was observed in oligodendroglial cell bodies, but not in neurons (arrows). Scale bar = 5 μm.

Article Snippet: Sections were immunostained with antibodies against α-syn (Chemicon, 1:250), NeuN (neuronal marker, Millipore, 1:1000), MAP2 (dendrites, Millipore, 1:250), GFAP (astroglial marker, Millipore, 1:500), Iba1 (microglia, Wako, 1:2000), p25 (oligodendrocytes), and horse anti-mouse IgG (Vector Laboratories) and imaged with an Olympus BX54 bright field digital microscope or a laser scanning confocal microscope.

Techniques: Negative Control, Fluorescence, Injection, Staining

Journal: Pathogens

Article Title: Porcine Alveolar Macrophages’ Nitric Oxide Synthase-Mediated Generation of Nitric Oxide Exerts Important Defensive Effects against Glaesserella parasuis Infection

doi: 10.3390/pathogens8040234

Figure Lengend Snippet: SH0165-induced activation of NF-κB signaling pathway-mediated NO generation in 3D4/21 cells. ( a ) Western blot showing time-dependent and bacterial MOI-dependent phosphorylation of the NF-κB p65 subunit in 3D4/21 cells in response to SH0165. ( b ) Western blot and comparison of p65 phosphorylation in response to SH0165 and HK-SH0165 (MOI 100) at the time points indicated. ( c ) Western blot analysis of IκB-α degradation in whole-cell lysates of 3D4/21 cells with or without SH0165 infection. ( d ) Real-time PCR analysis of NOS2 expression in 3D4/21 cells upon 12 h of SH0165 infection (MOI 100) with or without pretreatment of BAY11-7082 or CAY10657. ( e ) Intracellular NO generation at 12 h of infection at MOI of 100 was detected by the DAF-2 DA probe via fluorescence microscope and quantified with a fluorescence microplate reader. Scale bar, 100 μm. ( f ) Real-time PCR analysis of IRF1 expression in 3D4/21 cells upon SH0165 infection. ( g ) Cells were collected 24 h after siRNA transfection and real-time PCR analysis of IRF1 expression. NC, negative control. ( h ) IRF1 silencing by siRNA and was then infected with SH0165 or non-infected for 12 h. Real-time PCR analysis of IRF1 expression in 3D4/21 cells. Band densitometry was normalized to T-p65 or β-actin and shown as mean ± SD from three independent analyses. All real-time PCR data were normalized to GAPDH and shown as means ± SDs from three independent assays. Statistical differences were analyzed using Student’s t -tests (***, p < 0.001; **, p < 0.01; *, p < 0.05; NS: not significant).

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) in 0.1 M Tris-buffer saline (pH 7.5) containing 0.05% Tween-20 (TBST) for at least 1 h, and then probed overnight with appropriate primary antibodies against as follows: IκB-α, p-p65, p65 or β-actin (1:2000 in 5% BSA in TBST; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Comparison, Infection, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Transfection, Negative Control

Journal: Molecular cell

Article Title: Rad52 Restrains Resection at DNA Double-Strand Break Ends in Yeast

doi: 10.1016/j.molcel.2019.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mPEG 2OOO-DOPE , Avanti Polar Lipids , 880130C.

Techniques: Produced, Recombinant, Protease Inhibitor, Clone Assay, DNA Labeling, SYBR Green Assay, Western Blot, Fluorescence, Microscopy, Software, Real-time Polymerase Chain Reaction, Imaging, Hybridization

Journal: Cell Communication and Signaling : CCS

Article Title: The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation

doi: 10.1186/s12964-014-0053-y

Figure Lengend Snippet: Ectopic expression of BtpA in HeLa cells induces filopodia. HeLa cells were transiently transfected with plasmids encoding GFP-BtpA. Sixteen hours after transfection the cells were fixed and actin was stained with Rhodamine phalloidin. Images were taken using confocal fluorescence microscopy. Large image on top, Z-Stack of 16 merged images slices (1 μm depth) of HeLa cells, showing actin distribution (red) and one cell expressing GFP-BtpA (green). The smaller images below are individual image slices. Scale bar, 25 μm. Data are representative of 3 or more independent experiments.

Article Snippet: Monoclonal mouse anti-β-tubulin (Sigma Aldrich, T4026), mouse anti-FK2 (Enzo Life science, BML-PW8810), Texas Red anti-mouse (Vector Laboratories, TI-2000), Rhodamin phalloidin (Invitrogen R415), Rabbit anti-BtpA (a gift from Marty Roop, East Carolina University, Greenville), FITC anti-rabbit (Vector Laboratories, FI-2000) were used for immune labelling.

Techniques: Expressing, Transfection, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A) Circular tree representing the domain conservation relationship of human RNH1. Protein sequence alignments were made using MAFFT. A maximum–likelihood phylogenetic tree was generated using IQ-Tree with 1000 bootstrap replicates. Internal tree scale is shown with circular grid. (B) Structural alignment of protein domains. The domain information of selected proteins taken from Uniprot and represented using Illustrator for Biological Sequences (IBS) tool. (C) Human healthy bone marrow biopsies were stained with RNH1 antibody. Myeloid cells showing high RNH1 expression are indicated with arrows. Original magnification 10X and 100X. (D) Bone marrow biopsies from patients with confirmed inflammatory conditions were stained with RNH1 antibody. Myeloid cells with increased RNH1 expression are indicated with arrows. Original magnification 10X and 100X. (E) THP1 cells were stimulated with TLR2 ligand Pam3CSK4 (1 µg/ml) for 24 hours. Total protein lysates were isolated and analyzed by Western blot with indicated antibodies. Blots are representative of two independent experiments. (F) Mouse primary BMDMs were stimulated with TLR4 ligand LPS (1 µg/ml) for 24 hours. Total protein lysates were isolated and analyzed by Western blot with indicated antibodies. Blots are representative of two independent experiments.

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Sequencing, Generated, Staining, Expressing, Isolation, Western Blot

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A and B) PMA differentiated wildtype (WT) and RNH1-KO THP1 cells were stimulated with Nigericin (5 µM) for 1h or with MSU (500 µg) and Gramicidin A (30 µg) for 5h. (A) Supernatants were collected and IL-1β ELISA was performed. Data are means ± SEM of pooled data from three independent experiments. Statistical analyses were performed using a two-tailed t -test. (B ) Cell lysates and supernatants were analysed for pro- and cleaved-forms of caspase-1 and IL-1β by Western blot. Blots were representative of three independent experiments. (C) WT and RNH1-KO THP1 cells were stimulated with NLRP3 agonist Nigericin (5µM) for 18h. Supernatants were collected, and cell death was measured with LDH assay. Data are means ± SEM of pooled data from three independent experiments. Statistical analyses were performed using a two-tailed t -test. (D and E) RNH1-KO THP-1 cells transiently infected with GFP tagged control or RNH1 expressing lentivirus particles. (D) Cell lysates were analysed by Western blot to demonstrate the RNH1 reconstitution in RNH1-KO THP1 cells. Blots are representative of three independent experiments. (E) These cells were stimulated with Nigericin (5 µM) for 1h or with MSU (500 µg) or Gramicidin A (30 µg) for 5h. Supernatants were collected and IL-1β ELISA was performed. Data are means ± SEM of pooled data from three independent experiments. (F and G) Total cell lysates from THP1 cells constitutively expressing control or Flag-RNH1 were analysed by Western blot with indicated antibodies to demonstrate RNH1 overexpression (F) . Blots are representative of three independent experiments. These cells were stimulated with Nigericin (5 µM) for 1h or with MSU (500 µg) or Gramicidin A (30 µg) for 5h. Supernatants were collected and IL-1β ELISA was performed (G) . Data are means ± SEM of pooled data from three independent experiments. (H and I) Immunofluorescence microscopy analysis of ASC specks in THP1 cells stimulated with Nigericin (5 µM) for 1h. DNA staining is shown in blue (DAPI) and ASC staining is shown in red. Arrowheads indicate ASC inflammasome specks. (Scale bar: 100 μm) (H) . Quantification of ASC specks ( I ). Data are means ± SEM of pooled data analysing at least 10 fields from three independent experiments.

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Lactate Dehydrogenase Assay, Infection, Expressing, Over Expression, Immunofluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A and B) Undifferentiated WT and RNH1-KO THP1 cells were stimulated with TLR2 ligand Pam3CSK4 in a dose-( A ) and time-( B ) dependent manner as indicated. Total cell lysates analysed for pro-IL-1β and NLRP3 expression by Western blot. Blots are representative of three independent experiments. (C) Undifferentiated WT and RNH1-KO THP1 cells were stimulated with Pam3CSK4 (1µg/mL) for different time points as indicated. Total cell lysates analysed for NF-κB activation by Western blot with indicated antibodies. Blots are representative of three independent experiments. (D and E) Undifferentiated WT and RNH1-KO THP1 cells were stimulated with Pam3CSK4 (1µg/mL) for 6h and 18h. Supernatants were analyzed for TNF and IL-6 by ELISA, respectively. Data are means ± SEM of pooled data from three independent experiments. (F and G) PMA differentiated WT and RNH1-KO THP1 cells were transfected with AIM2 agonist poly dA:dT (5 µg) or NAIP/NLRC4 agonist cytosolic flagellin (600 ng) for 5h. Supernatants were collected and IL-1β ELISA was performed (F) . Data are means ± SEM of pooled data from three independent experiments. Statistical analyses were performed using a two-tailed t -test. Cell lysates and supernatants were analysed for pro- and cleaved-forms of caspase-1 and IL-1β by Western blot (G) . Blots are representative of three independent experiments.

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Expressing, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A) Total cell lysates from WT and RNH1-KO THP1 cells were analysed for pro-caspase-1 and ASC expression by Western blot. Blots are representative of two independent experiments. (B) qRT-PCR analysis for CASP-1 and ASC mRNAs from WT and RNH1-KO THP1 cells. mRNA levels are normalized to 18S rRNA expression. Data shown as means ± SEM from three independent experiments. (C-E) WT and RNH1-KO THP1 cells were treated with Actinomycin D or Cycloheximide or with the proteasome inhibitor MG 132 for indicated time duration. Cell lysates were analysed for pro-caspase-1 protein levels by Western blot. Blots are representative of three independent experiments. (F) HEK293T cells were treated with or without MG-132 and transfected with Flag-tagged casapase-1 with different concentration of GFP-tagged RNH1 plasmid as indicated. Cells were harvested and cell lysates were analyzed for caspase-1 by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (G) HEK293T cells were transfected with Flag-tagged OMI/HTRA2 plasmid with different concentrations of GFP-tagged RNH1 plasmid as indicated. Cells were harvested and cell lysates were analyzed by Western blot with indicated antibodies. Blots are representative of three independent experiments.

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Concentration Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A) Schematic showing design of Rnh1- floxed targeting vector (see methods for details) (B) DNA isolated from mouse ear biopsies was genotyped by PCR. Primers were designed to distinguish WT and floxed allele sequence. 314 bp size corresponds to WT, and 514 bp size corresponds to floxed gene. To detect Rnh1 deletion after Cre-recombination in Rnh1 fl/fl MxCre + mice (after one week of polyIC treatment), a third primer was used which amplifies 365 bp size (see methods). (C) Total protein lysates from BM cells of WT and Rnh1 -/ mice were analyzed by Western blot with the indicated antibodies. Blots are representative of 3 independent experiments. (D and E) BMDMs from WT and Rnh1 -/- mice primed with LPS (100 ng) for 3h and then cells were stimulated with Nigericin (5 µM) for 1h or transfected with poly dA:dT (5 µg) or flagellin (600 ng) for 5h. (D) Supernatants were collected and IL-1β ELISA was performed. Data are means ± SEM and representative of three independent experiments. Statistical analyses were performed using a two-tailed t -test. (E) Cell lysates and supernatants were analysed for pro- and cleaved-forms of caspase-1 and IL-1β by Western blot. Blots were representative of two independent experiments. (F) Schematic showing in vivo experimental setup. Bone marrow was transplanted from WT ( Rnh1 fl/fl ) or Rnh1 -/- (Rnh1 fl/fl MxCre + ) (CD45.2) into irradiated recipients (CD45.1). After 8 weeks of reconstitution mice were treated with Poly(I:C) (300 μg/mouse) once every two days for three doses. Four days later mice were used for the MSU peritonitis and LPS endotoxemia lethality models. (G) Post transplantation reconstitution levels were monitored after 8 weeks in the peripheral blood (PB) as indicated (n = 3 mice). Data are shown as mean ± SEM. P values were determined by 2way ANOVA with Sidak’s multiple comparisons test. (H) PB counts of WBC, neutrophils and monocytes in WT and Rnh1 -/- mice one week after polyIC injections. Data are shown are mean ± SEM. P values were determined by a two-tailed t -test (n = 3). (I and J) WT and Rnh1 -/- mice received intraperitoneal injection of MSU (1 mg/mouse) or sterile PBS. After 12h, peritoneal lavage fluid was taken and neutrophils infiltration analysed by FACS (I) . Peritoneal lavage supernatants were collected and IL-1β levels were quantified by ELISA (J) . Data shown as means ± SEM and P values were determined by a two-tailed t -test (n = 3-6 per group). (K) Kaplan–Meier survival curves of WT and Rnh1 -/- mice after LPS (10 mg/kg) treatment (n = 4 mice).

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Plasmid Preparation, Isolation, Sequencing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, In Vivo, Irradiation, Transplantation Assay, Injection

Journal: bioRxiv

Article Title: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients

doi: 10.1101/2021.04.12.438219

Figure Lengend Snippet: (A) Total cell lysates from peripheral blood buffy coats of ICU and general ward admitted COVID-19 patients were analysed for RNH1 protein levels by Western blot. Blots were repeated three times. (B) RNH1 protein levels from blots were quantified by ImageJ analysis for each patient and normalized with respective GAPDH protein levels. (C) Postmortem lung tissue from deceased persons with either COVID-19 or non-viral causes of death were stained for RNH1 and imaged using a Zeiss axioscan Z1. Subsequent image analysis was performed using Zeiss ZEN software to extract images at different magnifications. Insets show higher magnification of area indicated in the red boxes. Brown staining indicates RNH1 positive cells. (D) PMA differentiated WT and RNH1-KO THP1 cells were infected with SARS-CoV-2 pseudovirus for 24 hours. Infection efficiency was monitored by measuring GFP signal with fluorescence microscopy. Images are representative of two independent experiments. (Scale bar 25 µm). (E) Illustration of RNH1 mediated anti-inflammatory mechanisms. First, RNH1 could potentially inhibit NF-κB signaling through an unknown mechanism. Second, RNH1 regulates inflammasome activation by controlling caspase-1 protein levels via proteasome-mediated degradation.

Article Snippet: To construct full length GFP-RNH1 plasmid, a human RNH1-full coding sequence (ORIGEN RC 200082) insert was cloned into the entry vector pENTR4-GFP-C1 (W392-1, Addgene) and then recombined into the destination vector pLenti CMV Blast DEST (706-1, Addgene) plasmid, using the Gateway cloning method.

Techniques: Western Blot, Staining, Software, Infection, Fluorescence, Microscopy, Activation Assay