r pic k ettii  (ATCC)

Journal: Cell Biology and Toxicology

Article Title: The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway

doi: 10.1007/s10565-024-09888-z

Figure Lengend Snippet: CSN5 interacted with MALT1 to activate NF-κB signaling pathway. A Gene Ontology categories by Gene Ontology analysis of genes interacted with MALT1 in A549 cells. BP biological process, MF molecular function, CC cellular component. The COP9 signalosome was highlighted. B In A549 cells, CSN5 protein in MALT1 Co-IP protein mix detected by MS, arrow indicated the identified CSN5 peptide peak. C The interaction between CSN5 and MALT1 in A549 cells was detected by Co-IP assays. D-F The effects of MALT1 ( D ), MI-2 ( E ) and CSN5 ( F ) on NF-κB signaling pathway activation in A549 cells were detected by immunoblotting. G-H Dual-luciferase reporter assays were used to analyze NF-κB activation in A549 cells after transfection ( G, I ) or MI-2 treatment ( H ). J Rescue assays were performed with dual-luciferase reporter assays to detected the NF-κB activation in A549 cells. Each experiment was performed in triplicate and data are presented as mean ± SD. One-way ANOVA, Dunnett’s Multiple comparison test and LSD multiple comparison test were used to analyze the data (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Protein was extracted in cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (SelleckChem), separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes (Millipore, MA, USA), which incubated with primary antibodies against MALT1 (CST, 2494, Danvers, USA), CSN5 (Proteintech, 27511–1-AP), FBXO3 (Proteintech, 17803–1-AP), p-p65 (Absci, AB11014, OR, USA), Ub (Proteintech, 10201–2-AP), β-actin (Santa Cruz, 47778, CA, USA), and GAPDH (Santa Cruz, 47724).

Techniques: Co-Immunoprecipitation Assay, Activation Assay, Western Blot, Luciferase, Transfection, Comparison

Journal: Cell Biology and Toxicology

Article Title: The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway

doi: 10.1007/s10565-024-09888-z

Figure Lengend Snippet: CSN5 mediated MALT1 protein stability. A, B The protein level of MALT1 ( A ) and CSN5 ( B ) in A549 cells after transfection was detected by immunoblotting. C The mRNA expression of MALT1 in A549 cells transfected as described was determined by qPCR. D 24 h after transfection, A549 cells were treated with MG-132 (50 μM) for 6 h, then the protein level of MALT1 was detected by immunoblotting. E CHX chase assays were conducted to measure MALT1 protein stability in CSN5 silenced A549 cells. Transfected A549 cells were treated with 10 μg/ml CHX for different times (4, 8, 12 h) and collected for immunoblotting. F Transfected A549 cells were analyzed by Co-IP with anti-polyubiquitin antibody for IP and anti-MALT1 antibody for immunoblotting. G CSN5 promoted MALT1 K48-linked poly-ubiquitination. HA-tagged Ub, K48 only mutant and CSN5 siRNA were co-transfected into A549 cells. Co-IP and immunoblotting were performed to detect the ubiquitination of MALT1. Each experiment was performed in triplicate and data are presented as mean ± SD. One-way ANOVA and Dunnett’s Multiple comparison test were used to analyze the data (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Protein was extracted in cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (SelleckChem), separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes (Millipore, MA, USA), which incubated with primary antibodies against MALT1 (CST, 2494, Danvers, USA), CSN5 (Proteintech, 27511–1-AP), FBXO3 (Proteintech, 17803–1-AP), p-p65 (Absci, AB11014, OR, USA), Ub (Proteintech, 10201–2-AP), β-actin (Santa Cruz, 47778, CA, USA), and GAPDH (Santa Cruz, 47724).

Techniques: Transfection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Mutagenesis, Comparison

Journal: Cell Biology and Toxicology

Article Title: The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway

doi: 10.1007/s10565-024-09888-z

Figure Lengend Snippet: CSN5 mediated MALT1 stability through E3 ligase FBXO3. A In A549 cells, FBXO3 protein in MALT1 Co-IP protein mix detected by MS, arrow indicated the identified FBXO3 peptide peak. Potential E3 ligases of MALT1 were predicted by UbiBrowser. B The interaction between FBXO3 and MALT1 in A549 cells was detected by Co-IP assays. C The protein level of MALT1 in transfected A549 cells as described was detected by immunoblotting. D CHX chase assays were conducted to measure MALT1 protein stability in FBXO3 silenced A549 cells. Transfected A549 cells were treated with 10 μg/ml CHX for different times (4, 8, 12 h) and collected for immunoblotting. E Co-IP and immunoblotting assays determined the ubiquitination of MALT1 in transfected A549 cells. F FBXO3 promoted MALT1 K48-linked poly-ubiquitination. HA-tagged Ub WT plasmid or HA-tagged Ub K48 only plasmid and FBXO3 siRNA were co-transfected into A549 cells. Co-IP and immunoblotting were performed to detect the ubiquitination of MALT1. G The interaction between CSN5 and FBXO3 was confirmed using Co-IP assays in A549 cells. H Co-IP and immunoblotting assays determined the interaction between MALT1 and FBXO3 in transfected A549 cells. I CSN5 impaired the assembly of FBXO3 E3 ligase and the neddylation of CRL. Co-IP assays were performed to examine the protein binding to FBXO3 in A549 cells. J The siRNA or plasmid of CSN5 and FBXO3 were co-transfected into A549 cells and the MALT1 protein level was detected. K Co-IP and immunoblotting assays were performed to examine the ubiquitinated MALT1 in a rescue model of A549 cells. Each experiment was performed in triplicate. One-way ANOVA were used to analyze the data (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Protein was extracted in cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (SelleckChem), separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes (Millipore, MA, USA), which incubated with primary antibodies against MALT1 (CST, 2494, Danvers, USA), CSN5 (Proteintech, 27511–1-AP), FBXO3 (Proteintech, 17803–1-AP), p-p65 (Absci, AB11014, OR, USA), Ub (Proteintech, 10201–2-AP), β-actin (Santa Cruz, 47778, CA, USA), and GAPDH (Santa Cruz, 47724).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Plasmid Preparation, Protein Binding

Journal: Cell Biology and Toxicology

Article Title: The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway

doi: 10.1007/s10565-024-09888-z

Figure Lengend Snippet: The over-activated CSN5/FBXO3/MALT1 regulatory axis is correlated with the poor prognosis of NSCLC patients. A The mRNA level of MALT1, CSN5 and FBXO3 in NSCLC tissues (n = 16) and normal lung tissues (n = 14) was analyzed by GEO database (GSE40275). B Representative IHC images of MALT1 in tissue specimens. Quantification of MALT1 levels according to IHC scores in right panel. C The protein level of MALT1 in bronchial epithelium cell BEAS-2B and NSCLC cell lines was determined by immunoblotting. D Relapse free survival Kaplan–Meier curves of NSCLC patients based on the expression of MALT1 were analyzed via PrognoScan database (GSE31210) and the cut-point value of MALT1 expression was 0.81. E Overall survival rate of NSCLC patients with high CSN5 expression was lower than those with low CSN5 expression. The data was analyzed by PrognoScan database (GSE13213) and the cut-point value of CSN5 expression was 0.68. F Relapse free survival Kaplan–Meier curves of NSCLC patients based on the expression of FBXO3 were analyzed via PrognoScan database (GSE31210) and the cut-point value of FBXO3 expression was 0.64. G, H The correlation between MALT1 and CSN5 in normal lung tissues ( G ) and NSCLC tissues ( H ), respectively. The data was analyzed by GEPIA website based on TCGA database. I Simplified model of over-activated CSN5/FBXO3/MALT1 regulatory axis promoting NSCLC malignancy via NF-κB pathway. In NSCLC, overexpressed CSN removes NEDD8 from Cullin-RING to inhibit E3 ligase FBXO3 activity resulting in reduction of MALT1 ubiquitination which contributes to MALT1 stabilization and keeps NF-κB activating, while small molecular inhibitor MI-2 irreversibly inhibits it. Each experiment was performed in triplicate and data are presented as mean ± SD. One-way ANOVA and Paired-Samples T test were used to analyze the data (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Protein was extracted in cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (SelleckChem), separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes (Millipore, MA, USA), which incubated with primary antibodies against MALT1 (CST, 2494, Danvers, USA), CSN5 (Proteintech, 27511–1-AP), FBXO3 (Proteintech, 17803–1-AP), p-p65 (Absci, AB11014, OR, USA), Ub (Proteintech, 10201–2-AP), β-actin (Santa Cruz, 47778, CA, USA), and GAPDH (Santa Cruz, 47724).

Techniques: Western Blot, Expressing, Activity Assay

Journal: eLife

Article Title: ONC201/TIC10 enhances durability of mTOR inhibitor everolimus in metastatic ER+ breast cancer

doi: 10.7554/eLife.85898

Figure Lengend Snippet:

Article Snippet: Antibody , anti-TUFM (rabbit polyclonal) , Thermo Fisher Scientific , CatPA5-27511; RRID: AB_2544987 , WB (1:500).

Techniques: Transfection, Construct, Cell Viability Assay, Viability Assay, Software

Journal: Frontiers in Microbiology

Article Title: The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum

doi: 10.3389/fmicb.2023.1179676

Figure Lengend Snippet: Genome-wide identification and conservation analysis of the COP9 signalosome (Csn) complex in fungi. (A) Number of Csn subunits at the subphylum or phylum level. The evolutionary tree shows the relationships among the various fungal groups. The area of a circle is proportional to the total amount of the Csn subunits identified in each group. The number of species is indicated in parentheses. (B) Conservation of the Csn subunits in fungi. The eight Csn subunits in Aspergillus nidulans were used as query sequences for analysis.

Article Snippet: The GFP-Csn5 were immunoprecipitated from total proteins of tested strains using anti-GFP agarose beads (ChromoTek, Martinsried, Germany) and Flag-Csn1 was detected from proteins co-purified with GFP-Csn5 to verify the association between Csn5 and Csn1.

Techniques: Genome Wide

Journal: Frontiers in Microbiology

Article Title: The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum

doi: 10.3389/fmicb.2023.1179676

Figure Lengend Snippet: Protein–protein interactome of the Csn complex in F. graminearum . (A) Interactions between the Csn subunits. The interactions of pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T were used as positive and negative controls, respectively. Yeast transformants carrying the indicated constructs were plated onto selective plates supplemented without Leu/Trp/His/Ade to assay growth. (B) A model depicting the protein–protein interactions among the Csn subunits of the Csn complex. The black lines indicate the direct interaction. (C) Western blot showing the interaction between Csn5 and Csn6 by using a coimmunoprecipitation (co-IP) assay. Total proteins isolated from the strains bearing GFP-Csn5 and/or Flag-Csn6 (input) and the proteins eluted from the anti-GFP beads (elution) were detected using anti-Flag antibody. GAPDH was used as an internal control. (D) Western blot showing the interaction between Csn5 and Csn1 by using a co-IP assay. (E) Western blot showing the interaction between Csn5 and Csn2 by using a co-IP assay. (F) Western blot showing the interaction between Csn5 and Csn3 by using a co-IP assay. (G) Western blot showing the interaction between Csn5 and Csn4 by using a co-IP assay. (H) Western blot showing the interaction between Csn5 and Csn7 by using a co-IP assay.

Article Snippet: The GFP-Csn5 were immunoprecipitated from total proteins of tested strains using anti-GFP agarose beads (ChromoTek, Martinsried, Germany) and Flag-Csn1 was detected from proteins co-purified with GFP-Csn5 to verify the association between Csn5 and Csn1.

Techniques: Construct, Western Blot, Co-Immunoprecipitation Assay, Isolation

Journal: Frontiers in Microbiology

Article Title: The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum

doi: 10.3389/fmicb.2023.1179676

Figure Lengend Snippet: The localization of the Csn subunits in hyphae of F. graminearum . (A) Micrographs showing the localization of GFP-Csn5 in hyphae of F. graminearum after growth in liquid complete medium (CM) for 16 h. H1-mCherry was used to visualize the nuclei. Bar = 5 μm. A line scan graph was generated at the indicated position (arrow) to show the relative localization of GFP-Csn5 (green) and H1-mCherry (red). (B) Micrographs showing the localization of six Csn subunits in hyphae of F. graminearum . Bar = 5 μm.

Article Snippet: The GFP-Csn5 were immunoprecipitated from total proteins of tested strains using anti-GFP agarose beads (ChromoTek, Martinsried, Germany) and Flag-Csn1 was detected from proteins co-purified with GFP-Csn5 to verify the association between Csn5 and Csn1.

Techniques: Generated

Journal: Frontiers in Microbiology

Article Title: The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum

doi: 10.3389/fmicb.2023.1179676

Figure Lengend Snippet: Transcriptome profiles governed by Csn5 in F. graminearum. (A) Volcano plots showing genes in which the expression was significantly upregulated (red-colored) and downregulated (blue-colored) in the Δ csn5 mutant compared to the wild-type strain PH-1 ( P < 0.05, fold change ≥2.0) under nutrient-rich conditions [complete medium (CM)] or toxin induction conditions [trichothecene biosynthesis induction (TBI) medium]. (B) Venn diagrams showing the number of genes in which the expression was significantly upregulated (left panel) or downregulated (right panel) in the Δ csn5 mutant compared with the wild-type strain PH-1 under both nutrient-rich conditions and toxin induction conditions. (C) Enriched gene ontology (GO) terms in genes with downregulated transcripts in the Δ csn5 mutant under nutrient-rich conditions.

Article Snippet: The GFP-Csn5 were immunoprecipitated from total proteins of tested strains using anti-GFP agarose beads (ChromoTek, Martinsried, Germany) and Flag-Csn1 was detected from proteins co-purified with GFP-Csn5 to verify the association between Csn5 and Csn1.

Techniques: Expressing, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum

doi: 10.3389/fmicb.2023.1179676

Figure Lengend Snippet: Regulation of DON production by the Csn complex. (A) Heatmap comparing the relative transcriptional abundance of TRI genes in the wild-type strain or Δ csn5 mutant in three independent replicates using transcriptomic analysis. WT, wild type. MT = Δ csn5 mutant. The transcription levels of TRI genes in the replicate 1 of the wild-type strain were set to 1.0. (B) Bar charts showing the relative transcriptional abundance of TRI genes in the wild-type strain or indicated mutant strains using quantitative real-time PCR (qRT–PCR). (C) Bar charts showing DON production in 7-day-old TBI cultures of the indicated strains. (D) Western blot analysis of Tri1 protein levels in tested strains expressing Tri1-GFP with the anti-GFP antibody. GAPDH was used as an internal control. (E) Fluorescence signals of Tri1-GFP were detected in the tested strains after growth in liquid TBI medium for 48 h. Bar = 10 μm. Error bars indicate the standard deviation from three independent experiments. Statistical analysis was performed via Student’s t -test. A triple asterisk indicates statistical significance with p < 0.001.

Article Snippet: The GFP-Csn5 were immunoprecipitated from total proteins of tested strains using anti-GFP agarose beads (ChromoTek, Martinsried, Germany) and Flag-Csn1 was detected from proteins co-purified with GFP-Csn5 to verify the association between Csn5 and Csn1.

Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Fluorescence, Standard Deviation

Journal: Journal of AOAC International

Article Title: 3M™ Petrifilm Yeast and Mold Count Plate for the Enumeration of Yeasts and Molds in Dried Cannabis Flower: AOAC Official Method SM 997.02

doi: 10.1093/jaoacint/qsac114

Figure Lengend Snippet: Exclusivity results for the 3M Petrifilm YM Count Plates

Article Snippet: 34 , Ralstonia pickettii , ATCC 27511 , Clinical Isolate , –.

Techniques: Derivative Assay