unc93b1  (InvivoGen)

Journal: PLOS Biology

Article Title: Expression of intron-containing HIV-1 RNA induces NLRP1 inflammasome activation in myeloid cells

doi: 10.1371/journal.pbio.3003320

Figure Lengend Snippet: (A) Flow cytometry analysis (left panel) of MDMs infected with WT or mutant (M10 or M10-4xCTE) Lai∆envGFP/G viruses (MOI 1) in the presence of SIV3 + /Vpx VLPs, compared to mock (no virus infection). % infection (GFP + ) in MDMs was quantified at 3 dpi (right panel). (B) Immunoblotting analysis of whole cell lysates from MDMs infected with HIV-1 (WT, M10, or M10-4xCTE constructs) in the absence (untreated, DMSO) or presence of RT inhibitor (EFV, 1 μM) at 3 dpi. HIV-1 proteins (p55 Gag and p24 Gag ) were visualized using anti-HIV-1 IgG, β-actin was used as a loading control. (C) RT-qPCR quantification of unspliced (usRNA), and multiply-spliced (Tat/Rev) transcripts (msRNA) ( D ) in whole cell lysates from MDMs infected with WT, M10, or M10-4xCTE virus ± EFV, at 3 dpi compared to uninfected (mock) lysates. HIV-1 unspliced and spliced transcripts were quantified by RT-qPCR. Production of bioactive type-I IFN (E) , and secretion of IL-1β ( F ) in culture supernatants from MDMs infected with WT, M10, or M10-4xCTE virus ± EFV were harvested at 3 dpi and quantified using type I IFN bioassay or ELISA, respectively. (G, H) Transient knockdown of MAVS, STING and UNC93B1 by siRNA transfection in primary MDMs. Knockdown efficiency was quantified by RT-qPCR (G) at day 2 post-transfection and reported as relative expression normalized to that observed with scramble siRNA (siControl), and protein level analyzed by immunoblotting (H) . siRNA-transfected MDMs were infected with Lai∆envGFP/G (MOI 1) for 3 days (I–L) . Culture supernatants were harvested and analyzed at 3 dpi for p24 Gag (I) , IL-1β (J) , and IP-10 (K) secretion by ELISA. (L) Bioactive type I IFN in the culture supernatants from infected MDMs at 3 dpi quantified using interferon bioassay. The values were normalized to that of mock-infected control in each donor. The means ± SEM are shown, with each symbol representing an independent donor. The means ± SEM are shown, and each symbol represents an independent donor. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons (C, D) , or Dunnett’s post-test, comparing HIV-infected MDMs to uninfected (mock) control (E, F) , or with specific siRNAs to that from siControl (K, L) . P -values: **** < 0.0001; *** < 0.001; ** < 0.01; * < 0.05; ns: not significant ( p ≥ 0.05). The data underlying this figure can be found in .

Article Snippet: At day 2 post-transfection, cells were either infected with HIV-1 (as described above), or stimulated as follows to determine functional knockdown: AIM2 (cells were treated with ultra-pure LPS (100 ng/m, Invivogen) for 2 h, followed by transfection with linearized DNA (1 μg/mL) for 4 h); NLRP1 and CARD8 (cells were primed with Pam3CSK4 (0.5 μg/mL, InvivoGen) for 4 h, followed by stimulation with Val-boroPro (10 μM, InvivoGen) for 24 h; NLRP3 and caspase-1 (cells were primed with ATP (5 mM, Thermo Scientific) for 6 h, followed by activation with nigericin (10 μM) for 60 min; Caspase-4 (cells were transfected with ultra-pure LPS (5 μg/ml, InvivoGen) for 6 hrs; UNC93B1 (cells were treated with Resiquimod (5 μg/ml, Invivogen) for 24 h. At the indicated timepoints, culture supernatants were harvested for IL-1β and IP-10 ELISA, and cells were lysed for RNA or protein analysis.

Techniques: Flow Cytometry, Infection, Mutagenesis, Virus, Western Blot, Construct, Control, Quantitative RT-PCR, Bioassay, Enzyme-linked Immunosorbent Assay, Knockdown, Transfection, Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Distributions of mRNA in BRCA and identification of DEm-RNA association with immunity and prognostic of BRCA patients. A Volcano plot of Differently expressed mRNA B Venn diagram of immune-related genes and DEmRNAs associated with prognosis in BRCA patients C A heat map of UNC93B1 and its co-expressed mRNA. Significance identifier: ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques:

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Differential expression of UNC93B1 in pancancer and BRCA. A Differential expression of UNC93B1 in 33 tumours from the TCGA database B Differential expression of UNC93B1 in a variety of tumours from the paired TCGA database C Differential expression of UNC93B1 in unpaired BRCA samples D Differential expression of UNC93B1 in paired BRCA samples E Differential expression of UNC93B1 in the GSE45827 dataset. Significance identifier: *, p< 0.05; **, p<0.01; ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Quantitative Proteomics

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Clinical correlation analysis of UNC93B1 expression. A - G Expression of UNC93B1 in different patient groups with different clinicopathological factors H Overall survival curve of UNC93B1 from TCGA database I The ROC curve of UNC93B1. Significance identifier: *, p< 0.05; ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Subgroup prognostic analysis of survival and UNC93B1 expression. A - I Kaplan-Meier survival curves of UNC93B1 expression in relation to overall survival (OS) in different patient subgroups with different clinicopathological factors

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Functional enrichment analysis of UNC93B1 in BRCA. A Results of GO analysis B Results of KEGG analysis C - D Results of GSEA analysis. A positive abscissa indicates that UNC93B1 expression is positively correlated with this pathway; a negative abscissa indicates the opposite

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Functional Assay, Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: UNC93B1 expression and tumor immunity. A In the bar graph, UNC93B1 expression was correlated with 24 immune infiltration cells B - I UNC93B1 expression was positively correlated with 8 immune infiltration cells. Significance identifier: ns (no significance, p≥0.05); *, p< 0.05; **, p<0.01; ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Immunoinfiltration analysis of UNC93B1 in BRCA. A Subgroup comparison of the infiltration levels of a total of 17 immune cells in the UNC93B1 low- and high-expression groups B UNC93B1 was significantly associated with LAG-3 C UNC93B1 was significantly associated with HAVCR-2 D UNC93B1 was significantly associated with TNFRSF4 E UNC93B1 was significantly associated with PDCD1 F UNC93B1 was significantly associated with CTLA4 G UNC93B1 was significantly associated with CD274. Significance identifier: *, p< 0.05; **, p<0.01; ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Comparison, Expressing

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Evaluation of the expression of UNC93B1 in clinical breast cancer samples. A - B Detection of UNC93B1 and β-ACTIN expression in BRCA and adjacent tissues by Western blotting and quantitative analysis by Western blotting. The four proteins on the left are the WB results of adjacent tissues, and the four on the right are the WB results of BRCA tissues C - D Immunohistochemical images of UNC93B1 protein expression in normal tissue C versus BRCA D in Human Protein Atlas (HPA) data E Quantitative analysis of immunohistochemical images. Significance identifier: *, p< 0.05; ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: Discover Oncology

Article Title: UNC93B1: a novel immune−related prognostic biomarker in breast cancer

doi: 10.1007/s12672-025-03124-8

Figure Lengend Snippet: Effect of UNC93B1 knockdown on the proliferation and invasion of BRCA cells. A - B After transfection of siRNA, qPCR was performed to assess the level of UNC93B1 gene expression in MDA-MB-231 and SK-BR-3 cells C - D The proliferation of MDA-MB-231 and SK-BR-3 was examined by CCK-8 E - F The proliferation of MDA-MB-231 and SK-BR-3 was examined by colony-formation assays G - H The migration of MDA-MB-231 and SK-BR-3 was examined by transwell assays. Significance identifier: ***, p<0.001

Article Snippet: The siRNAs targeting UNC93B1 were designed, synthesized, and purchased from Sangon Biotech (Shanghai).

Techniques: Knockdown, Transfection, Gene Expression, CCK-8 Assay, Migration

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) Clinical images displaying skin rashes on the patient’s back (left) and hip (right). (B) Pedigree of the patient with a homozygous variant c.284G>T (NM_030930), p.R95L in UNC93B1 . (C) Validation of the homozygous variant in UNC93B1 using Sanger sequencing. (D) The evolutionary conservation of the arginine at position 95 in UNC93B1 across species. (E) Serum Levels of cytokines IL-6, IFN-α, and chemokine IP-10 in the patient (P) and healthy controls (HCs, n = 6) were detected by CBA. (F) Serum Levels of colony-stimulating factors M-CSF and G-CSF in the patient (P) and healthy controls (HCs, n = 6) were detected by ELISA. (G) Transcription level of NF-κB and type I IFN pathways in PBMCs from the patient (P) and healthy controls (HC, n = 3). Analysis of each sample was performed in duplicate. (H) qPCR analysis of NF-κB and type I IFN pathways related genes in PBMCs from the patient (P) compared with healthy controls (HCs, n = 5). (I) Uniform manifold approximation and projection (UMAP) visualization and marker-based annotation of 18 cell subtypes from the patient and healthy controls (HCs, n = 3). (J) UMAP visualization of the target pathway scoring in the patient and healthy controls (HCs, n = 3). Upper, pathway scoring based on genes on NF-κB signal transduction gene set in Gene Ontology database; bottom, pathway scoring based on genes on response to type I IFN gene set in Gene Ontology database. Scores were generated based on Seurat Addmodulescore method. (K) Expression levels of essential genes in NF-κB and type I IFN pathways in various immune cells among the patient and healthy controls (HCs, n = 3). (L) Enrichment of upregulated pathways of differential expressed gene in patient’s DCs based on GSEA.

Article Snippet: UNC93B1 (PA5-20510) antibody was from Invitrogen.

Techniques: Variant Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Marker, Transduction, Generated, Expressing

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) NF-κB luciferase assays in HEK293T cells co-transfected TLR7 (left) or TLR8 (right) with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. EV: empty vector; WT: wild-type UNC93B1; R95L: R95L mutant; HR: H412R mutant. (B) qPCR analysis of IL8 transcription level in HEK293T cells co-transfected TLR7 (left) or TLR8 (right) with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; ***, P < 0.001; two-tailed unpaired Student’s t test. (C) qPCR analysis of IL8 in HEK293T cells co-transfected TLR3 with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; two-tailed unpaired Student’s t test. EV: empty vector; WT: wild-type UNC93B1; R95L: R95L mutant; HR: H412R mutant. (D) NF-κB luciferase assay in HEK293T cells co-transfected TLR9 with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; *, P < 0.05; two-tailed unpaired Student’s t test. WT: wild-type UNC93B1; R95L: R95L mutant. (E) qPCR analysis of IL1B , IL6 , IL8 and TNF transcription levels in THP-1 expressing the WT or R95L mutant UNC93B1 treated with various TLRs agonists (n = 3 biological replicates). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. (F, G) Intracellular cytokine staining of Tnf in mouse RAW 264.7 expressing the WT or R95L mutant UNC93B1, and treated with various TLRs agonists or inhibitors. (H, I) qPCR analysis of NF-κB (H) and type I IFN (I) pathways related genes transcription in BMDCs from indicated mice treated with various TLRs agonists(n = 5 mice per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.

Article Snippet: UNC93B1 (PA5-20510) antibody was from Invitrogen.

Techniques: Luciferase, Transfection, Expressing, Two Tailed Test, Plasmid Preparation, Mutagenesis, Staining

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) Sanger sequencing showing the amino acid substitution Unc93b1, ENSMUST00000162708.7: c.283_285delinsCTC, p.R95L in homozygous knock-in mice. Wild type (+/+), heterozygous (+/R95L), and homozygous (R95L/R95L) are indicated. (B) Body weight of Unc93b1 +/+ , Unc93b1 +/R95L and Unc93b1 R95L/R95L mice. (C) Representative images of splenomegaly and normalized spleen weight to body weight (spleen index) of 12-wk-old mice (n = 5 per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; one-way ANOVA. (D) Plasma levels of anti-DNA, anti-RNA, ANA, CRP and C3 in the indicated mice were detected using ELISA (n = 5 per group). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA. (E) Plasma levels of Il-1β, Il-6 and chemokine Cxcl1 in the indicated mice were detected using ELISA ( Unc93b1 +/+ , n = 10; Unc93b1 +/R95L , n = 6; Unc93b1 R95L/R95L , n = 6). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; one-way ANOVA. (F) H&E staining of the spleen from indicated mice (left). Normalized white pulp was calculated (right). ( Unc93b1 +/+ , n = 10; Unc93b1 +/R95L , n = 6; Unc93b1 R95L/R95L , n = 6). Data represent mean ± SEM; *, P < 0.05; ***, P < 0.001; one-way ANOVA. (G) PAS staining of the kidney from indicated mice (left). Glomerulus area was calculated (right). ( Unc93b1 +/+ , n = 10; Unc93b1 +/R95L , n = 6; Unc93b1 R95L/R95L , n = 6).Data represent mean ± SEM; ***, P < 0.001; one-way ANOVA. (H) PAS staining of the lung and liver from indicated mice. (I) Short fasting blood glucose levels of the indicated mice (n = 8 per group). Data represent mean ± SEM; **, P < 0.01; two-tailed unpaired Student’s t test. (J) Top 15 enriched pathways of differential expressed genes in murine hepatocytes from the Unc93b1 +/+ , and Unc93b1 R95L/R95L mice. (K) Representative immunofluorescent images of IgG and C3 in glomerulus from the indicated mice(left). IgG (top) and C3 (bottom) staining in individual glomerulus was quantified (n = 30 glomerulus from three mice per group). Data represent mean ± SEM; ***, P < 0.001; one-way ANOVA. (L) qPCR analysis of NF-κB and type I IFN pathways related genes in the spleen, kidney, liver, and brain from the indicated mice (spleen, kidney and liver, n = 8-10 per group; brain,n = 4-7 per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; one-way ANOVA.

Article Snippet: UNC93B1 (PA5-20510) antibody was from Invitrogen.

Techniques: Sequencing, Knock-In, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) UMAP visualization and marker-based annotation of 28 cell subtypes from splenocytes of Unc93b1 R95L/R95L and Unc93b1 +/+ mice (n = 3 mice per group). NKT, natural killer T cell; gdT, gamma-delta T cell; Treg, regulatory T cell; MZ B, marginal zone B cell; Fo B, follicular B cell; DC, dendritic cell; MDP, myeloid dendritic progenitor. (B) Upregulated inflammatory signal in Unc93b1 R95L/R95L mice. Left, pathway scoring of NF-κB signal transduction gene set in Gene Ontology database; right, GSEA plot shows upregulated TNF signaling pathway in murine splenic T cells. Pathway score is calculated by the Seurat Addmodulescore method. (C) Upregulated autoimmune characteristics in Unc93b1 R95L/R95L mice. Left, pathway scoring of immunoglobulin production gene set in Gene Ontology database; right, GSEA plot shows upregulated SLE signaling pathway in murine splenic B cells. Pathway score is calculated by the Seurat Addmodulescore method. (D-G) Splenic germinal center B cells (GC B) (D), age-associated B cells (ABC) (E), marginal zone B cells (MZ B) (F), follicular B cells (F), and plasma cells (G) distribution are indicated by FACS plots and percentages (n = 11 mice per group). Data represent mean ± SEM; ***, P < 0.001; two-tailed unpaired Student’s t test. (H, I) Splenic CD4 + T cells (H) and CD8 + T cells (I) activation are indicated by FACS plots and percentages (n = 8 mice per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. (J) Flow cytometric analysis of splenic T cell populations from indicated mice (n = 6-8 mice per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. (K) Flow cytometric analysis of splenic NK, pDCs, mDCs and monocytes populations from indicated mice (NK, n = 6-7 mice per group; pDCs, mDCs and monocytes, n = 11 mice per group). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.

Article Snippet: UNC93B1 (PA5-20510) antibody was from Invitrogen.

Techniques: Marker, Transduction, Two Tailed Test, Activation Assay

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) The structure of TLR7 and UNC93B1 complex (PDB: 7CYN) shows H1 helix (residues 91-97) of UNC93B1 directly contacting two loop regions of the C-terminal LRR-CT domain of TLR7. Protein structural analysis was executed with Pymol. (B) Flag-immunoprecipitation and western blot analyses of UNC93B1 and TLR7 (left)/TLR8 (right) interaction in HEK293T cells. (C) TLR8-immunoprecipitation and western blot analyses of UNC93B1 and TLR8 interaction in HEK293T cells. (D) BioID and western blot analyses of UNC93B1 and TLR7 interaction in RAW 264.7 cells. (E) Immunoblots of TLR7 in endosomes and late endosomes/lysosomes from RAW 264.7 cells expressing the WT or R95L mutant UNC93B1. (F, G) Representative immunofluorescent images (F) and quantification of the colocalization between Tlr7 and Lamp1 or Eea1 in BMDMs from the indicated mice (G) (n = 14-18 cells per group). Data represent mean ± SEM; two-tailed unpaired Student’s t test. (H) RNA pull-down and western blot analyses of ssRNA binding affinity of Tlr7 in BMDCs. (I) Graphic model of how R95L mutation affect UNC93B1/TLR axis and downstream signaling.

Article Snippet: UNC93B1 (PA5-20510) antibody was from Invitrogen.

Techniques: Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Two Tailed Test, Binding Assay