nc_010612  (ATCC)

Journal: iScience

Article Title: Characterizing replisome disassembly in human cells

doi: 10.1016/j.isci.2024.110260

Figure Lengend Snippet: CUL2 LRR1 interacts with the replisome during S-phase (A) LRR1 interacts with replisome components on chromatin during S-phase. Chromatin extracts of FLAG-LRR1 expressing cells synchronized in S-phase by release from DTB were immunoprecipitated with FLAG M2 beads. Immunoprecipitated samples were analyzed by western blotting with indicated antibodies. (B) LRR1ΔVHL mutant, unable to interact with CUL2, can still interact with the replisome. Chromatin extracts of FLAG-LRR1 or FLAG-LRR1ΔVHL-expressing cells synchronized in S-phase by release from DTB were immunoprecipitated with FLAG M2 beads. Immunoprecipitated samples were analyzed by western blotting with indicated antibodies. The western blot signal was quantified for all proteins in IP samples and FLAG-signal in IP and input (chromatin fraction) and represented as a fold change over the WT FLAG-LRR1 underneath the western blot. (C) GINS was immunoprecipitated from chromatin extracts of FLAG-LRR1 or FLAG-LRR1ΔVHL-expressing cells, synchronized in S-phase (DTB release). Immunoprecipitated samples were analyzed by western blotting with indicated antibodies. The western blot signal was quantified for FLAG-signal in IP and input (chromatin fraction) and represented as a fold change over the WT FLAG-LRR1 underneath the western blot. (D) Endogenous mAC-tagged LRR1 can interact with replisome components on chromatin in S-phase. Chromatin extracts of mAC-LRR1-expressing cells, synchronized in S-phase (with lovastatin release), were enriched on GFP-Trap magnetic agarose. Immunoprecipitated samples were analyzed by western blotting with indicated antibodies. (E) CUL2 and endogenous LRR1 can interact with GINS on S-phase chromatin. Chromatin extracts of mAC-LRR1 expressing cells, synchronized in S-phase as in (D), were co-immunoprecipitated with GINS antibody after treatment with p97i (5 μM). Immunoprecipitated samples were analyzed by western blotting with indicated antibodies.

Article Snippet: For pulling down mAC-LRR1, 1 mg chromatin lysates were incubated with 25 μL GFP-Trap Magnetic Agarose (Chromotek) for 2 h at 4°C with rotation.

Techniques: Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Journal: iScience

Article Title: Characterizing replisome disassembly in human cells

doi: 10.1016/j.isci.2024.110260

Figure Lengend Snippet: CUL2 or LRR1 depletion leads to replisome retention on the chromatin (A) CUL2 downregulation by siRNA. U2OS cells were transfected with Non-T or CUL2 siRNA (two sequences used in combination; see Oligonucleotides in ) for 48 or 72 h. Whole-cell extract samples were then analyzed by western blotting with the indicated antibodies. (B) CDC45 accumulates on chromatin in S-phase upon depletion of CUL2. Asynchronous U2OS cells transfected with Non-T or CUL2 siRNA for 72 h were analyzed by immunofluorescence. Shown is the quantification of total chromatin-bound CDC45 intensity in EdU-positive (S-phase) cells ( n = 4) (AVG > 295 cells/sample). Red lines indicate the median (p=<0.0001, Two-tailed Mann-Whitney test). (C) Same as for (B) but in CENPF-positive (G2) cells ( n = 4) ( p = 0.0076, Two-tailed Mann-Whitney test) (AVG > 80 cells/sample). (D) Same as for (C) but quantification of the percentage of G2 cells with CDC45 signal above the median of Non-Tsi level ( n = 4) ( p = 0.0009, Two-tailed unpaired t test); mean value +/− SEM. (E) Degradation of mAC-LRR1 after 24 h of auxin treatment. Whole-cell extracts of HCT116-OsTIR1 cells expressing mAC-LRR1 were incubated with GFP-Trap Magnetic Agarose and samples analyzed by western blotting with indicated antibodies. (F) MCM7 accumulates on chromatin in G2 cells upon mAC-LRR1 depletion. HCT116 LRR1-mAC cells were treated with DOX for 48 h and ±IAA for 24 h and analyzed by immunofluorescence. Shown is the quantification of chromatin-bound MCM7 intensity in CENPF-positive (G2) cells ( n = 3) (AVG > 70 cells/sample). Red lines indicate the median (p=<0.0001, Two-tailed Mann-Whitney test). (G) Same as for (F) but quantification of the percentage of G2 cells with MCM7 signal above the median of -IAA cells ( n = 3) ( p = 0.0359, Two-tailed unpaired t test); mean value +/− SEM. (H) Ubiquitylation of MCM7 on chromatin is reduced in S-phase following LRR downregulation. HCT116 LRR1-mAC cells were treated with DOX for 48 h, transfected with HIS-Ubi plasmid, synchronized with STB and released for 6 h ± IAA. HIS-tagged proteins were isolated using the HIS pull-down assay and samples analyzed by western blotting with the indicated antibodies. Levels of MCM7 ubiquitylation were then quantified using ImageJ. Mean ± SEM ( n = 4) ( p = 0.0145, Two-tailed paired t test). (I) CMG components accumulate on chromatin in synchronized cells upon LRR downregulation. U2OS cells were transfected with Non-T or LRR1 siRNA (see Oligonucleotides in ), synchronized with DTB and then released for indicated time points, with addition of NZ at 8 h. Chromatin was extracted with CSK buffer and samples analyzed by western blotting with indicated antibodies. (J) CDC45 accumulates on chromatin in G2 cells upon LRR1 downregulation. Asynchronous U2OS cells transfected with Non-T or LRR1 siRNA for 72 h were analyzed by immunofluorescence. Shown is the quantification of total chromatin-bound CDC45 intensity in CENPF-positive (G2) cells ( n = 3) (p=<0.0001, Two-tailed Mann-Whitney test) (AVG > 60 cells/sample). Red lines indicate the median. (K) Same as for (J) but quantification of the percentage of G2 cells with CDC45 signal above the median of Non-Tsi control cells ( n = 3) ( p = 0.0417, Two-tailed unpaired t test); mean value +/− SEM. (L) MCM7 accumulates on G2 chromatin upon LRR1 downregulation. Whole-cell extracts of HEK293T cells inducibly expressing LRR1 shRNA (see ) for 6 days were analyzed by western blotting with indicated antibodies. Also shown is the representative immunofluorescence images of chromatin-bound pH3-S10 and MCM7 in G2-phase cells expressing LRR1 shRNA.

Article Snippet: For pulling down mAC-LRR1, 1 mg chromatin lysates were incubated with 25 μL GFP-Trap Magnetic Agarose (Chromotek) for 2 h at 4°C with rotation.

Techniques: Transfection, Western Blot, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Expressing, Incubation, Plasmid Preparation, Isolation, Pull Down Assay, Control, shRNA

Journal: iScience

Article Title: Characterizing replisome disassembly in human cells

doi: 10.1016/j.isci.2024.110260

Figure Lengend Snippet:

Article Snippet: For pulling down mAC-LRR1, 1 mg chromatin lysates were incubated with 25 μL GFP-Trap Magnetic Agarose (Chromotek) for 2 h at 4°C with rotation.

Techniques: Recombinant, Transfection, Imaging, Isolation, Immunoprecipitation, Sequencing, shRNA, Plasmid Preparation, CRISPR, Marker, Software, Cell Analysis

Journal: BMC Biotechnology

Article Title: Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

doi: 10.1186/1472-6750-11-94

Figure Lengend Snippet: Growth of bacterial isolates on lignin . Growth of P. norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 on a) HMW lignin fraction and b) LMW lignin fraction. Experiments were performed in 4-fold; mean values of CFUs are shown with error bars indicating the maximum deviation from the mean. * Last CFU count for Bacillus sp. LD003 was performed at 96 h instead of 120 h.

Article Snippet: The isolates, Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were deposited at the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) under the following numbers: [DSMZ: DSM 24563], [DSMZ: DSM 24571] and [DSMZ: DSM 24559], respectively.

Techniques:

Journal: BMC Biotechnology

Article Title: Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

doi: 10.1186/1472-6750-11-94

Figure Lengend Snippet: Growth of bacterial isolates on lignin monomers.

Article Snippet: The isolates, Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were deposited at the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) under the following numbers: [DSMZ: DSM 24563], [DSMZ: DSM 24571] and [DSMZ: DSM 24559], respectively.

Techniques:

Journal: BMC Biotechnology

Article Title: Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

doi: 10.1186/1472-6750-11-94

Figure Lengend Snippet: Decolourization zones in dye-containing plates . Decolourization zones in dye-containing plates after 72 h of incubation. a) LB with 25 mg/L Methylene Blue (MB); b) LB with 25 mg/L Azure B (AB); c) LB with 25 mg/L Toluidine Blue O (TB); d). MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Methylene Blue; e) MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Azure B; f) MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Toluidine Blue O; 1 - P. norimbergensis LD001, 2 - Pseudomonas sp. LD002, 3 - Bacillus sp. LD003. Experiment performed in duplicate.

Article Snippet: The isolates, Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were deposited at the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) under the following numbers: [DSMZ: DSM 24563], [DSMZ: DSM 24571] and [DSMZ: DSM 24559], respectively.

Techniques: Incubation

Journal: BMC Biotechnology

Article Title: Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

doi: 10.1186/1472-6750-11-94

Figure Lengend Snippet: Decolourization of ligninolytic indicator dyes . Decolourization (% of initial value) of ligninolytic indicator dyes in LB medium, 25 h after dye addition to exponentially growing cultures of P. norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003. Error bars indicate the maximum deviation from the mean of duplicate experiments. * indicates that dye adsorption to the cell pellet was observed after centrifugation. Dyes: Azure B (AB), Methylene blue (MB), Toluidene Blue O (TB), Malachite Green (MG), Congo red (CR), Xylidine ponceau (XP), Indigo Carmine (IC) and Remazol Brilliant Blue R (RBBR).

Article Snippet: The isolates, Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were deposited at the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) under the following numbers: [DSMZ: DSM 24563], [DSMZ: DSM 24571] and [DSMZ: DSM 24559], respectively.

Techniques: Adsorption, Centrifugation

Journal: BMC Biotechnology

Article Title: Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

doi: 10.1186/1472-6750-11-94

Figure Lengend Snippet: Preliminary identification of isolated strains from enrichment cultures by 16S rRNA gene sequencing.

Article Snippet: The isolates, Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were deposited at the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) under the following numbers: [DSMZ: DSM 24563], [DSMZ: DSM 24571] and [DSMZ: DSM 24559], respectively.

Techniques: Isolation, Sequencing