Journal: iScience

Article Title: Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation

doi: 10.1016/j.isci.2021.103354

Figure Lengend Snippet: Overexpression of Nfs1p restores DNA repair in cells lacking fumarase (A) Overexpression of Nfs1p from the multi-copy vector YEp13 under the control of its own promoter restored growth of diploid BY4743ΔWΔfum1 cells on plates containing 360mM HU: Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for 12 days at 28°C. (B) Restoration of DNA repair requires mitochondrial localization of Nfs1p: Overexpression of Nfs1p, but not Nfs1p lacking its mitochondrial targeting signal MTS, from the multi-copy vector PADNX under the control of the strong ADH1 promoter restored growth of haploid BY4742ΔWΔfum1 cells on plates containing 300mM HU. Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for six days at 28°C. (C) Cytosolic Fum1p interacts with cytosolic Nfs1p in the split-Ub assay in vivo: Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for six days at 28°C. Nub-Fum1ΔMTSp was expressed from the ADH1 promoter, while Nub-Nfs1ΔMTS was expressed from the NFS1 promoter. Fum1ΔMTS-Cub-RUra3p was expressed from the FUM1 promoter and Nfs1ΔMTS-Cub-RUra3p was expressed from the NFS1 promoter. The protein–protein interaction between Fum1p and Nfs1p in the cytosol is indicated by the absence of growth on the plate lacking uracil. (D) Fumarase interacts with Nfs1p in a GST pull-down assay in vitro: GST and GST-Nfs1ΔMTSp were purified with glutathione beads in the presence of H 6 -HA-Fum1ΔMTSp. Co-precipitated H 6 -HA-Fum1ΔMTSp was detected with an anti-HA antibody.

Article Snippet: Multi-copy library of Saccharomyces cerevisiae genomic DNA fragments in YEp13 , ATCC , Cat#37323.

Techniques: Over Expression, Plasmid Preparation, Control, Incubation, In Vivo, Pull Down Assay, In Vitro, Purification

Journal: iScience

Article Title: Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation

doi: 10.1016/j.isci.2021.103354

Figure Lengend Snippet: Nfs1S491 and I492 are required for the protein interaction of Nfs1p with Isu1/2p (A) Overexpression of Isu1/2p from the multi-copy vector YEp13 under the control of their own respective promoters restores DNA repair in nfs1S491P and I492T : Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for 12 days at 28°C. (B) Restoration of DNA repair requires mitochondrial localization of Isu1/2p: Overexpression of Isu1/2p, but not Isu1/2p lacking the mitochondrial targeting signal MTS, from the multi-copy vector PADNX under the control of the strong ADH1 promoter restored growth of nfs1S491 and nfs1I492T on plates containing 300mM HU: Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for six days at 28°C. (C) Nfs1S491 and I492 are required for the protein interaction of Nfs1p with Isu1p: Cells of the indicated genotype were 10-fold serially diluted, spotted onto the depicted plates, and incubated for six days at 28°C. Nub-Nfs1ΔMTSp was expressed from the NFS1 promoter and Isu1ΔMTS-CubRUra3p was expressed from the CUP1 promoter. Protein interaction between Nfs1p and Isu1p is revealed by the lack of growth on the plates lacking uracil.

Article Snippet: Multi-copy library of Saccharomyces cerevisiae genomic DNA fragments in YEp13 , ATCC , Cat#37323.

Techniques: Over Expression, Plasmid Preparation, Control, Incubation

Journal: iScience

Article Title: Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation

doi: 10.1016/j.isci.2021.103354

Figure Lengend Snippet:

Article Snippet: Multi-copy library of Saccharomyces cerevisiae genomic DNA fragments in YEp13 , ATCC , Cat#37323.

Techniques: Virus, Recombinant, Generated, Molecular Cloning, Software

Journal: PLoS Pathogens

Article Title: The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

doi: 10.1371/journal.ppat.1004446

Figure Lengend Snippet: (A) Interaction between ESAT-6 and β2M in a yeast two-hybrid system was studied by mating yeast strain AH1109 transformed with bait plasmid pGBKT7-ESAT-6 with yeast strain Y187 transformed with a human leukocyte cDNA prey library on a synthetic dropout plate (–Ade/–His/–Leu/–Trp). Prey cDNA was amplified by PCR using primers encompassing the cDNA insert in pACT2 and sequenced and identified to be β2M. The AH109 yeast strain transformed with pGBKT7-ESAT-6 and pACT2-β2M shows Ade and His interaction reporter activation on QDO plates. AH109 transformed with pGBKT7 and pACT2-β2M was used as a negative control while AH109 transformed with pGBKT7-p53 and pGADT7-T was used as a positive control for the yeast two-hybrid screening. (B) Untagged β2M was cloned along with His-tagged ESAT-6 in the pETDuet vector system and was transformed into E. coli BL21 cells. The transformed cultures were induced with IPTG and the over-expressed proteins were purified using TALON resin. Purified proteins were separated on a 16% Tris-Tricine SDS-PAGE and visualized by silver staining. Lane 1 is a molecular weight marker. (C) β2M with no N-terminal signal sequence (ESAT-6:β2M NSS) and the full length β2M with the N-terminal signal sequence (ESAT-6:β2M WSS) were cloned in pETDuet vector along with 6× His-tagged ESAT-6. Clones were over-expressed in E. coli BL21. Protein expression was induced by addition of IPTG and over-expressed proteins were purified using metal affinity TALON resin. Purified proteins were separated on 16% Tris-Tricine SDS-PAGE and transferred onto nitrocellulose membranes and probed with anti-β2M Ab to detect β2M and anti-His Ab to detect ESAT-6. The bands corresponding to β2M (upper panel) and ESAT-6 (lower panel) were visualized by chemiluminescence. (D) GST pre-cleared THP-1 macrophage extract was mixed and incubated with Glutathione-agarose bead bound-GST or GST-ESAT-6 and washed. The bound proteins were eluted and resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted for β2M protein using rabbit anti-human β2M Ab and HRP conjugated anti-rabbit Ab. Blots were visualized by chemiluminescence. Whole cell extracts from THP-1 cells was used as positive control for β2M expression. (E) In another experiment, purified recombinant 6× His-tagged ESAT-6 was incubated with THP-1 macrophage lysate and immunoprecipitated (IP) with anti-His Ab bound to protein A/G agarose beads. Control immunoprecipitation was carried out without the addition of His-tagged ESAT-6 protein (IP control). The eluted protein mixture is resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted (IB) for β2M protein using a rabbit anti-human β2M Ab and HRP conjugated anti-rabbit secondary Ab and the blots were visualized by chemiluminescence. Lane 1 is input control. (F) Direct interaction of β2M with ESAT-6 was monitored using a BIACORE 3000 Biosensor where β2M was immobilized on the sensor chip and recombinant ESAT-6 at different concentrations was injected in the running buffer. The changes in the refraction index at the surface due to interactions between immobilised β2M and fluid phase ESAT-6 were detected and recorded as RU (Resonance Units). Curves generated from the RU trace were evaluated using a curve-fitting algorithm. ESAT-6 was found to bind specifically to β2M and no binding was observed in a control cell which did not have any immobilized β2M. Results are representative of three different experiments.

Article Snippet: Yeast cell strain AH109 transformed with the bait plasmid pGBKT7-ESAT-6 was mated with yeast strain Y187 transformed with a human leukocyte cDNA prey library obtained from Clontech (Clontech Laboratories, USA).

Techniques: Transformation Assay, Plasmid Preparation, Amplification, Activation Assay, Negative Control, Positive Control, Two Hybrid Screening, Clone Assay, Purification, SDS Page, Silver Staining, Molecular Weight, Marker, Sequencing, Expressing, Incubation, Recombinant, Immunoprecipitation, Injection, Generated, Binding Assay

Journal:

Article Title: Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

doi: 10.1128/MCB.23.6.1968-1982.2003

Figure Lengend Snippet: WT PITX2a can form dimers. (A) The amino acid numbering for the schematic corresponds to the PITX2a isoform. The black region represents the homeodomain (HD). Mated yeast culture (5 μl) was spotted on SD/−His/−Leu/−Trp plates with 0 to 10 mM 3-AT (−HIS) and SD/−Ade/−His/−Leu/−Trp plates (−HIS −ADE) and incubated at 30°C for 5 days. The SD/−Leu/−Trp auxotrophic medium was used in all plates to maintain bait and prey selection. The β-galactosidase activity was assayed from mated yeast colonies by using chemiluminescent detection. The vector interaction with the PITX2a bait or prey was set to 1. The data represent results from four to six separate colonies obtained from two to three separate matings and represent the mean β-galactosidase activity (relative to that of vector) ± the standard error of the mean. (B) Schematic representation of the PITX2 constructs used as baits in the yeast-two hybrid analysis. The mated yeast cultures were spotted on −HIS plates with 0 to 2.5 mM 3-AT and −HIS −ADE plates; β-galactosidase activity was measured as described in the legend for panel A. The data represent results from four to six colonies from two to three separate matings. ND, not determined. (C) The expression of bait constructs used in panels A and B were confirmed by Western blotting by using the anti-GAL4 DBD antibody. Full-length PITX2a bait and molecular weight markers are indicated.

Article Snippet: The bait and prey cDNAs were cloned by standard PCR techniques into the GAL4 DNA binding domain (DBD) fusion vector (pGBKT7) (Clontech) and the GAL4 activation domain (AD) fusion vector (pGADT7) (Clontech), respectively. pGBKT7 and pGADT7 vectors allow growth under tryptophan (−Trp) and leucine (−Leu) auxotrophic selection, respectively.

Techniques: Incubation, Selection, Activity Assay, Plasmid Preparation, Construct, Expressing, Western Blot, Molecular Weight

Journal:

Article Title: Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

doi: 10.1128/MCB.23.6.1968-1982.2003

Figure Lengend Snippet: K88E mutant interacts more strongly with WT PITX2a. The indicated baits and the preys PITX2a K88E (A) or PITX2a T68P (B) or Pit-1 (C) were mated as described in the legend to Fig. ​Fig.11 and plated on SD/−His/−Leu/−Trp plates with 0 to 10 mM 3-AT (−HIS) and/or SD/−Ade/−His/−Leu/−Trp plates (−HIS −ADE) to measure the strength of interaction. As in Fig. ​Fig.1,1, β-galactosidase activity was also measured. The data represent results from four to six separate colonies obtained from two to three separate matings and represent the mean β-galactosidase activity (relative to that of the vector) ± the standard error of the mean. (D) Western blot of the bait constructs shown in panel C with anti-GAL4 DBD antibody. Full-length PITX2a is indicated. (E) Background activity of PITX2a, K88E, and T68P baits with the prey vector was determined as described in the legend to Fig. ​Fig.1A.1A. ND, not determined.

Article Snippet: The bait and prey cDNAs were cloned by standard PCR techniques into the GAL4 DNA binding domain (DBD) fusion vector (pGBKT7) (Clontech) and the GAL4 activation domain (AD) fusion vector (pGADT7) (Clontech), respectively. pGBKT7 and pGADT7 vectors allow growth under tryptophan (−Trp) and leucine (−Leu) auxotrophic selection, respectively.

Techniques: Mutagenesis, Activity Assay, Plasmid Preparation, Western Blot, Construct

Journal: Cell

Article Title: Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted strategy for bacterial vaginosis treatment

doi: 10.1016/j.cell.2024.07.029

Figure Lengend Snippet:

Article Snippet: Yeast Extract Solution (100mL) , Gibco , 18180-059.

Techniques: Virus, Cloning, Molecular Cloning, Mutagenesis, Expressing, Plasmid Preparation, Recombinant, Electron Microscopy, Purification, Saline, Reverse Transcription, Clarification Assay, Viscosity, Gel Extraction, Transformation Assay, Electroporation, RNA Sequencing, Sequencing, Software, Transmission Assay, Microscopy

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Molecular cloning of hSsu72 following yeast two-hybrid screen with RbΔK11 as a bait. ( A ) Schematic representation of hSsu72 clones recovered from the yeast two-hybrid screen with RbΔK11 as bait. The approximate size of clone no. 113 was deduced from fragment size; the exact size of the other clones was determined by DNA sequencing. ( B ) Sequence alignment and amino acid conservation in human, mouse and yeast Ssu72 proteins. The human and mouse proteins are almost identical with the exception of the two indicated conserved substitutions. Red denotes identical residues; orange, similar; black, non-conserved. Note that the N-terminus is generally more conserved than the C-terminus. The PPase domain and the putative LxCxE are indicated.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Molecular Cloning, Two Hybrid Screening, Clone Assay, DNA Sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Interaction of pRb and hSsu72 in vitro and in vivo . ( A ) COS7 cells were transfected with plasmids expressing Myc-tagged pRb, Myc-RbΔ22ΔC and/or Flag-tagged Ssu72 as indicated. Four percent of protein lysates were analyzed directly by western blotting (lanes 1–5). The remaining lysates were immunoprecipitated with anti-Myc antibodies, washed extensively and immunoblotted with anti-Flag antibodies to reveal co-immunoprecipitated Flag-Ssu72. Ssu72 was co-sedimented only in the presence of pRb (lane 9) but not RbΔΔ22Δc (lane 10) or in the absence of Rb (lane 8). Asterisk in both panels denotes uncharacterized proteins. IP, immunoprecipitation; IB, immunoblot. ( B–C ) GST-pull down experiments demonstrating that interaction of pRb and hSsu72 is not mediated by the LxCxE-binding site in pRb (B–C) or the LxCxE motif in Ssu72 (C). In GST-RbLxCxEmut, the LxCxE-binding domain is disrupted by I753A, N757A and M761A substitutions ( , ). hSsu72 LxCxE contains a L T C E E to L T G E E substitution. Similar substitution of the central cystein in the LxCxE motifs in adenovirus E1A and human HSP75 completely disrupted the interaction of these proteins with pRb . The indicated proteins were expressed in COS7 cells by transfecting the corresponding expression vectors. Lysates were mixed with the indicated GST-fusion proteins, extensively washed and immunoblotted with antibodies reactive to HA, Flag or large Tag, which is constitutively expressed in COS7 cells. Note that interaction of pRb with HDAC and Tag but not Ssu72 is disrupted by mutations in the LxCxE-binding domain in pRb. ( D ) Schematic representation of the GST-Rb fusion protein and various deletion mutants used in the following panel. ( E ) GST-pull down experiments of the indicated GST-Rb fusion proteins with transfected HA-hSsu72 or endogenous Tag in COS7 cells. Deletions in the A or B regions (GST-RbΔA and (GST-RbΔ22) or C-terminal domain (GST-RbΔC), reduce but did not completely inhibit binding to hSsu72; double deletions of exon 22 and in the C-terminus (GST-RbΔCΔ22) abrogated pRb- hSsu72 interaction. Coomassie staining of the GST-fusion proteins is shown. Inputs in all panels were 4%.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: In Vitro, In Vivo, Transfection, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Staining

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Human hSsu72 interacts with TFIIB and yeast Pta1. ( A ) Interaction of hSsu72 with TFIIB. COS7 lysates were mixed with GST-hSsu72 or GST proteins immobilized on beads, washed extensively and immunoblotted with antibodies reactive to TFIIB. GST-hSsu72 but not GST efficiently precipitated endogenous TFIIB. ( B ) Interaction of hSsu72 with yeast Pta1. COS7 cells transfected with pcDNA3-Myc-Ssu72 or vector alone were lysed and mixed with GST-Rb or GST-Pta1 proteins, washed extensively and immunoblotted with antibodies reactive to a Myc-specific epitope. Both human Rb and yeast Pta1 efficiently interacted with human Ssu72. ( C ) Hat-hSsu72 or Hat-hSsu72Δ1-12, containing an in-frame deletion that removes the PPase domain, was purified on Talon resins, mixed with the indicated GST-fusion proteins, washed extensively and immunoblotted with polyclonal antibodies reactive to hSsu72. Pta1, pRb and TFIIB interacted with both Hat-hSsu72 and Hat-hSsu72Δ1-12.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Transfection, Plasmid Preparation, Purification

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Structural organization of mammalian ssu72 genes. ( A ) Structural organization of human ssu72 on chromosome 1. The intron/exon boundaries were deduced from the genomic sequence. Exon 1 denotes only the coding region (transcription start site is unknown). ( B ) Location, conservation and sequences around the translation initiation sites in ssu72 and its intronless pseudogenes. ( C ) Structural organization of mouse ssu72 on chromosome 4 and two related pseudogenes, which are likely non-functional due to internal deletions/mutations.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Sequencing, Functional Assay

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Identification of ssu72 transcripts and Ssu72 protein. ( A ) Aliquots containing 50 μg of total RNA from the indicated cell lines were separated by electrophoresis through a 1% agarose/formaldehyde gels, northern blotted and probed with 32 P-radiolabeled human ssu72 cDNA as a probe. Note a doublet with variable intensity in the different cell types. Gapdh was used as internal RNA loading control. ( B ) Western blot analysis of indicated cell lines with rabbit polyclonal antibodies raised against GST-hSsu72. Note a single band of ∼30 kDa in all lines. α-tubulin was used as internal protein loading control.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Electrophoresis, Northern Blot, Control, Western Blot

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Regulation of mammalian Ssu72 subcellular localization. ( A ) Shown are duplicate COS7 cultures transiently transfected with a nuclear marker, Myc-Stra13 and a cytoplasmic marker, Myc-luciferase. Nuclear and cytoplasmic fractions were prepared, separated on polyacrylamide–SDS gels and immunoblotted with anti-Myc antibody to determine the efficiency of the fractionation procedure and anti-hSsu72 antibody to reveal subcellular localization of endogenous Ssu72. ( B ) Immunofluorescent localization of ectopically expressed untagged or Myc-tagged hSsu72 proteins using anti-hSsu72 or anti-Myc specific antibodies, respectively. Nuclei were labeled by DAPI staining. ( C ) Schematic structure of Myc- hSsu72 and Myc-NLS-hSsu72 deletion mutants. ( D ) Cytoplasmic localization of two representative Myc-hSsu72 deletion mutants, Δ16-40 and Δ64-119. All other Myc-hSsu72 deletion mutants shown in (C) also resided in the cytoplasm (data not shown). ( E ) Immunofluorescent analysis of COS7 cells ectopically expressing Myc-NLS-hSsu72 in which hSsu72 is fused to the SV40 Tag NLS. About 45% of transfected cells exhibited complete nuclear localization (top), 45% cytoplasmic localization (bottom); the rest (10%) were found throughout the cell (data not shown). ( F ) Immunofluorescent analysis of COS7 cells ectopically expressing the same two deletion mutants, Δ16-40 and Δ64-119 (D), fused to SV40 TAg NLS (Myc-NLS-hSsu72). Both deletion mutants were completely nuclear. All other Myc-NLS-hSsu72 deletion mutants shown in (C) also exhibited exclusive nuclear localization (data not shown). Secondary fluorescent antibodies used were Rhodamine-conjugated secondary anti-rabbit antibody (A, top) and FITC-conjugated secondary anti-mouse antibody (all other panels).

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Transfection, Marker, Luciferase, Fractionation, Labeling, Staining, Expressing

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Dynamic regulation of mouse ssu72 expression during development. ( A ) Aliquots containing 20 μg of total RNA from the indicated mouse adult tissues were subjected to northern blot analysis with 32 P-labeled mssu72 cDNA probe. As loading control, the blots were re-hybridized with gapdh and the levels of 28S and 18S ribosomal RNAs revealed by EtBr staining. (B–F) Radioactive section in situ hybridization analysis of ssu72 during mouse embryogenesis. ( B ) Sagittal section of an E10.5 embryo showing widespread expression most prevalent in the nervous system. ( C ) Bright field (BF) and dark field images of coronal sections of E13.5 embryos hybridized with sense or anti-sense mouse ssu72 as indicated. At this stage, ssu72 expression becomes restricted to the developing gut and nervous system, with high expression in TG, DRG and spinal cord. ( D ) Bright and dark field images of sagittal sections of E13.5 embryos with high expression in lens, TG and the CNS around the lateral ventricle. ( E and F ) Sagittal sections of E17.5 and E18.5 fetuses showing exclusive and high expression in the nervous system, primarily in DRG of the PNS, and intestine. Other tissues including heart, liver, lung and muscles do not express significant levels of mouse ssu72 .

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Expressing, Northern Blot, Labeling, Control, Staining, In Situ Hybridization, Muscles

Journal: Nucleic Acids Research

Article Title: Conserved and specific functions of mammalian ssu72

doi: 10.1093/nar/gki171

Figure Lengend Snippet: Human ssu72 does not rescue ssu72 -lethal mutation in yeast. ( A ) A ssu72 (+/−) diploid yeast (RF79977) transformed with the yeast ssu72 locus (including promoter and coding region in pRS316 vector), the human ssu72 gene under control of a galactose-inducible promoter (pYES) or parental vectors were induced to sporulate. Upon sporulation, ssu72 − spores died, giving rise to two ssu72 + colonies. Expression of yeast Ssu72 rescued the ssu72 lethal defect resulting in four colonies per tetrad, two of which contained the ssu72 − neo R allele as revealed by replica plating onto G418-plates (data not shown). In contrast, hSsu72 expressing and the parental vectors failed to rescue, yielding only two clones per tetrad, which were both G418-sensitive (i.e. expressing wild-type endogenous yeast ssu72 allele). ( B ) Western blot analysis of RF79977 yeast ssu72 (+/−) transformants grown in the presence of galactose showing high expression of the human Ssu72 in yeast.

Article Snippet: For complementation analysis, ssu72 (+/−) diploid yeast strain [ATCC-4021990; ( )] was transformed with human or yeast ssu72 expression vectors using lithium acetate.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Control, Expressing, Clone Assay, Western Blot

Journal: Scientific Reports

Article Title: Locus specific engineering of tandem DNA repeats in the genome of Saccharomyces cerevisiae using CRISPR/Cas9 and overlapping oligonucleotides

doi: 10.1038/s41598-018-25508-3

Figure Lengend Snippet: Analysis of tandem DNA repeats assembled at the YMR262 locus. (A) Southern blot analysis of genomic DNA from wild type YPH499 and edited strains containing DNA repeats of 165 bp G/C rich or G/C neutral repeats. Molecular weight (MW) is indicated in kb. In some clones, secondary rearrangements can be observed, leading to two repeated arrays of different sizes (indicated by *). NS denotes on non specific hybridization present in all clones including with YPH DNA lane 1). (B) Beeswarm plots showing the size distribution of tandem repeats assessed by Southern blotting and Sanger sequencing for each type of repeat. The horizontal black line in each plots marks the median size of the arrays. Sequences of the analyzed clones are available in Supplementary Figs – .

Article Snippet: The Saccharomyces cerevisiae strain YPH499 (ATCC # 204679) was used as a parental strain throughout the study.

Techniques: Southern Blot, Molecular Weight, Clone Assay, Hybridization, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 activates AP-1-dependent promoters by inducing c-Jun transactivation independent of FHL2. (A) Protein levels of FHL2 and p57 in different cell lines. 100 μg of total protein extract were analyzed by immunoblotting for FHL2 and p57 using a mouse monoclonal FHL2- and a rabbit polyclonal p57-specific antibody. CEM, human leukemic; HEL, human erythroleukemic; HL-1, mouse cardiac muscle; WM35, human melanoma; 293, human embryonic kidney; MCF-7, human breast cancer; HRT-18, human colon cancer; U2OS, human osteosarcoma; HeLa, human cervix carcinoma. The unspecific band at 70 kD ( ∗ ) after probing for p57 was used to judge equal loading. (B) Knockdown of FHL2 using four different doxycyclin-inducible shRNAs in stable pools of lentivirally transduced HRT-18 cells. Western blot analysis of extracts obtained from HRT-18 cells. The expression of shRNAs was induced by the addition of doxycycline (1 μg/mL) for 72 h. Cells expressing shRNA targeting luciferase mRNA (shLuc) served as a control. The FHL2-specific antibody revealed efficient (sh215, sh718) and less efficient (sh428, sh598) inhibition of FHL2 expression. GAPDH served as a loading control. (C) Regulation of an artificial AP-1-dependent promotor (schematic representation top panel) by p57. HRT-18.sh215 cells either untreated (– Doxy) or treated with 1 μg/mL doxycycline (+ Doxy) for 72 hours were transfected with 5xTRE-Luc reporter gene construct and 10 ng pUbi-Rluc (expressing renilla luciferase as a transfection control) together with the p57 expression vectors as indicated. A total of 48 h after transfection, cells were harvested and a Dual luciferase assays performed. Relative promoter activities were expressed as firefly luciferase activities normalized for renilla-luciferase activities. Shown are the mean values ± SD from six independent experiments. (D) Gal4-dependent luciferase reporter gene experiments comparing the effect of p57 on c-Jun and c-Fos transactivation. 293 cells with stably integrated Gal4-dependent luciferase reporter gene (293FR) were transfected with DNA-expression plasmids for Gal-DBD, Gal-Jun or Gal-Fos and p57. Schematic representation of the reporter construct is shown in the top panel. In addition, 10 ng pUbi-Rluc expressing renilla luciferase were cotransfected as control. A total of 30 h after transfection, cells were extracted and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (–) for Gal-Jun and Gal-Fos which are set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 15, top: 100 to 2,250). Shown is the mean ± SD from three independent experiments. (E) Gal4-dependent luciferase reporter gene experiments analyzing effect of p57 on c-Jun transactivation. HRT-18 cells with stably integrated Gal4-dependent luciferase reporter gene (HRT-18FR) were transfected with DNA-expression plasmids for Gal-Jun, p57 and the indicated shRNAs. pUbi-Rluc (expressing renilla luciferase) was cotranfected as a transfection control. shco was used as a control shRNA not targeting any known mRNA. 72 hours after transfection, cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (“–”/“shco”) cells which is set to 1. Shown is the mean ± SD from three independent experiments. Level of significance is indicated. ** = Highly significant, n.s. = not significant.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Western Blot, Expressing, shRNA, Luciferase, Inhibition, Transfection, Construct, Stable Transfection, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: Strong activation of Gal-Jun activity is specific for p57 and requires both, the N -terminal and C -terminal domains of p57. (A) Gal4-dependent luciferase reporter gene experiments comparing the effect of the Cip/Kip-family members p57, p27, and p21 on c-Jun transactivation. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57, p27 or p21. pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. To represent minor activation, the y-axis is split into two segments (bottom: 0 to 15, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments. (B) Similar experiment to (A) comparing p57 and cyclin/CDK-binding deficient p57 mutant. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57 or the cyclin/CDK-binding deficient p57-CK- mutant (CK-) and pUbi-Rluc. A total of 30 h after transfection, cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from four independent experiments. (C) Schematic representation of p57 and its N -terminal and C -terminal domains. Key structural or functional regions of human p57 are indicated. Numbers above indicate positions of amino acid flanking distinct regions. Amino acid including the N -terminal (p57-Nt) and C -terminal (p57-Ct) domains are shown in brackets. (D) Experiment as described above (A) analyzing the contribution of p57-domains to the activation of Gal-Jun by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun and p57 or the indicated p57 domain mutants. A total of 30 hours after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 100, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Activation Assay, Activity Assay, Luciferase, Expressing, Transfection, Binding Assay, Mutagenesis, Functional Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: JNK-independent activation of c-Jun by p57. (A) Schematic of human wildtype c-Jun, Gal-Jun and Gal-Jun4A mutant. Functional domains and phosphorylation sites in the transactivation domain (TAD) are indicated. A Gal4-DNA binding domain (DBD) fusion in frame to the aminoterminus of c-Jun or mutant c-Jun4A ranging from amino acids 1–223 is used in reporter gene experiments. (B) Gal4-dependent luciferase reporter gene experiments analyzing the role of the four major phosphorylation sites in the TAD of c-Jun in the activation by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun or the TAD phosphosite mutant Gal-Jun4A in absence or presence of p57 or the constitutive active upstream kinase of JNKs (ΔMEKK). pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) Gal-Jun which was set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 2.5, top: 50 to 2,000). Shown are the results from three independent experiments. (C) p57 does not induce serine 63 or serine 73 phosphorylation of c-Jun. Immunoblot-analysis investigating the effect of p57 on c-Jun TAD-phosphorylation. 293 cells were transfected with expression vectors for c-Jun and p57 (both HA-tagged) or the constitutive active kinase ΔMEKK. Extracts were prepared 40 h after transfection and subjected to immunoblot-analysis by the LiCor Odyssey system to visualize expression of HA-Jun and Ser 63 and Ser 73 phosphorylation simultaneously. Mixture of Ser 63 and Ser 73 phospho-specific rabbit polyclonal and anti HA mouse monoclonal antibodies were used as first antibodies followed by a mixture of goat derived secondary antibodies coupled with fluorochromes for 680 (anti mouse) or 780 nm (anti rabbit). Three images are shown. Upper: 700 nm channel, phospho-Ser 63/73; middle: 800 nm channel, HA; lower: merged pseudo-colors from channel 700 (red) and 800 (green). Note that molecular weight of HA-Jun is shifted in the presence of the constitutive active kinase ΔMEKK and appears yellow in the merged image.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Activation Assay, Mutagenesis, Functional Assay, Binding Assay, Luciferase, Expressing, Transfection, Western Blot, Derivative Assay, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 might recruit transcription repressors and activators to c-Jun. (A) Gal4-dependent luciferase reporter gene experiments analyzing the effect of p57-domain coexpression on p57-induced c-Jun-activation. Coexpression of p57-Ct may titrate out activators and coexpression of p57-Nt may titrate out repressors of c-Jun activation by p57. 293FR cells were cotransfected with expression plasmids for Gal-Jun, pUbi-Rluc, and p57 wildtype together with vectors expressing p57-Ct or p57-Nt or the related aminoterminal CDK-inhibitory domain of p27 (p27-Nt). A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from three independent experiments. (B) Mechanistic models for p57 in stimulating Gal-Jun transactivation function and a schematic of a putative transcription complex. (C) Model how p57 and FHL2 might act at endogenous promoters at AP-1 sites (TRE). Question mark in (B) and (C) indicates that there is no experimental proof for a direct interaction of p57 with the depicted proteins. For details see text.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Luciferase, Activation Assay, Expressing, Transfection

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: ( A ) Schematic diagram of binary activation in a population of cells. Higher doses of stimulation increase the probability, but not the magnitude, of activation by each cell. ( B ) β-Glucan stimulation of THP-1 WT and BCL10 -KO monocytes transduced with an NF-κB transcriptional reporter. Monocytes were stimulated for 24 hr then measured for T-Sapphire expression via flow cytometry. The graph shows the mean ± standard deviation (SD) fraction of cells positive for NF-κB expression of three biological replicates. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of THP-1 WT cells upon stimulation with 10 μg/ml β-glucan. ( C ) HEK293 WT and BCL10 -KO cells were transduced with an NF-κB transcriptional reporter and stimulated with PMA for 24 hr then measured for T-Sapphire expression via flow cytometry. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of HEK293T WT cells upon stimulation with 5 ng/ml PMA. Shown are means ± SD of three biological replicates. ( D ) THP-1 BCL10 -KO monocytes were transduced with a doxycycline-inducible BCL10-mEos3.2 construct. Cells were imaged for BCL10 expression 24 hr after Dox induction. Images show BCL10 puncta only in cells treated with β-glucan for 16 hr (see also ). In contrast, BCL10 E53R does not form puncta regardless of stimulation. Scale bar: 10 µm. ( E ) Quantification of the number of THP-1 cells with BCL10 puncta after β-glucan stimulation. Each dot represents the average of 4 independent experiments with more than 50 cells each. Shown are means ± SD. ( F ) Endogenous untagged CARD9 and BCL10 form high molecular weight species in THP-1 cells stimulated with β-glucan for 24 hr. After treatment, cells were lysed, and the protein extracts were resolved by size exclusion chromatography followed by capillary immunodetection. Figure 1—source data 1. Full image of protein immunodetection for CARD9 in lysates of THP-1 cells treated with β-glucan. Figure 1—source data 2. Full image of protein immunodetection for CARD9 in lysates of untreated THP-1 cells. Figure 1—source data 3. Full image of protein immunodetection for BCL10 in lysates of THP-1 cells treated with β-glucan. Figure 1—source data 4. Full image of protein immunodetection for BCL10 in lysates of untreated THP-1 cells.

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , American Type Culture Collection , # CRL-3216 , .

Techniques: Activation Assay, Transduction, Expressing, Flow Cytometry, Standard Deviation, Construct, High Molecular Weight, Size-exclusion Chromatography, Immunodetection

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: ( A ) Schema of the BCL10 and MALT1 loci indicating the respective sites targeted by guide RNA for CRISPR-Cas9-mediated knockout approaches. After transfection of plasmids containing Cas-9 expression with the respective guide RNA, we verified the correct KO by targeted deep sequencing and western blot. ( B ) Capillary immunodetection of BCL10 protein in lysates of THP-1 cells. We selected two independent clones to perform experiments and confirm complete ablation of BCL10 expression after CRISPR-Cas9-mediated KO. ( C ) Capillary immunodetection of BCL10 and MALT1 proteins in 293T cells. We selected two clones that completely lost the expression of BCL10 and MALT1. ( D ) Top, schematic of the lentiviral construct integrated into THP-1 BCL10 -KO cells to reconstitute the expression of BCL10 fused to mEos3.2. Bottom, capillary immunodetection of BCL10 in cells following doxycycline (1 µg/ml) treatment, showing tight control over BCL10 expression. Figure 1—figure supplement 2—source data 1. Full image of protein immunodetection for BCL10 and actin in lysates of THP-1 WT and BCL10-KO cells. Figure 1—figure supplement 2—source data 2. Full image of protein immunodetection for BCL10, actin and MALT1 in lysates of HEK293T WT, BCL10-KO, and MALT1-KO cells. Figure 1—figure supplement 2—source data 3. Full image of protein immunodetection for BCL10 and actin in lysates of THP-1 WT cells and BCL10-KO cells reconstituted with BCL10-mEos3.2.

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , American Type Culture Collection , # CRL-3216 , .

Techniques: CRISPR, Knock-Out, Transfection, Expressing, Sequencing, Western Blot, Immunodetection, Clone Assay, Construct, Control

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: ( A ) Schematic of the lentivirus construct to reconstitute BCL10 expressed as a fusion to mScarlet. ( B ) Capillary immunodetection of BCL10 levels in WT, KO, and reconstituted 293T cells. The latter were stimulated with 1 µg/ml Dox for 24 hr. Lysates were generated from 2 million cells in each case. ( C ) Quantification of BCL10 protein levels from three independent experiments as shown in ( B ). BCL10 intensities were normalized to that of actin in each lane. Reconstituted cells express approximately endogenous levels of BCL10, fused to mScarlet. Statistical comparisons were made using unpaired t -test. Shown are means ± standard deviation (SD) of three measurements. ( D ) Capillary immunodetection of BCL10 in 293T WT cells, THP-1 cells, and primary human fibroblasts. Lysates were generated from 1 million cells in each case. ( E ) Quantification of BCL10 protein levels from three independent experiments as shown in ( D ). BCL10 is expressed to the same level in these diverse human cells. Statistical comparisons were made using unpaired t -test. Shown are means ± SD of three measurements. ( F ) Left, flow cytometry histograms of anti-BCL10 staining for the indicated cell lines. 293T BCL10 -KO cells reconstituted with BCL10-mScarlet were stimulated with 1 µg/ml Dox for 24 hr. Right, median BCL10 intensity of three independent experiments. Wildtype and BCL10-mScarlet expressed to approximately the same level. Shown are means ± SD of three measurements. Figure 4—figure supplement 3—source data 1. Full image of protein immunodetection for BCL10 and actin in lysates of HEK293T WT cells and BCL10-KO cells reconstituted with BCL10-mScarlet. Figure 4—figure supplement 3—source data 2. Full image of protein immunodetection for BCL10 and actin in lysates of HEK293T WT, THP-1, fibroblasts, and HEK293T BCL10-KOcells.

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , American Type Culture Collection , # CRL-3216 , .

Techniques: Construct, Immunodetection, Generated, Standard Deviation, Flow Cytometry, Staining

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: ( A ) Plot of p65 nuclear translocation and BCL10-mScarlet intensities from HEK293T BCL10KO cells reconstituted with BCL10-mScarlet expressing EYFP-p65. Cells were treated with Dox 1 µg/ml for 16 hr then treated with PMA 10 ng/ml. Images were captured after 4.5 hr of PMA treatment. n = 221. ( B ) HEK293T BCL10KO cells reconstituted with BCL10-mScarlet expressing EYFP-p65. Cells were treated with Dox 0.05 µg/ml for 16 hr then treated with PMA 10 ng/ml. Images were captured after 4.5 hr of PMA treatment. n = 157. ( C ) Images of HEK293T BCL10-KO cells reconstituted with BCL10-mScarlet expressing EYFP-p65. Cells were stimulated with dox for the respective duration and were tracked for BCL10 assembly status as well as p65 localization. Arrows indicate representative cases of spontaneous nucleation of BCL10-mScarlet and the corresponding EYFP-p65 nuclear translocation. Scale bar: 50 µm. ( D ) Quantification of the percentage of cells containing BCL10-mScarlet polymers. The analysis includes three independent measurements with over 300 cells quantified for each condition. Shown are means ± standard deviation (SD).

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , American Type Culture Collection , # CRL-3216 , .

Techniques: Translocation Assay, Expressing, Standard Deviation

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , American Type Culture Collection , # CRL-3216 , .

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Simple Western, Recombinant, Software