Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Heatmap of log 2 FC values for ubiquitylated peptides in MDS-L FBXO11-KO versus WT cells. Shown are hits with a FC of greater than |0.5|. Known RNA-binding proteins are labeled in red. Data were reanalyzed from ref. . ( B ) Scatter plot of the results from co-IP and MS identification of endogenous FBXO11 complexes in F-36P cell nuclear fractions. Shown are the –log( P value) against the FC enrichment of individual proteins identified in the FBXO11 IP versus the IgG control IP. The blue box highlights proteins with a P value of less than 0.05 and a FC of greater than 1.5. The P value was derived by G test. ( C ) STRING network analysis of FBXO11-interacting proteins identified by MS. Only input nodes are shown, with the threshold cutoff of medium confidence at 0.7. The thickness of the lines connecting nodes indicates the relative strength of evidence supporting the protein-protein interactions. ( D ) Strategy for the FBXO11 substrate-focused CRISPR/Cas9 screen, in which the gRNA library encompasses all of the differentially ubiquitylated peptides identified from the ubiquitin proteomics experiment represented in A . ( E ) Summary table of significant hits from the MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout) analysis of the CRISPR screen in D , demonstrating selective enrichment or depletion of guides in sgCTRL or sgFBXO11 colonies. ( F ) Schematic overview of the integrated multiomics approach to identify relevant candidate FBXO11 substrates meeting the listed criteria, revealing NPM1 and HNRNPU. ( G ) FC in individual gRNA read counts for NPM1 and HNRNPU in the colony-forming assay (day 10) versus initial representation (day 0) in the CRISPR screen. The FC for individual guides was compared between sgCTRL and sgFBXO11 experimental groups, shown on the graphs. n = 6 guides per gene, in 2 independent biological replicates of the CRISPR screen. * P < 0.05, by 2-tailed, paired t test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: RNA Binding Assay, Labeling, Co-Immunoprecipitation Assay, Control, Derivative Assay, Protein-Protein interactions, CRISPR, Ubiquitin Proteomics, Genome Wide, Knock-Out

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of overexpressed FLAG-FBXO11 isoforms in HEK293T cells. Blotting was done for endogenous NPM1 and H2A. Endogenous NPM1 IPs were probed for FBXO11 and NPM1. ( B ) Immunoblots of co-IP FLAG-FBXO11 complexes of FBXO11 and NPM1. ( C ) Left: Input immunoblots for FLAG-FBXO11, NPM1, ubiquitin-GFP, and H2A in HEK293T cells. IPs of NPM1 were performed to detect ubiquitylation by FBXO11. Right: NPM1 versus IgG control IP, immunoblotted for GFP-ubiquitin. n = 3. ( D ) Densitometry for NPM1 poly-ubiquitin bands from C . The area quantified is 75 kDa and above. n = 3. * Q < 0.05, by Kruskal-Wallis ANOVA corrected for multiple comparisons by the Benjamini method. ( E ) Schematic depicting K248-Ub of NPM1 in its C-terminal core. The ubiquitin (Ub) footprint was identified by MS, and . ( F ) HEK239T cells were transfected with NPM1-GFP or NPM1-K248R-GFP fusions. Red outlines indicate GFP-bright regions annotated in QuPath. Scale bars: 10 μm. ( G and H ) Annotated regions quantified for circularity ( G ) and aspect ratio ( H ). n = 2. Dots represent individual NPM1 + regions.* P < 0.05 and ** P < 0.01, by 2-tailed Mann-Whitney U test. ( I ) Confocal images of human CD34 + cells expressing sh CTRL or sh FBXO11 mCherry constructs. Images are pseudocolored for FBXO11 (magenta), NPM1 (green), and DAPI (blue). Scale bars: 5 μm. ( J ) NPM1-bright objects per cell. * P < 0.05, by 2-tailed Mann-Whitney U test. ( K ) Circularity values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( L ) Aspect ratio values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( M ) Relative MFI values for FBXO11 signal and NPM1 signal on a per-cell basis. **** P < 0.0001, by 2-tailed t test. ( N ) Human CD34 + cells stained for FBXO11 (green) and NPM1 (magenta). Colocalization (white arrowheads) where green and magenta overlap. Scale bars: 5 μm. ( O ) Pearson correlation values for NPM1 and FBXO11 signals in the nucleolus (NPM1-bright) and nucleoplasm (NPM1-dim). Each dot represents 1 cell, with greater than 100 cells at ×63. **** P < 0.0001, by 2-tailed Mann-Whitney U test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Control, Transfection, MANN-WHITNEY, Expressing, Construct, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Schematic of splicing reporter delivered to F-36P cells. ( B ) Immunoblot for FBXO11 in HEK293T lysates with an increasing transfected plasmid dose of FBXO11-long and FBXO11-short, compared with endogenous hnRNPR and SYNCRIP. ( C ) Percentage of DsRed + cells normalized to reporter-positive cells in B measured by FACS at 48 hours. * Q < 0.05 and ** Q < 0.01, by multiple 2-tailed t tests corrected for multiple comparisons. ( D ) Representative flow plots of FBXO11-long and FBXO11-short reporter-positive cells. ( E ) DsRed-eGFP + F-36P cell percentages normalized to reporter-positive cells. n = 3 independent experiments; n = 3 wells per group. ** P < 0.01, by 2-tailed t test. ( F ) FC in DsRed + cells in sgFBXO11 versus sgNT, in the context of parental F-36P cells or F-36P cells with NPM1 overexpression. ** Q < 0.01. ( G ) Representative flow plots of sgFBXO1 versus sgNT in F-36P cells overexpressing NPM1. ( H ) log 2 FC of FBXO11 expression in healthy control cells or MDS CD34 + samples, grouped by common splicing factor mutations. SF WT, splicing factor WT. * Q < 0.05, by 1-way ordinary ANOVA corrected for multiple comparisons using the Benjamini method. ( I ) Number of significant alternative splicing events determined by rMATS analysis. ( J ) Top: Schematic of junction reads versus skipped reads, used to determine exon inclusion levels for splicing events. Bottom: Exon inclusion levels for putative FBXO11-dependent splicing events in SLC22A16 and AFTPH in MDS CD34 + samples that were null for any splicing factor mutation. Each dot represents 1 patient.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Transfection, Plasmid Preparation, Over Expression, Expressing, Control, Alternative Splicing, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Workflow to isolate CD34 + cell fractions from healthy donor ( n = 6) and MDS patient ( n = 13) samples, to normalize to 25,000 live cells per replicate tube, and perform total quantitative proteomics by DIA of peptide spectra. Illustration was created in BioRender.com. ( B ) Volcano plot of differentially detected proteins in MDS samples versus healthy controls. Each data point indicates 1 protein, with coordinates reflecting the –log 10 of the P value against its log 2 FC for patients with MDS ( n = 13) versus healthy donors ( n = 6). Color-coding indicates a P value cutoff of less than 0.05 and a log 2 FC cutoff >|0.5|. ( C ) Heatmap of the top 400 differentially expressed proteins in MDS versus healthy controls. Unsupervised hierarchical clustering of patient samples was performed using ClustVis. Each column represents 1 replicate tube from the indicated patient samples below the map. R1, run 1; R2, run 2. ( D ) GO molecular function analysis of cluster 2 proteins, the largest cluster downregulated in MDS. ( E ) GO molecular function analysis of cluster 8 proteins, the largest cluster upregulated in MDS. ( F ) Schematic depicting the data integration performed using Cytoscape to quantify the FBXO11 interactome in primary MDS HSPCs. Schematic was created in BioRender.com. ( G ) Resultant data visualization of the integrated proteomics analysis performed in F . Blue indicates down in MDS; red indicates up in MDS. ( H ) Normalized enrichment scores from GSEA of the significantly differentially expressed proteins in the MDS proteome versus healthy controls.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Quantitative Proteomics

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Upstream regulator analysis of FBXO11-interacting proteins performed using IPA software. MYC was identified as a significant upstream regulator of the FBXO11 interactome ( P = 1.07 × 10 15 ). Shown is the MYC mechanistic network in hierarchical format, with FBXO11-interacting proteins in blue. ( B ) Integrative genomics viewer gene track of FBXO11 from MYC ChIP-Seq results in ENCODE. ( C and D ) FC of MYC binding to FBXO11 promoter sequences, calculated from MYC ChIP-qPCR in F-36P and MDS92 cells. TLR2 agonist (CU-T12-9) treatment resulted in a complete loss of MYC expression. * Q < 0.05, by 2-way ANOVA corrected for multiple comparisons using the Benjamini method. ( E ) TLR2 peptide intensity determined by quantitative proteomics from MDS patient sample replicates in which TLR2 peptides were detected. Each dot indicates 1 replicate. **** P < 0.0001, by 2-tailed Mann Whitney U test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Software, ChIP-sequencing, Binding Assay, ChIP-qPCR, Expressing, Quantitative Proteomics, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of FBXO11, NPM1, and H3 with densitometry. Each sample was normalized to H3, and then to controls. ( B ) Densitometric values of FBXO11 and NPM1 from A . Pearson correlation for all pairs of values and line of best fit with a 95% CI. ( C ) Cell fitness assay using primary CD34 + cells. FC in indel percentages at the end of the assay versus the initial read. For double knockout, the indel percentage tracks NPM1 edits. * Q < 0.05 and ** Q < 0.01, by 2-group specific longitudinal mixed-effects analysis corrected for multiple comparisons. ( D ) Number of colonies. * Q < 0.05, ** Q < 0.01, and *** Q < 0.001, by multiple t tests corrected for multiple comparisons. n = 3. ( E ) Number of colonies. n = 62 with 3 wells per assay. * Q < 0.05 and ** Q < 0.01, by t tests on pooled replicates corrected for multiple comparisons. ( F ) Growth curve of F-36P GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( G ) Growth curve of MDS92 GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( H ) Schematic of the RUNX1-driven MDS mouse model on an inducible Mx1-Cre + Fbxo11 +/+ or Fbxo11 +/– background. ( I ) Percentage of GFP + peripheral blood cells isolated from Fbxo11 +/+ RUNX1-GFP or Fbxo11 +/– RUNX1-GFP transplants. n = 7–10 mice per group. **** P < 0.0001, by fixed-effects (type III) analysis. ( J ) Percentage of GFP + mononuclear cells isolated from BM aspirates of transplant recipients at 11 weeks. n = 8–9 mice per group. * P -linear < 0.05, by 2-tailed t test. ( K ) Percentage contribution to the LSK, Lin - /Sca1 + /Kit + /SLAM + (signaling lymphocyte activation molecules) (LSK-SLAM + ) gate of immunophenotypically defined HSPCs in surviving RUNX1 transplant recipients. n = 6 mice per group. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP-GM, granulocyte-monocyte biased multipotent progenitors; MPP-MkE, megakaryocyte-erythroid biased multipotent progenitors; MPP Ly, lymphoid-biased multipotent progenitor. Q > 0.05, by t tests corrected for multiple comparisons (no significant differences were detected). ( L ) Percentage contribution of RUNX1-GFP + common myeloid progenitor (CMP), GMP, and megakaryocyte-erythrocyte progenitor (MEPs) in the Lin – c-kit + HSPC compartments. n = 6 mice per group. * P < 0.05, by multiple unpaired tests, t test for groups with normal distribution, or Mann-Whitney U test. ( M ) Differential CBCs in mice 16 weeks after pIpC. * P < 0.05, by unpaired, 2-tailed t for groups with normal distribution or Mann-Whitney U test. ( N ) Strategy of Nup98-Hoxd13 -driven MDS mouse model with sh CTRL or sh Fbxo11 vectors. n = 6–10 mice per group. ( O ) Western blot for FBXO11, NPM1, and ACTIN in c-kit + cells from Nup98-Hoxd13 + mice, transduced with lentiviral sh CTRL or sh Fbxo11 . ( P ) CBC in Nup98-Hoxd13 transplants at 2 months. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.005, by unpaired, 2-tailed t test for groups with normal distribution or Mann-Whitney U test. ( Q ) BM cellularity of Nup98-Hoxd13 mice. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( R ) Representative H&E-stained femur cells from Q . Original magnification, ×10. Scale bar: 200 μm.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Double Knockout, Plasmid Preparation, Isolation, Activation Assay, MANN-WHITNEY, Transduction, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Map of FBXO11 mutations identified by whole-exome sequencing of the MSK-IMPACT cohort of patients with hematologic malignancies. ( B ) Alpha-fold predicted structure of FBXO11 with structural features and mutations within myeloid diseases mapped in the disordered N-terminus. ( C ) Variant allele frequencies plotted along the amino acid residues affected by FBXO11 mutations; FBXO11 functional domains are boxed. N-term., N-terminus; ZnF-UBR, zinc finger-ubiquitin-protein ligase E3 component n-Recognin 1. ( D ) Prediction of intrinsically unstructured proteins 2 (IUPRED2) score of amino acid residues in FBXO11-long. The blue arrowhead indicates the initial methionine residue in FBXO11-short. ( E ) Representative confocal images of FBXO11-long and FBXO11-short expressed in HEK293T cells. Scale bars: 10 μm. ( F ) Quantification of signal distribution of FBXO11-long + and FBXO11-short + cells using the SD of FLAG-FBXO11 signal across each nucleus. ** P < 0.01, by 2-tailed, unpaired Mann-Whitney U test. ( G ) GSEA analysis of RNA-Seq data ( GSE156708 ) from MDS-L cells that were FBXO11-KO compared with MDS-L parental cells, cells with reexpression of FBXO11-long cDNA, or cells with reexpression of FBXO11-short cDNA. The size of circle indicates the number of genes in the set.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Sequencing, Variant Assay, Functional Assay, Ubiquitin Proteomics, Residue, MANN-WHITNEY, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: Patient characteristics associated with FBXO11 mutations identified in myeloid malignancies

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD activates Nrf2 signaling in response to LTA. MH-S cells were pretreated with GBD for 30 min and then stimulated with LTA. (A) Confocal images indicating Nrf2 nuclear translocation 3 h poststimulation. Scale bar, 50 µm. (B) Time-course of Nrf2 mRNA expression after LTA stimulation (0, 2, 4, 6, and 8 h). (C) Nrf2 mRNA expression was determined through reverse transcription-quantitative PCR 2 h poststimulation, as described in the materials and methods section. (D) Western blot analysis of Nrf2 protein levels 3 h after LTA stimulation. Data are presented as means±SD. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 h or DMSO; ## P<0.01 and ### P<0.001 vs. DMSO+LTA. GBD glabridin; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Translocation Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD increases HO-1 expression through Nrf2 signaling under LTA challenge. MH-S cells were pretreated with GBD for 30 min and then stimulated with LTA. (A) Confocal images of HO-1 expression 6 h poststimulation. Scale bar, 50 µm. (B) Time-course of HO-1 mRNA expression after LTA stimulation (0, 2, 4, 6, and 8 h). (C) HO-1 mRNA expression was determined by reverse transcription-quantitative PCR. (D) HO-1 protein expression analyzed through western blotting 6 h poststimulation. Data are presented as means±SD (n=4). *P<0.05 and ***P<0.001 vs. 0 h or DMSO; ### P<0.001 vs. DMSO+LTA. GBD glabridin; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: ML385 inhibits GBD-induced Nrf2 activation in LTA-stimulated MH-S cells. MH-S cells were pretreated with DMSO (0.1%), GBD (20 µM), or ML385 (5 µM) for 30 min and then stimulated with LTA. (A) Confocal microscopy of Nrf2 nuclear localization 3 h poststimulation. Scale bar, 10 µm. (B) Quantification of Nrf2 nuclear accumulation based on nuclear mean fluorescence intensity. (C) Nrf2 mRNA expression 2 h poststimulation, analyzed through reverse transcription-quantitative PCR. Data are presented as means ± SD. ***P<0.001 vs. DMSO+LTA; ## P<0.01 vs. GBD+LTA; †† P<0.01 and ††† P<0.001 vs. ML385+LTA. GBD glabridin; Nrf2, nuclear factor erythroid 2-related factor 2; LTA, lipoteichoic acid; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Activation Assay, Confocal Microscopy, Fluorescence, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: Pharmacological and genetic inhibition of Nrf2 attenuates GBD-induced HO-1 expression. MH-S cells were pretreated with the indicated compounds for 30 min and then stimulated with LTA. (A) Immunofluorescence analysis of HO-1 expression 6 h poststimulation. Scale bar, 10 µm. (B) Immunofluorescence image evaluation of HO-1 mean fluorescence intensity. (C) HO-1 mRNA expression 6 h poststimulation, determined through RT-qPCR. (D and E) Effect of Nrf2 knockdown on GBD-induced HO-1 expression. MH-S cells were transfected with control siRNA (siNC) or Nrf2 siRNA (siNrf2) for 6 h, followed by the indicated treatments. HO-1 mRNA levels were measured by RT-qPCR. Data are presented as means ± SD. ***P<0.001 vs. DMSO+LTA (B-C) or si-Ctrl+DMSO (D and E); ## P<0.01 and ### P<0.001 vs. GBD+LTA (B-C) or si-Ctrl+DMSO+LTA (E); †† P<0.01 and ††† P<0.001 vs. ML385+LTA (B-C) or si-Ctrl+GBD+LTA (E). Nrf2, nuclear factor erythroid 2-related factor 2; GBD, glabridin; LTA, lipoteichoic acid; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription-quantitative PCR; si, short interfering; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Inhibition, Expressing, Immunofluorescence, Fluorescence, Quantitative RT-PCR, Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: GBD inhibits LTA-induced macrophage migration through Nrf2 activation. MH-S cells were pretreated with the indicated compounds for 30 min and then stimulated with LTA. (A) Representative wound healing images 0, 6, and 24 h after LTA stimulation. (B) Quantification of wound closure. (C) Representative images of Transwell migration at 6 h and 24 h after LTA stimulation. Migrated cells on the lower surface of the membrane were fixed, stained with crystal violet and images captured under an inverted light microscope. Scale bar, 200 µm. (D) Quantification of migrated cells. Data are presented as means ± SD. ***P<0.001 vs. DMSO; ### P<0.001 vs. DMSO+LTA; † P<0.05, †† P<0.01 and ††† P<0.001 vs. GBD+LTA; ‡‡ P<0.01 vs. ML385+LTA. GBD, glabridin; LTA, lipoteichoic acid; Nrf2, nuclear factor erythroid 2-related factor 2; DMSO, dimethyl sulfoxide.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Migration, Activation Assay, Membrane, Staining, Light Microscopy

Journal: Molecular Medicine Reports

Article Title: Glabridin attenuates LTA-induced alveolar macrophage migration via activation of the Nrf2/HO-1 pathway

doi: 10.3892/mmr.2025.13758

Figure Lengend Snippet: Schematic of GBD-induced modulation of macrophage migration in vitro . Upon LTA stimulation, MH-S cells increase ROS production, activating Nrf2 and upregulating HO-1. GBD thus modulateS cell migration by increasing Nrf2 nuclear translocation and HO-1 expression. GBD, glabridin; LTA, lipoteichoic acid; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1.

Article Snippet: Cells were then transfected with either a small interfering (si)RNA targeting Nrf2 [ Nfe2l2 siRNA: 5′-AGCAUUUUAACAUGUUAACAG-3′ (sense) and 5′-GUUAACAUGUUAAAAUGCUAU-3′ (antisense)] or a negative control siRNA [si-Ctrl: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and reverse 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); cat. no. HY-RS09246; MedChemExpress], using a transfection reagent (cat. no. HY-K2017, MedChemExpress) and 50 nM siRNA diluted in serum-free medium, according to the manufacturer's instructions.

Techniques: Migration, In Vitro, Translocation Assay, Expressing

Journal: bioRxiv

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.1101/2025.11.07.687309

Figure Lengend Snippet: Induced recruitment of USP15 preferentially stabilises a cell surface pool of CTLA4. A, B. Extracellular (A) and lytic (B) HiBiT assay to assess cell surface and total CTLA4 levels respectively, performed on FF-USP15 and CS cells stably expressing HiBiT-CTLA4-FH treated ± doxycycline (Dox; 0.1 µ g/ml) for 6 h, then treated for 18 h ± A/C (500 nM). Error bars show SD from three independent, colour-coded experiments each performed as technical triplicates. Statistical significance was determined using two-way ANOVA with uncorrected Fisher’s LSD. ****P < 0.0001 and **P < 0.01. C. FF-USP15 and CS cells stably expressing HiBiT-CTLA4-FH were transfected with either USP8- or non-targeting (NT1) siRNA for 48 h, then treated for 6 h with doxycycline (Dox; 0.1 µ g/ml), and supplemented for another 20 h ± A/C (500 nM). Cells were lysed and samples subjected to a pulldown using TUBEs to enrich ubiquitylated proteins, followed by western blotting with anti-HA. Representative western blot depicting the Ub-HiBiT-CTLA4-FH signal. IB: Immunoblot. CTRL: Control beads. D. Treatment schedule and quantification of data represented in C. Shown is the the Ub-HiBiT-CTLA4-FH enriched in the TUBE pulldown normalised to HiBiT-CTLA4-FH in the input samples. Error bars show SD from three independent colour-coded experiments. Statistical significance was determined using two-way ANOVA with uncorrected Fisher’s LSD. ****P<0.0001. E. USP15 RapTag in action. Left panel: At steady state, cell surface CTLA4 is rapidly internalised and sorted in a ubiquitin-dependent manner for degradation in the lysosome. Middle panel: A/C-induced recruitment of active USP15 deubiquitylates CTLA4 and thereby opposes its internalisation whilst also promoting recycling, resulting in a net increase in cell surface CTLA4. Right panel: Inactive USP15 fails to deubiquitylate CTLA4 and is itself destabilised as a bystander by sorting with CTLA4 to the lysosome.

Article Snippet: Stable HEK293 HiBiT-CTLA4-FH Flp-In T-REx FF-USP15 WT and CS cells were transfected with 40 nM non-targeting (NT1) or USP8 targeting (oligo 1) siRNA using Lipofectamine RNAiMAX (13778030; Invitrogen) according to manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Control, Ubiquitin Proteomics