Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: EGR1 binds at the PKD1 proximal promoter. a The percentage of recovery relative to input chromatin was measured using qPCR with primers flanking the putative EGR1 binding site. The binding of EGR1 at the EGR1 consensus of the PKD1 promoter was confirmed by the recovery of immunoprecipitated fragments for all 3 conditions tested after PMA-induced differentiation, no treatment (GM + Nil), AngII (GM + AngII) and AngII/pioglitazone (GM + A + P). Undifferentiated THP-1 cells (GM only) express just detectable levels of EGR1, and therefore, this condition was used as a negative control of EGR1 recovery from the EGR1 binding locus and for comparison with the other conditions. b Non-specific background recovery was very low, as measured by ChIP using an IgG antibody. c Primers designed around a non-EGR1-specific locus within the proximal promoter of PKD1 also showed very low recovery, confirming the specificity of the EGR1 recovery. The data represent the means ± standard deviation from 3 independent experiments with p values as indicated.

Article Snippet: From the resulting sonicated whole cell extract, a portion (1/38.5) was kept as input, and the rest was incubated overnight with Dynal Protein G magnetic beads (Life Technologies) coated either with 10 μg of EGR1 antibody (44D5; Cell Signaling Technology, New England Biolabs, Hitchin, UK) or normal rabbit IgG control antibody (2,729; Cell Signaling Technology, New England Biolabs).

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Comparison, Standard Deviation

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Analyses of available ChIP-seq data reveal a conserved EGR1 binding peak within the PKD1 proximal promoter of multiple cell lines. a Duplicate EGR1 binding peak coordinates for PKD1 were extracted from NCBI GEO (accession number GSE32465; part of the ENCODE project). Overlapping peaks were merged and graphically aligned to genomic loci (hg19) using R code based on Gviz and ggplot2 packages. A common conserved EGR1 binding peak within the PKD1 proximal promoter was identified in the above cell lines. The widths in the bar graphs indicate the position and heights proportional to the average ‘score’ values provided by ENCODE data. b Sequence of the conserved EGR1 binding peak within the PKD1 proximal promoter. c Four out of the above 5 cell lines also harbour EGR1 binding peaks within the PKD2 promoter.

Article Snippet: From the resulting sonicated whole cell extract, a portion (1/38.5) was kept as input, and the rest was incubated overnight with Dynal Protein G magnetic beads (Life Technologies) coated either with 10 μg of EGR1 antibody (44D5; Cell Signaling Technology, New England Biolabs, Hitchin, UK) or normal rabbit IgG control antibody (2,729; Cell Signaling Technology, New England Biolabs).

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Expression of PKD1 and EGR1 in THP-1 cells. Relative RNA expression of PKD1 (a) and EGR1 (b). a, b The THP-1 cells were induced for 4 hours with PMA (t = 0), then the PMA was washed out and AngII (GM + AngII), AngII/pioglitazone (GM + A+P) or no treatment control (GM + Nil) were applied for the time points indicated on the x-axis. No treatment control THP-1 cells after 24 h were set as reference for comparison with other treatments/time points (relative expression = 1). Data represent the means ± standard error of the mean from 3 independent experiments with p values as indicated. Rel. = Relative; arb. = arbitrary. c Representative staining after 48 h of treatment. Polycystin-1 expression was assessed by immunocytochemistry under the same conditions. Polycystin-1 was localised at the cell periphery (arrowheads). The data are representative of 3 independent experiments, with i-iii representing 100% magnifications of the boxed areas in corresponding merged images; scale bars as indicated.

Article Snippet: From the resulting sonicated whole cell extract, a portion (1/38.5) was kept as input, and the rest was incubated overnight with Dynal Protein G magnetic beads (Life Technologies) coated either with 10 μg of EGR1 antibody (44D5; Cell Signaling Technology, New England Biolabs, Hitchin, UK) or normal rabbit IgG control antibody (2,729; Cell Signaling Technology, New England Biolabs).

Techniques: Expressing, RNA Expression, Control, Comparison, Staining, Immunocytochemistry

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Expression of PKD1 and EGR1 in EGR1 shRNA lentivirally transduced THP-1 cells. Relative expression of PKD1 (a) and EGR1 (b). The THP-1 cells were induced for 12 h with PMA (t = 0), then PMA was washed out and no treatment control (Nil), AngII (AngII) or AngII/pioglitazone (A + P) were applied for the time points indicated on the x-axis. No treatment control THP-1 cells after 24 h were set as reference for comparison with other treatments/time points (relative expression = 1). Data represent the average ± standard error of the mean from 3 independent experiments with p values as indicated. Rel. = Relative; arb. = arbitrary.

Article Snippet: From the resulting sonicated whole cell extract, a portion (1/38.5) was kept as input, and the rest was incubated overnight with Dynal Protein G magnetic beads (Life Technologies) coated either with 10 μg of EGR1 antibody (44D5; Cell Signaling Technology, New England Biolabs, Hitchin, UK) or normal rabbit IgG control antibody (2,729; Cell Signaling Technology, New England Biolabs).

Techniques: Expressing, shRNA, Control, Comparison

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Effect of Egr1 deficiency on changes in aortic diameter in CaCl 2 -induced aneurysm formation

Article Snippet: From the resulting sonicated whole cell extract, a portion (1/38.5) was kept as input, and the rest was incubated overnight with Dynal Protein G magnetic beads (Life Technologies) coated either with 10 μg of EGR1 antibody (44D5; Cell Signaling Technology, New England Biolabs, Hitchin, UK) or normal rabbit IgG control antibody (2,729; Cell Signaling Technology, New England Biolabs).

Techniques:

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Nsp1 N-terminal and central domain mutants are defective for ribosome and mRNA binding (A) HEK293T cells were transfected with plasmids expressing WT or the indicated mutant 3xFLAG-Halo-tagged nsp1. Nsp1 was immunoprecipitated (IP) using ⍺-FLAG beads and coIP of ribosomal proteins RACK1, RPS2, RPS3, and RPS24 was monitored by western blotting, with vinculin serving as a loading control. Input lanes contain 1/10 of the amount of protein used for the IPs. (B) Equilibrium binding measurements of fluorescently labeled WT (blue), R124A,K125A (red), and R99A (green) nsp1 to purified ribosomes. Data represent a total of 3 biological replicates. (C) HEK293T cells were co-transfected with HBB-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 constructs. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were extracted and nLuc mRNA was quantified by qRT-PCR. The mRNA values were then normalized to the values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) HEK293T cells were co-transfected with a 3xFLAG-Halo-tagged nsp1 plasmid or empty vector control, together with a plasmid expressing either GFP with a 5′ stem loop (GFP+SL) or a control GFP lacking the stem loop (GFP). Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating GFP+SL or GFP mRNAs were quantified by qRT-PCR. The mRNA values were then normalized to those obtained from the empty vector control. The bars represent the mean value of the replicates and error bars represent standard deviation. (E) The levels of GFP+SL and GFP mRNA present in the input samples from (D) were quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 (empty vector control) set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; unpaired t test. See also , , and . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Labeling, Purification, Plasmid Preparation, Construct, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Transfection, Luciferase, SYBR Green Assay, Primer Extension Assay, Clone Assay, Software

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Proteins identified to associate with CHIKV nsP3 HVD. ( A ) The organization of CHIKV genome, the domains of nsP3 and schematics of constructs for expressing EGFP-CHIKVnsP3HVD and non-tagged EGFP in Flp-In T-REx cells; ( B ) Immunoblot analysis using anti-EGFP antibodies and immunoprecipitated (IP) samples obtained from the parental Flp-In T-Rex cells and transgenic T-REx-EGFP and T-REx-EGFP-CHIKVnsP3HVD cell lines; ( C ) Interaction network of proteins associated with the HVD of CHIKV nsP3 was created using STRING database; for this analysis proteins that showed at least 2-fold enrichment in SILAC-based quantitative assay were chosen.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Construct, Expressing, Western Blot, Immunoprecipitation, Transgenic Assay, Multiplex sample analysis

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Mapping of CD2AP-binding site in HVD of CHIKV nsP3. ( A ) Sequences of two consecutive potential SH3 domain ligands (M1, M2) in HVD of CHIKV nsP3 (wt) and positions of the mutations that were introduced into EGFP CHIKVnsP3HVD expression constructs: Δ9, Δ16, P423A, R428A, Δ16 + P423A, Δ16 + R428A; “_” designates deleted residue; ( B ) Flp-In T-REx cells were transfected with plasmids expressing CHIKVnsP3HVD harbouring mutations indicated above the panel; 24 h later immunoprecipitation (IP) via EGFP-capturing beads was carried out, obtained samples were analysed by immunoblot for the presence of CD2AP and EGFP CHIKVnsP3HVD.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Binding Assay, Expressing, Construct, Residue, Transfection, Immunoprecipitation, Western Blot

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Binding of CD2AP by nsP3 HVDs of different alphaviruses. ( A ) Western blot analysis of immunoprecipitated samples obtained from Flp-In T-Rex, T-Rex-EGFP, T-Rex-EGFP-CHIKVnsP3HVD and T-Rex-EGFP-VEEVnsP3HVD cells with anti-EGFP antibody; ( B ) Network analysis of cellular proteins captured by VEEV nsP3 HVD performed as described for C; ( C ) M1 and M2 like elements identified via multiple sequence alignment in nsP3 HVD of different Old and New World alphaviruses. Underline indicates CHIKV CD2AP-binding M2 motif identified in this study or a motif similar to it; ( D ) Flp-In T-REx cells were transfected with plasmids expressing EGFP fused to HVD from the indicated Old World alphaviruses; 24 h p.t. cells were lysed and EGFP-specific magnetic beads were used to pull down proteins; obtained samples were probed for presence of CD2AP using antibody against CD2AP. Blot developed using anti-EGFP antibody is shown as recombinant protein expression/capture control.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Sequencing, Transfection, Expressing, Magnetic Beads, Recombinant, Control

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Motif M2 of SFV nsP3 HVD associates with multiple proteins.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques:

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Effects of mutations in SH3 domain ligands on CHIKV RNA infectivity and CHIKV replication. ( A ) BHK-21 cells were electroporated with 1 μg of in vitro transcribed RNAs of CHIKV (wt or bearing mutation in nsP3 HVD); the infectivity of the transcripts was analysed by ICA. Mutant virus RNA infectivity is presented as a percentage of wild-type virus RNA, mean values together with standard error of three independent experiments are shown; ( B ) Growth curve of wt CHIKV or its mutant variants in BHK-21 cells infected at MOI of 0.1. Mean values of four replicates with standard deviation are shown. * denotes p < 0.05 (according to Student’s t -test), in comparison with wt virus.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Infection, In Vitro, Mutagenesis, Virus, Standard Deviation, Comparison

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: Interaction of CHIKV or SFV nsP3 with CD2AP in infected cells. HOS cells were infected with wt or mutant CHIKV or SFV at MOI of 10 and lysed at 14 (CHIKV) or 8 (SFV) hpi to carry out immunoprecipitation (IP) using antisera against nsP3 of CHIKV or SFV, respectively. Cell lysates and IP samples were probed for nsP3, CD2AP and actin.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Infection, Mutagenesis, Immunoprecipitation

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: CD2AP co-localisation with CHIKV and SFV nsP3 in infected cells. HOS cells were infected with CHIKV wt or CHIKVmutM2 ( A ) or SFV wt or SFVmutM2 ( B ) at an MOI of 1. At 16 ( A ) or 8 h p.i. ( B ), cells were fixed and stained for nsP3 (green) and CD2AP (red), line scan images are given below the confocal images. Results are representative of three independent experiments.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Infection, Staining

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: dsRNA in wt CHIKV and SFV infected cells co-localisation with CD2AP. HOS cells infected with CHIKV wt or CHIKVmutM2 ( A ) or SFV wt or SFVmutM2 ( B ) at an MOI of 1 were fixed at 16 ( A ) or 8 h pi ( B ). Thereafter the cells stained for dsRNA (green) and CD2AP (red), line scan image is given below. Results are representative of three independent experiments.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Infection, Staining

Journal: Viruses

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

doi: 10.3390/v10050226

Figure Lengend Snippet: SH3KBP1 co-localises with wt CHIKV and SFV nsP3 protein. HOS cells were infected with CHIKV wt or CHIKVmutM2 ( A ) or SFV wt or SFVmutM2 ( B ) at an MOI of 1. At 16 ( A ) or 8 h pi ( B ), cells were fixed and stained for nsP3 (green) and SH3KBP1 (red) and line scan images are given. Results are representative of two independent experiments.

Article Snippet: Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary).

Techniques: Infection, Staining

Journal: Biosensors & Bioelectronics

Article Title: Paper-based immunoassay based on 96-well wax-printed paper plate combined with magnetic beads and colorimetric smartphone-assisted measure for reliable detection of SARS-CoV-2 in saliva

doi: 10.1016/j.bios.2021.113909

Figure Lengend Snippet: A) Study of the suitability of the reagent-free approach: sigmoidal curve obtained in drop (•) by adding 10 μL of TMB + 10 μL MAb-HRP, sigmoidal curve obtained after pre-loaded 10 μL of TMB and by adding 10 μL of MAb-HRP ( Δ ); B) Selection of MBs volume. 5, 10, and 20 μL of MBs, 200 μL of MAb-HRP anti-SARS-CoV-2, 2 μg/mL (in PBS + 0.05% Tween 20) + 300 μL of sample tested (negative control or 1 μg/mL S protein; C) Study of MAb-HRP concentration. 10 μL of MBs, 200 μL of MAb-HRP anti SARS-CoV-2, 1, 2, and 4 μg/mL (in PBS + 0.05% Tween 20) + 300 μL of sample tested (negative control or 1 μg/mL S protein; D) Calibration images obtained using the chosen parameters for S protein detection in buffer and in untreated saliva. The mean value (n = 3) with the corresponding standard deviation and the image of paper plate were reported for each measurement.

Article Snippet: Dynabeads® Pan Mouse IgG pre-coated with anti-Mouse IgG able to bind monoclonal antibody (supplied as a suspension containing 4 × 10 8 beads/mL in phosphate buffered saline, pH 7.4) was from Life Technologies (USA).

Techniques: Selection, Negative Control, Concentration Assay, 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: 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: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Composition and DNA binding affinity of the Flag–TIN2 complexes. ( A ) Experimental plan for characterization of Flag–TIN2 containing Shelterin complexes. Extracts made from HeLa cells transfected with Flag–TIN2 are incubated with the [ 32 P]-labeled probe, after which Flag–TIN2 containing complexes are immunoprecipitated with magnetic beads coated with the anti-Flag M2 antibody. Amount of probe recovered can then be used as a measure of the affinity of each probe for Shelterin complexes (Left arm). Alternatively, the isolated complexes can be released by incubation with an excess of 3XFLAG peptide, after which the eluted complexes are analyzed by native electrophoresis on composite gel containing TAM buffer (Right arm). Exposing the complexes to supershifting antibodies prior to the electrophoresis allows determination of the composition of the complexes. ( B ) Graphical representations of the probes D4 and D4-nTel. Shaded areas represent telomeric DNA. The probes carry four binding sites (ds-TTAGGGTTA motifs) for the Myb domain of TRF2 followed by a telomeric (D4) or non-telomeric (D4-nTel) 3′-overhang. [ 32 P]-phosphate was at the 5′-end of the bottom strand. ( C and D ) Subunit composition of the isolated Flag–TIN2 complexes. Extracts of HeLa cells transfected with (+) or without (–) Flag–TIN2 or Flag-TRF2 were incubated with [ 32 P]-probe D4. Protein/DNA complexes containing the Flag-tagged proteins were captured with beads coated with the M2 antibody and released by incubation with an excess of 3XFLAG peptide. After exposure to the indicated antibodies, the eluted complexes were resolved by native electrophoresis in a composite gel containing TAM buffer. Short arrow indicates positions of the supershifted Shelterin/DNA complex. ( E ) Saturation binding curve of the interaction of Flag–TIN2 complexes with probe D4. Extracts of HeLa cells transfected with Flag–TIN2 were incubated with increasing concentrations of probes D4 or D4-nTel, after which point Flag–TIN2 complexes were recovered with beads coated with either the anti-Flag antibody (M2 Ab) or normal mouse IgG (normal IgG). Scattered plot shows the amount of probe recovered (in fmoles) as a function of the initial probe concentration (pM). Dotted line shows fitting of the data to either a linear curve (Black, D4-nTel) or a one site saturation binding curve (Gray, D4). Nonlinear regression of the D4 data allowed determination of the dissociation constant ( K d = 1.5 ± 0.3 nM) and maximum number of binding complexes ( B max = 2.3 ± 0.2 fmol/reaction).

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Binding Assay, Transfection, Incubation, Labeling, Immunoprecipitation, Magnetic Beads, Isolation, Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Characterization of the DNA binding specificity of complex T2. ( A and C ) Graphical representations of the structures of all probes tested. Probes were designed to harbor different end structures and to contain different number of ds-TTAGGGTTA motifs. Shaded areas represent telomeric DNA. ( B ) DNA binding by complex T2 requires a functional POT1 binding site. Probes described in A were incubated with no extracts (lane 4) or with nuclear extracts of HT1080-Vector (lane 5) or HT1080-TRF2 cells (lanes 1–3 and 6–13), after which point the protein/DNA complexes were resolved by native electrophoresis in composite gels containing TAM buffer. Five minutes prior to loading, antibodies were added to samples in lanes 7 (anti-Flag M2 antibody) and 8 (Normal mouse IgG). Lanes 1–3 were overexposed to allow detection of the signal of probe A2. Short arrow indicates positions of the supershifted complex T2. ( D ) DNA binding by complex T2 requires at least one Myb binding site. Probes described in C were incubated with no extracts (lane 1) or with nuclear extracts of HT1080-Vector (lane 2) or HT1080-TRF2 cells (lanes 3–8), after which point the protein/DNA complexes were resolved by native electrophoresis in composite gels containing TAM buffer. Five min prior to loading, antibodies were added to samples in lanes 4 (anti-Flag M2 antibody) and 5 (Normal mouse IgG). Short arrow indicates positions of the supershifted complex T2. ( E ) Densitometric quantification of the T2/DNA complexes detected in (D).

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Binding Assay, Functional Assay, Incubation, Plasmid Preparation, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Characterization of the DNA binding specificity of Flag–TIN2 complexes. ( A ) Graphical representations of the structures of all tested probes. Probes were designed to harbor different end structures and to contain different number of ds-TTAGGGTTA motifs. Shaded areas represent telomeric DNA. ( B ) DNA binding specificity of Flag–TIN2 Complexes. Extracts of HeLa cells transfected with no DNA (Mock) or with Flag–TIN2 (+ Flag–TIN2) were incubated with the [ 32 P]-labeled probes described in A, after which Flag-tagged protein/DNA complexes were captured with the M2 antibody and the amount of radioactivity recovered was counted. Results in triplicates show the percent of each probe recovered by the M2 antibody (mean ± standard deviation; n = 3). The percent of probe C2 recovered from the Mock-transfected cell extract defines the value of the background (<0.1%). ( C ) DNA binding specificity of Flag–TIN2/TRF2 ΔB complexes. Extracts of HeLa cells transfected with TRF2 ΔB alone (TRF2 ΔB ) or with TRF2 ΔB plus Flag–TIN2 (TRF2 ΔB + Flag–TIN2) were incubated with the [ 32 P]-labeled probes described in A, after which Flag-tagged protein/DNA complexes were captured with the M2 antibody and the amount of radioactivity recovered was counted. Results in triplicates show the percent of each probe recovered by the antibody (mean ± standard deviation; n = 3). The percent of probe C2 recovered from the TRF2 ΔB alone-transfected cell extract defines the value of the background (<0.2%).

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Binding Assay, Transfection, Incubation, Labeling, Radioactivity, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Transfected Flag-TRF2 ΔB forms a complex that binds selectively to telomeric DNA fragments that carry a 3′-overhang. ( A ) Graphical representation of probes 4M-3′, 4M-bl and 4M-5′. Shaded areas represent telomeric DNA. The probes carried four binding sites for the Myb domain of TRF2 (ds-TTAGGGTTA motifs numbered 1–4) followed by different end structures: 3′-telomeric overhang (4M-3′), blunt (4M-bl) or 5′-telomeric overhang (4M-5′). [ 32 P]-phosphate was at the 5′-end of the top strand. ( B ) TRF2 constructs used in transient transfection. Full-length TRF2 contains a basic domain that binds ss-DNA independently of sequence (Basic), a TRF homology region (TRFH/Dimerization) that serves as dimerization interface, a domain of interaction with TIN2 (aa 350–367), and a Myb domain that binds to ds-TTAGGGTTA (Myb). Interaction with TIN2 allows the recruitment of TRF2 in Shelterin complexes. In these complexes, TPP1 and TIN2 act scaffolds linking TRF2 to the ss-DNA binding protein POT1, which binds to 5′-TTAGGGTTAG-3′. TRF2 ΔB lacks amino acids 2-44 containing the sequence-independent ss-DNA binding domain of TRF2. TRF2 ΔB (R361P) contains an additional mutation that blocks the association of TRF2 ΔB with TIN2. All constructs were tagged at the N-terminus with the Flag epitope. ( C ) Western blot analysis of HeLa cells transiently transfected with the different TRF2 ΔB constructs. Whole cell extracts prepared for EMSA (50 µg) were probed with the anti-Flag M2 antibody. ( D ) Transfected TRF2 ΔB is in a complex that binds selectively to telomeric DNA fragments carrying a 3′-overhang. Top panel: WCEs from mock-transfected (lane 1) and TRF2 ΔB -transfected HeLa cells (lanes 2–4) were incubated with the indicated [ 32 P]-probes (4M-3′, 4M-blunt, 4M-5′) and the protein/DNA complexes that formed were resolved by electrophoresis in TAM buffer. T2, TRF2-containing complex T2. NS, non-specific band. Bottom panel: after incubation of the extracts with the indicated probes, protein/DNA complexes containing Flag-TRF2 ΔB were captured using magnetic beads coated with an anti-Flag antibody, were eluted with an excess of 3XFLAG peptide, and were subsequently resolved by electrophoresis in TAM buffer. ( E ) Effects of R361P mutation on formation of complex T2. HeLa cells were mock-transfected or transfected with Flag-TRF2 ΔB or Flag-TRF2 ΔB (R361P). WCEs were prepared and incubated with probe 4M-3’, after which point protein/DNA complexes were resolved by electrophoresis in a native polyacrylamide gel containing TAM buffer. Complex T2 failed to be detected in extracts of HeLa cells transfected with the R361P mutant.

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Transfection, Binding Assay, Construct, Sequencing, Mutagenesis, FLAG-tag, Western Blot, Incubation, Electrophoresis, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Complex T2 formed by Flag-tagged TRF2 is a member of the Shelterin family. ( A ) Western blot analysis of HT1080-TRF2 and HT1080-Vector cells. WCEs prepared for EMSA (50 µg each) were analyzed by western blotting to detected total TRF2 (anti-TRF2 antibody) and the stably transfected Flag-TRF2 (anti-Flag M2 antibody). Membranes were re-probed with an antibody against β-actin. ( B ) Complex T2 formed by Flag-TRF2 contains the Shelterin scaffolding component TIN2. Extracts of HT1080-TRF2 cells were incubated with the indicated probes, after which point protein/DNA complexes were resolved by native electrophoresis in a composite gel containing TAM buffer. Five minutes prior to loading, the indicated antibodies were added to samples in lanes 4–8 (1 µg each of anti-Flag M2 antibody, normal mouse IgG, or antibodies against TIN2, c-Fos, or Vimentin). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( C ) Loss of Shelterin components prevents formation of complex T2. HT1080-TRF2 cells were transiently transfected with siRNA smartpools against TPP1, TIN2, POT1 or TRF1 or with non-targeting (NT) siRNA. Nuclear extracts prepared from HT1080-Vector cells (Vector) or from HT1080-TRF2 cells (TRF2) transfected with the different siRNA were incubated with probe 4M-3′, after which protein/DNA complexes were separated by native electrophoresis in composite gel containing TAM buffer. As expected, complex T2 was more abundant in cells expressing Flag-TRF2 (lane 1 versus 2), was selective for telomeric DNA fragments that carried a 3′-overhang (lane 7 versus 8), and was supershifted by the anti-Flag antibody (lane 9 versus 10). Complex T2 was reduced in HT1080-TRF2 cells transfected with siRNA smartpools directed against TIN2, TPP1 or POT1 (lanes 3–5 versus lane 2). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( D ) Relative abundance of the TPP1, TIN2, POT1 and TRF1 mRNA in the siRNA-transfected cells. For each transfection, abundance of the targeted mRNA was measured by real-time PCR, comparing cells transfected with the targeting and non-targeting (NT) siRNA. For each mRNA, abundance in cells treated with the non-targeting siRNA was set to 1. To show that targeting was specific, TRF2 and GAPDH mRNAs were also measured, both of which found to be unaffected by the different siRNA treatments.

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Scaffolding, Incubation, Electrophoresis, Expressing, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Modeling the interactions of Shelterin complexes with ds- and ss-telomeric DNA. ( A ) Simultaneous interactions of Shelterin complexes with both ds- and ss-telomeric DNA. Binding of these multi-subunit complexes to DNA requires a binding site for POT1 (5′-TTAGGGTTAG-3′) and at least one binding site for the Myb domains of either TRF1 or TRF2 (ds-TTAGGGTTA motif). Once bound to telomeric DNA, other Myb domains present in the complexes could further stabilize the protein/DNA complex by establishing additional contacts with ds-telomeric DNA. B) Implications of this model on the role(s) played at telomeres by the Shelterin complexes. The DNA binding specificity of Shelterin complexes would be expected to recruit these complexes to regions of telomeres where ss- and ds-telomeric DNA are present in close proximity, including the 3′-telomeric overhang (i), telomeric DNA bubbles (ii), and the D-loop at the base of T-loops (iii). At these locations, Shelterin complexes would be ideally positioned to control the accessibility of the 3′-telomeric overhang (i) to telomerase and T-loop forming machinery. Unoccupied Myb domains in complexes bound to the 3′-telomeric overhang could potentially be available to mediate long-range interactions with upstream ds-telomeric DNA (dotted arrow). These long-range interactions could help initiate the formation of T-loops. Finally, through their simultaneous interactions with both ds- and ss-telomeric DNA, Shelterin complexes could help stabilize pre-existing D-loops and T-loops (iii) as well as telomeric DNA bubbles (ii). Stabilization of these bubbles could promote T-loop formation if the stabilized bubbles are persisting long enough to have a chance to collide with the 3′-telomeric overhang.

Article Snippet: Rabbit polyclonal antibodies were against TRF2 (H-300, Santa Cruz Biotech.), TRF1 (ab1423, Abcam Inc.), RAP1 (A300-306A, Bethyl Lab. Inc.) and Actin (I-19, Santa Cruz Biotech.).

Techniques: Binding Assay, Control

Journal: Cell Regeneration

Article Title: Coordination of EZH2 and SOX2 specifies human neural fate decision

doi: 10.1186/s13619-021-00092-6

Figure Lengend Snippet: EZH2-deficient hESCs fail to differentiate into NPCs and subtype neuron/glia cells. a Schematic of the default neural differentiation and random differentiation strategy for human embryonic stem cells (hESCs) (more detail information in “Methods” sections). b Morphology of the wild-type (WT) H1 and EZH2 −/− hESCs during neural differentiation at day 0, day 9, day 16 and rosette-like cells. EZH2-deficient hESCs fail to form rosette-like cells. Scale bar, 50 μm. c Immunostaining for the NPC markers PAX6, SOX2, and NESTIN in WT rossettes and EZH2-mutant cells under neural differentiation. Scale bar, 50 μm. d FACS analysis of PAX6 + cells at day8 and day16 during neural differentiation in the indicated cells. The data represent the mean ± SD from three independent replicates ( n = 3). Significance was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. e Morphology of the undifferentiated WT NPCs and EZH2-mutant NPCs and their differentiated cells at day 7, day 14 and day 28 during random differentiation. Scale bar, 100 μm. f Immunostaining for the marker of neuron MAP2 and the marker of astrocyte GFAP in the indicted differentiated cells at day 28 from NPCs. Scale bar, 50 μm. Quantity data from MAP2 + or GFAP + cells were analyzed. Significance level was determined by unpaired two-tailed Student’s t-tests. ***, P < 0.001. The data represent the mean ± SD (standard deviation) from three independent replicates ( n = 3). All error bars throughout the figure represent the SD (standard deviation) from three independent replicates ( n = 3)

Article Snippet: The sonicated fragments from these samples were co-incubated with magnetic beads (Dynabeads protein A and G (1:1)) (Invitrogen) and 5 μg corresponding specific antibody (anti-H3K27me3 antibody (Diagenode, C15410069), anti-EZH2 antibody (CST, 5246) or anti-SOX2 antibody (R&D, AF2018), respectively) with rotation overnight at 4 °C.

Techniques: Immunostaining, Mutagenesis, Two Tailed Test, Marker, Standard Deviation

Journal: Cell Regeneration

Article Title: Coordination of EZH2 and SOX2 specifies human neural fate decision

doi: 10.1186/s13619-021-00092-6

Figure Lengend Snippet: EZH2 −/− hESCs aberrantly express meso/endoderm genes during neural induction. a qRT-PCR analysis of the expression of the pluripotent genes OCT4 / NANOG , the NPC genes SOX2 / SOX1 / PAX6 / FOXG1 , and meso-endoderm genes CDH1 / ZIC1 / PAX3 / ERBB3 / TFAP2A / FOXD3 / BMP4 / KDR / CXCL12 / FOXH1 / KRT8 / KRT18 / KRT19 / VEGFA / CLDN6 at day 0, day 8 and day 16 during neural differentiation. Wild-type H1 hESCs served as controls. The data represent the mean ± SD (standard deviation) from three independent replicates ( n = 3). Significance was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. *, P < 0.05. b Left panel, heatmap of up- or downregulated genes in EZH2-mutant and wild-type cells at day 8 of neural differentiation (EZH2 −/− -D8 and WT-D8, respectively). Right panel, GO analysis for up- or downregulated genes in EZH2-mutant cells at day 8 of neural differentiation. c . Heatmap analysis for genes mentioned in Fig. 2a

Article Snippet: The sonicated fragments from these samples were co-incubated with magnetic beads (Dynabeads protein A and G (1:1)) (Invitrogen) and 5 μg corresponding specific antibody (anti-H3K27me3 antibody (Diagenode, C15410069), anti-EZH2 antibody (CST, 5246) or anti-SOX2 antibody (R&D, AF2018), respectively) with rotation overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test, Mutagenesis

Journal: Cell Regeneration

Article Title: Coordination of EZH2 and SOX2 specifies human neural fate decision

doi: 10.1186/s13619-021-00092-6

Figure Lengend Snippet: EZH2 suppresses meso-endoderm genes while releases neural genes in hESC neural differentiation. a Left panel, heatmap of up- or downregulated genes in wild-type cells at day 8 (WT Day 8) compared with WT cells at day 0 (WT Day 0) neural differentiation. Right panel, GO analysis for up- or downregulated genes in wild-type cells at day 8 (WT Day 8). b Signal densities of H3K27me3-associated genes and EZH2-associated genes in the indicated wild-type (WT) or EZH2-mutant cells at day 0 and day 8 of neural differentiation from ChIP-seq data. c Signal densities for H3K27me3 or EZH2 enrichment from wild-type cells at day 0 and day 8 ChIP-seq data for upregulated genes in wild-type cells at day 8 (WT Day 8). d Signal densities of H3K27me3 and EZH2 enrichment from WT-Day 8 ChIP-seq data for upregulated genes in EZH2-mutant cells at day 8 of neural differentiation. e Genomic views of the H3K27me3 modification and EZH2 enrichment for NPC genes and meso-endoderm genes in wild-type cells at day 0 and day 8 from ChIP-seq data

Article Snippet: The sonicated fragments from these samples were co-incubated with magnetic beads (Dynabeads protein A and G (1:1)) (Invitrogen) and 5 μg corresponding specific antibody (anti-H3K27me3 antibody (Diagenode, C15410069), anti-EZH2 antibody (CST, 5246) or anti-SOX2 antibody (R&D, AF2018), respectively) with rotation overnight at 4 °C.

Techniques: Mutagenesis, ChIP-sequencing, Modification

Journal: Cell Regeneration

Article Title: Coordination of EZH2 and SOX2 specifies human neural fate decision

doi: 10.1186/s13619-021-00092-6

Figure Lengend Snippet: Coordination of EZH2 and SOX2 in neural lineage fate decision. a Signal densities heatmap of SOX2-ChIP-seq data indicating regions between WT ESCs and day 8 cells in neural differentiation (WT-Day 8). b Venn diagram for EZH2-binding genes and SOX2-binding genes in WT ESCs. c Signal densities of SOX2 and EZH2 enrichment for EZH2/SOX2 co-binding genes in WT ESCs. d GO analysis for EZH2/SOX2 both binding 678 genes in WT ESCs. e Left panel, Signal densities of SOX2 and EZH2 enrichment from WT-Day 8 ChIP-seq data for EZH2 lost genes, SOX2 lost genes or EZH2/SOX2 keeping genes in wild type cells at day 8 compared with day 0 in neural differentiation (WT-Day 8 and WT-Day 0, respectively). Right panel, GO analysis for EZH2 lost genes, SOX2 lost genes or EZH2/SOX2 keeping genes in WT-Day 8 cells. f RNA expression level in wild-type ESCs and day 8 cells in neural differentiation (WT-Day 8) for EZH2 lost genes, SOX2 lost genes or EZH2/SOX2 keeping genes. g RNA expression level in wild-type and EZH2 −/− cells at day 8 in neural differentiation for EZH2-bound and SOX2-bound genes, respectively. h The model for coordination of EZH2 and SOX2 in neural lineage fate decision from hESCs

Article Snippet: The sonicated fragments from these samples were co-incubated with magnetic beads (Dynabeads protein A and G (1:1)) (Invitrogen) and 5 μg corresponding specific antibody (anti-H3K27me3 antibody (Diagenode, C15410069), anti-EZH2 antibody (CST, 5246) or anti-SOX2 antibody (R&D, AF2018), respectively) with rotation overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, RNA Expression

Journal: Cell Regeneration

Article Title: Coordination of EZH2 and SOX2 specifies human neural fate decision

doi: 10.1186/s13619-021-00092-6

Figure Lengend Snippet: EZH2 promotes the proliferation of human neural progenitor cells (NPCs) in vitro . a Schematic of the default neural differentiation and random differentiation strategy for human embryonic stem cells (hESCs) with over-expression of EZH2. b Western blot for EZH2-FLAG proteins in the EZH2-overexpressed hES cells. c Morphology of the wild-type (WT) H1 and EZH2 over-expressed hESCs under NPC differentiation conditions at day 0 and day 16. Scale bar, 200 μm. d Immunostaining for the pluripotent marker OCT4 and the NPC markers SOX2/NESTIN in wild-type (WT) and EZH2 overexpressed rosette-like NPCs. Scale bar, 50 μm. e qRT-PCR analysis for the expression of EZH2 , the NPC genes SOX2 / SOX1 / PAX6 , and meso-endoderm genes ZIC1 / KRT18 / VEGFA / DLX5 at day0, day8, day 16 of neural differentiation. Significance was determined using unpaired two-tailed Student’s t-tests. The data represent the mean ± SD (standard deviation) from three independent replicates ( n = 3). **, P < 0.01. f Left panel, morphology of indicated NPCs maintained as neural spheres at passage 2 (P2) or passage 4 (P4). Scale bar, 200 μm. Right panel, proliferation curve of the indicated NPCs at different passages. Significance was determined using unpaired two-tailed Student’s t-tests. *, P < 0.05. **, P < 0.01. The data represent the mean ± SD (standard deviation) from three independent replicates ( n = 3). g Apoptosis assay in the indicated cells at passage 2 (P2) or passage 4 (P4). PI- and/or annexin V-positive cells were analyzed by FACS. The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments ( n = 3). h qRT-PCR analysis for the expression of EZH2 , the NPC genes SOX2 / SOX1 / PAX6 , and meso-endoderm genes ZIC1 / KDR / DLX5 / TFAP2A / PAX3 / VEGFA in the indicated NPCs at passage 4 (P4). The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments ( n = 3). i Morphology of NPCs and their subtype neuron/glia cells with over-expression of EZH2. Scale bar, 50 μm. j Immuno-staining analysis for SOX2/NES, MAP2/GFAP in NPCs with forced EZH2 expression during random differentiation. The numbers of MAP2 + or GFAP + cells were analyzed. The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments ( n = 3)

Article Snippet: The sonicated fragments from these samples were co-incubated with magnetic beads (Dynabeads protein A and G (1:1)) (Invitrogen) and 5 μg corresponding specific antibody (anti-H3K27me3 antibody (Diagenode, C15410069), anti-EZH2 antibody (CST, 5246) or anti-SOX2 antibody (R&D, AF2018), respectively) with rotation overnight at 4 °C.

Techniques: In Vitro, Over Expression, Western Blot, Immunostaining, Marker, Quantitative RT-PCR, Expressing, Two Tailed Test, Standard Deviation, Apoptosis Assay

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Nsp1 N-terminal and central domain mutants are defective for ribosome and mRNA binding (A) HEK293T cells were transfected with plasmids expressing WT or the indicated mutant 3xFLAG-Halo-tagged nsp1. Nsp1 was immunoprecipitated (IP) using ⍺-FLAG beads and coIP of ribosomal proteins RACK1, RPS2, RPS3, and RPS24 was monitored by western blotting, with vinculin serving as a loading control. Input lanes contain 1/10 of the amount of protein used for the IPs. (B) Equilibrium binding measurements of fluorescently labeled WT (blue), R124A,K125A (red), and R99A (green) nsp1 to purified ribosomes. Data represent a total of 3 biological replicates. (C) HEK293T cells were co-transfected with HBB-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 constructs. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were extracted and nLuc mRNA was quantified by qRT-PCR. The mRNA values were then normalized to the values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) HEK293T cells were co-transfected with a 3xFLAG-Halo-tagged nsp1 plasmid or empty vector control, together with a plasmid expressing either GFP with a 5′ stem loop (GFP+SL) or a control GFP lacking the stem loop (GFP). Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating GFP+SL or GFP mRNAs were quantified by qRT-PCR. The mRNA values were then normalized to those obtained from the empty vector control. The bars represent the mean value of the replicates and error bars represent standard deviation. (E) The levels of GFP+SL and GFP mRNA present in the input samples from (D) were quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 (empty vector control) set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; unpaired t test. See also , , and . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Labeling, Purification, Plasmid Preparation, Construct, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Transfection, Luciferase, SYBR Green Assay, Primer Extension Assay, Clone Assay, Software

Journal: Stem Cells International

Article Title: Extracellular Vesicle-Loaded Oncogenic lncRNA NEAT1 from Adipose-Derived Mesenchymal Stem Cells Confers Gemcitabine Resistance in Pancreatic Cancer via miR-491-5p/Snail/SOCS3 Axis

doi: 10.1155/2023/6510571

Figure Lengend Snippet: Verification of the binding between NEAT1 and miR-491-5p in PCa cells. (a) The binding site between NEAT1 and miR-491-5p in PCa cells predicted by the StarBase database. (b) The colocalization of NEAT1 and miR-491-5p detected by FISH (scale bar: 25 μ m). (c) The relationship between miR-491-5p expression and the prognosis of PCa patients (44 patients with high miR-491-5p expression and 132 patients with low miR-491-5p expression) analyzed by Ualcan database. (d) miR-491-5p expression in dasatinib-sensitive PCa cell lines and dasatinib-resistant PCa cell lines analyzed in GSE59357 ( n = 3). (e) Luciferase activity of NEAT1-WT/MUT analyzed by dual-luciferase reporter gene assay. (f) Enrichment of miR-491-5p by NEAT1 in SW1990 cells treated with anti-Ago2 or anti-IgG detected by RIP. (g) RNA pull-down assay for a biotinylated NEAT1 and an NC probe, miR-491-5p enrichment detected by RT-qPCR. (h) NEAT1 expression in SW1990 cells treated with overexpression vector or shRNA-mediated NEAT1 measured by RT-qPCR. (i) miR-491-5p expression in SW1990 cells treated with overexpression vector or shRNA-mediated NEAT1 measured by RT-qPCR. (j) miR-491-5p expression in SW1990 cells cocultured with EVs from sh-NEAT1-treated ADSCs measured by RT-qPCR. ∗ p < 0.05 vs. SW1990 cells transduced with mimic NC or inhibitor NC, SW1990 cells treated with Bio-NC-probe, Vector, or PBS; # p < 0.05 vs. SW1990 cells transduced with sh-NC or SW1990 cells cocultured with EVs from ADSCs transduced with sh-NC. All data were presented as mean ± standard deviation. Comparisons of data between two groups were analyzed by unpaired t -test, and comparison of data among multiple groups was tested by one-way analysis of variance, followed by Tukey's post hoc test. Cell experiments were independently repeated three times.

Article Snippet: The Argonaute 2 antibody (anti-Ago2, 67934-1-lg, 1 : 1000) and the normal IgG antibody (anti-IgG, 30000-0-AP, 1 : 1000, Proteintech Group Inc., IL) were incubated with magnetic beads (370-12D, Invitrogen) at 4°C for 1 h. Then, the cell lysates were incubated with the magnetic beads at 4°C overnight.

Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Reporter Gene Assay, Pull Down Assay, Quantitative RT-PCR, Over Expression, Plasmid Preparation, shRNA, Transduction, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Genome-wide p53 binding after various treatments. ( A ) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. ( B ) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. ( C ) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. ( D ) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Genome Wide, Binding Assay, Western Blot, ChIP-sequencing, Control

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Motif analysis and distribution of p53-binding sites. ( A ) Consensus p53-binding motifs identified from the top 1000 p53-binding sites for each treatment. ( B ) Characterization of p53-binding sites based on the type of sequence bound: zero spacer, consensus motif containing two decamers with no spacer sequence as shown in (A); 1–15 spacer, two decamers with a spacer sequence of length 1–15 nt; half site, one decamer; no motif, no evidence of zero spacer, 1–15 spacer, or a half site. ( C ) Frequency of p53-binding sites containing zero spacer (red), 1–15 spacer (blue), and half sites (green). ( D ) Box-plot depicting distribution of ChIP-over-Input fold enrichment for p53-binding sites containing zero spacer, 1–15 spacer, half site and no motif. Asterisk indicates statistically significant ( P < 10 −7 ).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Gene expression analysis following various treatments. ( A ) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. ( B ) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5 . ( C ) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. ( D ) Breakdown of expression changes of genes with a p53-binding site near the TSS or ( E ) in the intragenic region. ( F ) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Gene Expression, Expressing, Control, Microarray, Binding Assay

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Correlation between p53 binding, changes in gene expression and histone modifications H3K4me3 and H3K27me3 at 24 h after DXR, Nutlin or DMSO treatments. The heat map represents fold changes in mRNA levels quantified using qRT-PCR, as well as fold changes in p53, H3K4me3 and H3K27me3 occupancy quantified using ChIP-qPCR (see also Supplementary Figures S1D and S2A–C ). Presented are results for 36 genes that were bound by p53 and two genes ( GAPDH and MYOD1 ) that were not bound by p53. Genes are clustered into groups depending on whether the p53 target site contains a consensus p53-binding motif containing two decamers with no spacer (zero spacer), two decamers with a spacer sequence (spacers), or one decamer (half site) or no motif. Fold changes for gene expression were normalized to β-2-microglobulin, whereas p53, H3K4me3 and H3K27me3 ChIP-qPCRs were normalized to the negative controls GAPDH, MYOD1 and GAPDH, respectively. ( A ) DXR/NT, ( B ) Nutlin/DMSO and ( C ) DMSO/NT.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Gene Expression, Quantitative RT-PCR, ChIP-qPCR, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with ( A ) no shRNA, ( B ) scramble (control) shRNA, ( C ) p53shRNA 3755 or ( D ) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH . mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, shRNA, Expressing, Control, Quantitative RT-PCR, Positive Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Proposed models for p53-DNA interaction at half-sites (1 decamer) and full sites (two decamers) with and without a spacer. The minimal p53-binding unit is a half site composed of the consensus decamer RRRCWWGYYY (except for the NT control) based on binding to half sites (decamers) and full sites (two decamers) with a spacer. The binding models assume that p53 exists as a dimer and tetramers are formed following p53 dimer binding to a target sequence. p53-bound half sites as well as full sites containing a spacer are much less frequent across the genome than full sites with zero spacer. ( A ) Efficient tetramer binding at full sites with zero spacer. Strong interactions between dimers and target sequences can occur when the dimers bind to a pair of decamers with no spacer. The dark grey ovals indicates possible changes in p53 (such as configuration) that enable the strong binding. ( B ) Binding to a half site, which is much less efficient than to a full site with no spacer, could be due to weaker interactions of the decamer sequence with just one p53 dimer or with a dimer of dimers, where one dimer makes contact with the decamer sequence, whereas the second dimer interacts non-specifically with nearby DNA. ( C ) Binding to a full site with a spacer is proposed to occur through p53-decamer interactions that are described for the half sites in (B) with the additional possibility of weak interactions between the two bound p53 dimers (lower left panel).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Control, Sequencing

Journal: Molecular Systems Biology

Article Title: TNF stimulation primarily modulates transcriptional burst size of NF‐κB‐regulated genes

doi: 10.15252/msb.202010127

Figure Lengend Snippet: A NF‐κB can recruit a variety of binding partners to target promoters, including the chromatin modifying enzyme p300, the elongation complex P‐TEFb, and components of transcriptional machinery. NF‐κB target genes with a variety of functions were chosen for this study. B Induction of NF‐κB targets in Jurkat T cells in response to 20 ng/ml TNF treatment for 1, 2, and 4 h as measured by RT–qPCR. Target values were normalized to GAPDH and are reported as fold change relative to basal expression. Data are presented as mean ± standard deviation (SD) of three biological replicates. C Enrichment of RelA before and 30 and 60 min after treatment with 20 ng/ml TNF as measured by ChIP‐qPCR and shown as % input (non‐IP control). Data are presented as mean ± SD of three biological replicates. D Maximum intensity projections of smFISH fluorescence microscopy z‐stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia , Tnfaip3 , Tnf , and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. E Histograms of transcripts per cell for target genes (blue) overlaid with probability density plots (red) generated from smFISH data. Cells were combined from three replicates ( Nfkbia , Tnfaip3 , Tnf , and Il6 ) or one replicate ( Il8 , Csf2 ). F, G Bar graphs of mean (F) and CV (G) of smFISH distributions for the indicated genes. Error bars indicate bootstrapped 95% confidence intervals (CIs) for the samples in (E). Significant differences indicated by non‐overlapping CIs. H Ratio of enrichment of total histone H3 to acetylated H3 (AcH3) in Jurkat T cells at the indicated target promoters quantified by ChIP‐qPCR. Data are presented as mean of % input (non‐IP control) ± SD of three biological replicates. I Graph of log 10 (mean) vs log 10 (CV 2 ) of basal mRNA distributions measured in Jurkat T cells (black), HeLa cells (red), or murine bone marrow–derived macrophages (green) for endogenous genes and four latent HIV LTR integrations. Gray shading indicates 95% CI of the linear regression for the basal trend line. Poisson trend line indicated by dashed line. HeLa data from Lee et al and HIV LTR data from Wong et al .

Article Snippet: Anti‐NF‐κB p65 rabbit monoclonal , Cell Signaling Technology , 8242.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Standard Deviation, ChIP-qPCR, Control, Fluorescence, Microscopy, Staining, Labeling, Generated, Derivative Assay

Journal: Molecular Systems Biology

Article Title: TNF stimulation primarily modulates transcriptional burst size of NF‐κB‐regulated genes

doi: 10.15252/msb.202010127

Figure Lengend Snippet:

Article Snippet: Anti‐NF‐κB p65 rabbit monoclonal , Cell Signaling Technology , 8242.

Techniques: Saline, Plasmid Preparation, Protease Inhibitor, Magnetic Beads, SYBR Green Assay, Oligonucleotide Synthesis, Purification, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Excess glucose availability promotes GLUT1 clearance from the plasma membrane (A) HeLa cells were cultured for 24 h in media with no glucose, then switched to high glucose media (25 mM) for the indicated amount of time. Biotin-labeling was performed at the post-glucose shift time points. Following biotinylation, labeled cells were lysed and surface proteins were affinity purified with NeutrAvidin beads (Thermo Scientific). Analysis was performed by SDS-PAGE and immunoblot with antibodies that recognize GLUT1, Na + /K + ATPase, and GAPDH. (B,C) Quantification of captured GLUT1 (B) and Na + /K + ATPase (C) for the experiment shown in (A) was performed over multiple biological replicates (n ≥ 3). GLUT1 measurements were taken of the whole lane using FIJI. Immunoblots for biological replicate experiments are provided in . (D) HeLa cells stably expressing mCherry-CaaX (red) were cultured in no glucose media for 24 h (top row) then shifted to high glucose (25 mM) for 24 h (bottom row), at which point the samples were fixed for immunofluorescence detection with GLUT1 antibody (green). Zoomed images provided in the far right column correspond to the yellow dashed-line inset boxes in the "MERGE" image to the left. (E) Quantification of co-localization shown in (D) was measured by Pearson correlation on Softworx software (n = 30 cells), p = 3.75 × 10 -8 . (F) HeLa cells stably expressing GLUT1-GFP (green) were cultured using the conditions indicated in (D). Prior to imaging, cells were pulse-labeled with FM4-64 (red), a lipophilic tracer dye that inserts into the outer leaflet of the cell membrane. Live cells were incubated on ice in 8 μM cold FM4-64 for ∼5 min before imaging. Zoomed images provided in the far right column correspond to the yellow dashed-line inset boxes in the "MERGE" image to the left. (G) Quantification of the results shown in (F). Pearson correlation coefficient was measured using Softworx software (n = 30 cells), p = 1.69 × 10 -24 . For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Clinical Proteomics, Membrane, Cell Culture, Labeling, Affinity Purification, SDS Page, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Software, Imaging, Incubation

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Glucose-stimulated clearance of GLUT1 results in trafficking to lysosomes (A) Imaging of endogenous GLUT1 was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with GLUT1 antibody (green) and LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization (as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 1 × 10 -5 . (B) Imaging of stably expressed GLUT1-GFP (green) was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 1 × 10 -6 . (C) Imaging of stably expressed GLUT1-FLAG, which harbors a FLAG tag on its first exofacial loop, was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with FLAG antibody (green) and LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 0.004. For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Imaging, Cell Culture, Immunofluorescence, Software, Stable Transfection, FLAG-tag

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Characterization of the GLUT1 trafficking itinerary stimulated by excess glucose availability HeLa cells expressing the three versions of GLUT1 described in Figure 2 were cultured in media lacking glucose for 24 h then either fixed or switched to high glucose media and fixed at the indicated time points. Cells were then probed by immunofluorescence for the endosomal proteins VPS35 (A-B) or CD63 (C-D) (red). In each case, co-localization was analyzed for endogenous GLUT1 (left, green), GLUT1-GFP (middle, green), or GLUT1-FLAG (right, green). Co-localization of GLUT1 signal with VPS35 (B) and CD63 (D) was quantified over the glucose stimulation time course. Co-localization measurements were made in Softworx using Pearson correlation coefficient (n = 30 cells). ∗∗ indicates p < 0.002. (E) Summarized profile of the GLUT1 trafficking itinerary stimulated by excess glucose availability. Heat maps showing co-localization of endogenous GLUT1 (top), GLUT1-GFP (middle), and exofacial GLUT1-FLAG (bottom) with different markers along the endocytic/endosomal trafficking route. For each time point and each marker, at least 21 measurements were made of the Pearson coefficient of correlation using Softworx software. The color in each box is weighted based on the average Pearson coefficient (n ≥ 21) at the indicated time point. (F) HeLa cells harboring a doxycycline-inducible dominant-negative VPS4 variant (VPS4 E228Q -HA) were cultured in no glucose media + 1 μg/ml doxycycline for 24 h then fixed or switched to high glucose media + doxycycline and fixed at the indicated time point. Cells were then imaged for immunofluorescence detection of HA (red) and GLUT1 (green). VPS4 E228Q is a dominant-negative mutant that accumulates on late-endosomal compartments responsible for sorting cargo into intraluminal vesicles. GLUT1 puncta that co-localize with, and are surrounded by, VPS4 E228Q -HA are marked with white arrows. (G) Quantification of the experiments represented in (F) by measuring the Pearson coefficient of correlation (n = 30 cells) using Softworx software. ∗∗ indicates p < 0.001. (H) HeLa cells stably expressing both GLUT1-GFP (green) and doxycycline-inducible VPS4 E228Q -HA were cultured as described in (F) and then imaged for immunofluorescence detection of HA (red). GLUT1-GFP puncta that co-localize with, and are surrounded by, VPS4 E228Q -HA are marked with white arrows. (I) Quantification of the experiments represented in (H) by measuring the Pearson coefficient of correlation (n = 30 cells) using Softworx software. ∗∗ indicates p < 0.02. For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Expressing, Cell Culture, Immunofluorescence, Marker, Software, Dominant Negative Mutation, Variant Assay, Stable Transfection

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: The clathrin-binding motif and PY motifs of TXNIP are required for glucose-mediated GLUT1 trafficking to lysosomes (A) HeLa cells stably expressing a doxycycline-inducible expression vector were cultured using the conditions described in Figure 1 D and, for the induced samples, additionally treated with 1 μg/ml doxycycline for the last 24 h before fixation. Cells were fixed and imaged for immunofluorescence detection of GLUT1 (green) and LAMP1 (red), a marker of lysosomal compartments. Zoomed images provided in the bottom row correspond to the blue dashed-line inset boxes of the top row. (B) Quantification of the experiments represented in panel (A) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition shown. (C) HeLa cells and txnip knockout equivalents (clone 2) stably expressing GLUT1-GFP (green) were cultured as indicated in (A) then fixed for immunofluorescence detection of LAMP1 (red), a marker of lysosomal compartments. (D) Quantification of the experiments represented in panel (C) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition shown. (E) Schematic representation of TXNIP illustrating the predicted arrestin fold domain (yellow), the clathrin-binding motif (orange), and the two PY motifs (green). (F) Complementation analysis of HeLa cells stably expressing GLUT1-GFP (green) with the txnip gene knocked out via CRISPR/Cas9. The knockout cells were stably transfected with either an empty vector or a vector expressing wild-type TXNIP, a clathrin-binding mutant ( cb ), or a py motif mutant ( py ) expressed from a doxycycline-inducible promoter. Cells were cultured as indicated in Figure 1 D with doxycycline added the last 24 h to induce expression of the indicated protein. Cells were fixed and imaged by immunofluorescence for detection of LAMP1 (red), a marker of lysosomal compartments. (G) Quantification of the results shown in panel (F) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) for each condition indicated. The dashed line and area shaded in red indicate the average Pearson’s coefficient and standard deviation (respectively) for the condition in which there is no glucose and TXNIP expression is not induced. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM. All measurements of Pearson coefficient of correlation were performed using Softworx software.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Binding Assay, Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunofluorescence, Marker, Knock-Out, CRISPR, Transfection, Mutagenesis, Standard Deviation, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: TXNIP is dispensable for GLUT1 ubiquitin modification (A) HeLa cells stably expressing GLUT1-GFP were stably transfected with either empty vector (pINDUCER20) or vector expressing wild type, clathrin-binding mutant ( cb ), or py motif mutant (py) TXNIP under the control of Tet-on gene expression system. 1 μg/ml doxycycline was added to induce expression of TXNIP for 24 h prior to collection of cell lysate. Cell lysates were incubated with recombinant WWP1-FLAG at 4 ° C overnight then WWP1-FLAG was pulled down using αFLAG magnetic beads. Elution was performed using FLAG peptide. Lysates and eluates were resolved by SDS-PAGE and analyzed by immunoblot. Immunoblotting of GAPDH was performed as a loading control. (B) HeLa cells stably expressing GLUT1-GFP were transiently transfected with either a wild type or py mutant TXNIP-FLAG expression plasmid. When cells reached 100% confluence, they were collected in lysis buffer and incubated with αFLAG magnetic beads for 1 h at 4 ° C with rotation. TXNIP-FLAG was eluted with FLAG peptide and samples were resolved by SDS-PAGE then analyzed by immunoblot. GAPDH was used as a loading control. (C) HeLa cells stably expressing GLUT1-GFP and a dox-inducible clathrin-binding mutant TXNIP were transiently transfected with either wild-type WWP1-FLAG or a mutant WWP1-FLAG with all 4 ww domains mutated. TXNIP CB was induced with 1 μg/ml doxycycline 24 h before collecting lysates. Cells were then collected in lysis buffer and lysates were incubated with αFLAG magnetic beads for 1 h at 4 ° C with rotation. WWP1 was eluted using FLAG peptide and samples were resolved by SDS-PAGE and analyzed by immunoblot. GAPDH was used as a loading control. (D) HEK293T cells stably expressing FLAG-Ub were split into either 1) regular 25 mM glucose DMEM media, 2) DMEM media with no glucose, or 3) no glucose DMEM media and switched to 25 mM glucose media 2 h before collection. All cells were transiently transfected with a GLUT1-GFP expression plasmid. When cells reached 100% confluency, sample 3 cells were switched to high glucose (25 mM) DMEM media and lysates were collected 2 h later then incubated with magnetic FLAG affinity beads for 1 h at 4 ° C with rotation. FLAG-Ub was eluted using FLAG peptide; samples were resolved by SDS-PAGE, and analyzed by immunoblot. α-Tubulin was used as a loading control. (E) Quantification of the eluate GLUT1 signal for three biological replicates (n = 3) of the experiment shown in (D). ∗∗ indicates a significant difference (p < 0.05) compared to the no glucose condition (lane 2). (F) txnip knockout HeLa cells stably expressing constitutive GLUT1-FLAG and a dox-inducible TXNIP expression plasmid were transiently transfected with HA-Ub. 24 hours before collecting lysates, cells were either mock-treated (sample 2) or treated with 1 µg/ml doxycycline (sample 3) to induce TXNIP expression. As a control, HeLa cells with a stably integrated empty vector (i.e., endogenous TXNIP but no GLUT1-FLAG expression) were also analyzed (sample 1). Lysates were incubated with magnetic αFLAG affinity beads for 1 h at 4°C with rotation and eluted using FLAG peptide. Samples were resolved by SDS-PAGE and analyzed by immunoblot. GAPDH was used as a loading control. (G) Quantification of the eluate HA-Ub signal in four biological replicates (n = 4) of the experiment shown in (F). (H) HeLa cells stably expressing constitutive GLUT1-FLAG and dox-inducible TXNIP vectors were transiently transfected with either empty vector, HA-Ub, and/or WWP1 as indicated in the figure. 24 hours after inducing TXNIP with 1 μg/ml doxycyxline, cells were collected and lysed. Lysates were incubated with magnetic αFLAG affinity beads for 1 h at 4 ° C with rotation. GLUT1-FLAG was eluted with FLAG peptide and samples were resolved by SDS-PAGE then analyzed by immunoblot. GAPDH was used as a loading control. (I) Quantification of HA-Ub signal for at least three biological replicates (n ≥ 3) of the experiments shown in (H). Double asterisk (∗∗) indicates a significant difference (p < 0.05) compared to the empty vector control. All p-values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Ubiquitin Proteomics, Modification, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Control, Gene Expression, Incubation, Recombinant, Magnetic Beads, SDS Page, Western Blot, Lysis, Knock-Out

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Mapping of cytosolic lysines required for lysosomal trafficking of GLUT1 (A) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R) were cultured in media lacking glucose for 24 h then cultured for another 24 h in fresh media lacking glucose (“no glucose”) or shifted to fresh media with high glucose (25 mM) for 24 h (“high glucose”) prior to fixation and imaging for immunofluorescence detection of LAMP1 (red). Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (B) Quantification of the results shown in (A) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. ∗∗ indicates p < 1x10 -5 . (C) Schematic of GLUT1 illustrating the primary amino acid sequence of N-terminal and C-terminal cytosolic tails. Lysine residues in the N-terminal and C-terminal cytosolic tails are highlighted in red. A similar schematic illustrating the lysine residues in the major cytosolic loop is shown in Figure S11 B. (D) HeLa cells stably expressing either wild-type GLUT1-GFP, GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R), GLUT1-GFP with the 6 lysines on the major cytosolic loop mutated to arginine (6K loop →R ), or GLUT1-GFP with the 5 cytosolic lysines outside of the major loop mutated to arginine (5K tails →R) were cultured as in (A) prior to fixation and imaging for immunofluorescence detection of LAMP1 (red). Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (E) Quantification of the results shown in panel D was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. The dashed line and area shaded in red indicate the average Pearson’s coefficient and standard deviation for the WT GLUT1-GFP co-localization with LAMP1 under glucose-starved conditions. All measurements of Pearson coefficient of correlation were performed using Softworx software. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Stable Transfection, Expressing, Cell Culture, Imaging, Immunofluorescence, Sequencing, Standard Deviation, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: TXNIP-mediated trafficking of GLUT1 requires its cytosolic lysine residues (A) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R) were stably transfected with a doxycycline-inducible vector harboring wild-type TXNIP. Cells were cultured as indicated in Figure 1 D prior to fixation and imaging for immunofluorescence detection of LAMP1 (red), a marker of lysosomal compartments. Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (B) Quantification of the results shown in panel A was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. (C) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R GLUT1) were stably transfected with a doxycyclin-inducible vector harboring wild-type TXNIP. Cells were cultured as indicated in Figure 1 D. Prior to imaging, cells were placed on ice and switched to cold (4°C) buffer containing the lipophilic tracer dye FM4-64 (8 μM) (red) in order to label the plasma membrane. Live cells were imaged in cold buffer immediately to ensure retention of FM4-64 at the plasma membrane. Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (D) Quantification of the results shown in panel C was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM. All measurements of Pearson coefficient of correlation were performed using Softworx software.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Imaging, Immunofluorescence, Marker, Clinical Proteomics, Membrane, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet:

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Purification, Produced, Recombinant, Infection, Stable Transfection, Expressing, Diagnostic Assay, CRISPR, Plasmid Preparation, Software