Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin transgene inhibits DNA sensing-induced innate immune response. A. Schematic of cloning process. B-G. Cells were transfected with plasmid DNA (pDNA) using TransIT-2020. B. 2.5 μg and 7.5 μg of pDNA encoding Poxin or mutant Poxin (mtPoxin) were transfected into 293T cells, respectively, and FLAG-tagged proteins were detected. C. Activation of STING, TBK1, and IRF3 was analyzed by western blotting. Quantitative analysis of p-IRF3 expression level is shown. D. H151 was pre-treated 1 h before pDNA transfection, and protein expression levels were assessed after 24 h. E. Cells were transfected with pEGFP-N1, and protein expression levels were detected. F. mRNA levels of IFNB1 and ISG15 were analyzed by RT-qPCR (n = 3). G. IFN-β secretion levels were quantitatively analyzed using ELISA. (Representative western blots are shown.).

Article Snippet: Quantitative analysis of secreted human IFN-β was performed following manufacturers' protocol (luex-hifnbv2, Invivogen).

Techniques: Cloning, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin protects NK-92 cells from pDNA transfection-induced innate immunity. A. Cells were transfected by electroporation with 10 μg pDNA, and mRNA dynamics were analyzed by RT-qPCR (n = 3). B. IFN-β secretion levels were quantitatively analyzed using ELISA. C. Expression levels of innate immune signaling proteins were detected by western blotting. (Representative data shown). D. Quantitative analysis of p-STING and p-TBK1 expression levels.

Article Snippet: Quantitative analysis of secreted human IFN-β was performed following manufacturers' protocol (luex-hifnbv2, Invivogen).

Techniques: Transfection, Electroporation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: HSPA6 is induced by RIG-I-like receptors and negatively regulates type-I interferon signaling

doi: 10.1007/s00018-026-06147-8

Figure Lengend Snippet: HSPA6 is upregulated in expression by IFN signaling-competent MDA5. A . Volcano plot of RNA-seq data comparing gene expression between WT and GOF MDA5 expressed HEK293 cells. B . HEK293T cells were transfected with plasmids expressing WT or GOF MDA5. 24 h later, cells were lysed for RNA detection by RT-qPCR and protein detection by western blotting. EV, empty vector. C . HEK293T cells were mock-treated or treated with recombinant IFNβ; at a final concentration of 10 ng/ml for 24 h. RNA was purified, and gene expression was tested by RT-qPCR. D . HEK293T cells were transfected with plasmids expressing MDA5. 24 h later, cells were lysed for RNA detection. The qPCR primer-targeting regions on AL590385.23 was indicated in green. E . HEK293T cells were transfected with MDA5 expressing plasmid. 6 h post transfection, cells were transfected with poly I: C at a final concentration of 0.8 ng/μ;l. 18 h later, cells were lysed for RNA measurement. F . Cells were transfected with GOF MDA5 expressing plasmid and harvested at indicated hours post transfection. RNA was measured by RT-qPCR. G , H . Act.D was added at a final concentration of 5 μ;g/ml 24 h post transfection of plasmids indicated. 24 h post treatment, cells were lysed for RNA measurement. I , J . HEK293T cells were infected with EMCV at an MOI of 0.01. 24 h post infection, cells were lysed for RNA measurement. K . RNA levels of IFNβ; and HSPA6 in epithelial cells were shown upon SARS-CoV infection. The data was obtained from GEO database (accession number: GSE17400 ). For B-E, G-J, data are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. For F, data are means ± SD of two independent experiments

Article Snippet: The recombinant human IFNβ (Sino Biological, 10704-HNAS) was dissolved in PBS at a stock concentration of 1.0 mg/ml as the manufacture’s instruction suggested.

Techniques: Expressing, RNA Sequencing, Gene Expression, Transfection, RNA Detection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Recombinant, Concentration Assay, Purification, Infection

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT-derived mRNA enhances translation and reduces immune activation. ( A ) Flow cytometry analysis of eGFP expression in HEK293T cells transfected with eGFP mRNA synthesized using either dsDNA or NiLoT, as used in Fig. . Fluorescence intensity was measured at 18 h post-transfection. Cells treated with Lipofectamine TM 3000 alone served as mock controls. Mean fluorescence intensity was normalized to the mock condition. Statistical comparisons were performed using two-tailed, unpaired t -test; ** P < .01 and **** P < .0001. ( B ) Fluorescence microscopy images showing eGFP expression in cells transfected with NiLoT- or dsDNA-derived eGFP mRNA. ( C ) Quantification of IFN-β secretion by ELISA in HEK293T cells transfected with NiLoT- or dsDNA-based eGFP mRNA. Cells treated with Lipofectamine TM 3000 alone served as mock controls, and cells treated with poly(I:C) served as positive controls for immune activation. Statistical comparisons were performed using two-tailed, unpaired t -test; * P < .05, ** P < .01, and *** P < .001. ( D ) Flow cytometry analysis of eGFP expression in HEK293T and THP-1 cells transfected with m 1 Ψ-modified eGFP mRNA synthesized using either dsDNA or NiLoT. Fluorescence intensity was measured at 18 h post-transfection. Cells treated with Lipofectamine 3000 alone served as mock controls. Mean fluorescence intensity was normalized to the mock condition. Statistical comparisons were performed using two-tailed, unpaired t -test; **** P < .0001. ( E ) Fluorescence microscopy images showing eGFP expression in HEK293T and THP-1 cells transfected with NiLoT- or dsDNA-derived eGFP mRNA. ( F ) Quantification of IFN-β secretion by ELISA in HEK293T and THP-1 cells transfected with m 1 Ψ-modified eGFP mRNA synthesized from either NiLoT or dsDNA templates. Cells treated with poly(I:C) were included as positive controls for innate immune activation. ELISA measurements were performed 24 h post-transfection. Statistical comparisons were performed using a two-tailed, unpaired t -test; * P < .05 and ** P < .01.

Article Snippet: After 18 h, 500 μl of supernatant was analyzed using a human IFN-β ELISA Kit (Cusabio, Wuhan, China, #CSB-E09889h).

Techniques: Derivative Assay, Activation Assay, Flow Cytometry, Expressing, Transfection, Synthesized, Fluorescence, Two Tailed Test, Microscopy, Enzyme-linked Immunosorbent Assay, Modification

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Schematic workflow for targeted shRNA screening in RAW264.7 macrophages to identify lysosomal antiviral factors by assaying IAV viral infectivity using NS1-GFP fluorescence as a readout. ( B ) Ranking of the target genes based on the fold change of normalized fluorescence from cells expressing the indicated shRNAs in comparison to control cells expressing shRNA against luciferase. Data were mean ± SD, n = 3 biological replicates per knockdown, two independent shRNAs were evaluated for each gene. ( C ) Immunoblot analysis of the indicated Lamtor proteins in cells depleted of LAMTOR components using shRNA. Actin was used as a loading control. ( D ) Representative micrographs of RAW macrophages depleted of LAMTOR components following infection with IAV PR8-NS1-GFP at an MOI of 0.25 for 24 h. Scale bars: 100 µm. A minimum of four independent fields of view was obtained for each sample. ( E ) Schematic diagram depicting the importance of a lipidation site within the N-terminal region of Lamtor1 in controlling the LAMTOR lysosomal localization. 3A, non-lysosomal G2A/C3A/C4A mutant Lamtor1, WT wildtype. ( F ) Immunoblot analysis of the indicated proteins in Lamtor1 knockout (KO) mouse RAW264.7 macrophage lines reconstituted with vector control or FLAG-tagged human WT and 3A mutant LAMTOR1 accordingly. Actin was used as a loading control. ( G ) Time-course analysis of IFN-β secretion from Lamtor1 KO, WT, and 3A cells after stimulation with Poly(I:C) (30 µg ml −1 ), R848 (0.5 µg ml −1 ), CpG-B (1 µM), LPS (0.5 µg ml −1 ), or cGAMP (5 µg ml −1 ), as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group for each time point; two-way repeated measures ANOVA followed by Dunnett’s multiple comparisons test, adjusted P value as indicated. ( H ) Innate immune signaling in WT and 3A cells in response to PRR stimulation. Cells were lysed and analyzed for the levels of the indicated proteins and phosphorylation status of S6K1 (T389), TFEB (S122), TBK1 (S172), IRF-3 (S396), and STING (S365). ( I ) qRT-PCR analysis of Ifnb1 expression in Lamtor1 KO, WT, and 3A cells after 14 h of stimulation with PRR agonists. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s multiple comparisons test, adjusted P value as indicated. Mock-treatment controls were shared across PRR stimulation conditions. ( J ) Principal component analysis (PCA) of the RNA-seq transcriptome of LAMTOR1 WT and 3A cells in the presence or absence of the indicated PRR agonists. n = 3 biological replicates per group, differentially expressed genes (DEGs) with a false discovery rate (FDR) < 0.05, by DESeq2. ( K , L ) Venn diagram ( K ) and heatmap ( L ) of the transcriptome revealing the overlap of DEGs in IFN response between LAMTOR1 WT and 3 A cells across different inducers of innate immunity. ( M – O ) mRNA expression levels of Irf7 ( M ), Cxcl10 ( N ), and Mx2 ( O ) in WT and 3A cells after 14 h of stimulation with PRR agonists. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( P ) Immunoblot analysis of ISGs in WT and 3A cells in response to PRR stimulation. Representative results for A – I , M – P from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: shRNA, Infection, Fluorescence, Expressing, Comparison, Control, Luciferase, Knockdown, Western Blot, Mutagenesis, Knock-Out, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Quantitative RT-PCR, RNA Sequencing

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Schematic diagram showing the importance of two structural motifs (NIV and LVV) in Lamtor1 for LAMTOR-Rag interaction. NIV, N64A/I66A/V68A mutant Lamtor1; LVV, L154A/V155A/V156A mutant Lamtor1. ( B , C ) Schematic representation of LAMTOR-Rag GTPases ( B ) and close-up view of the protein–protein interaction interface surrounding the NIV or LVV motif of Lamtor1, modified from PDB:6U62 ( C ). ( D ) Immunofluorescence analysis of HeLa cells stably expressing FLAG-tagged WT, NIV, and the LVV mutant LAMTOR1. Cells were fixed and immunostained for FLAG and endogenous LAMP2. Micrographs are representative of two independent experiments. Scale bars: full size, 20 µm; insets: 2 µm. ( E ) Interaction between endogenous Rag GTPases and FLAG-tagged human LAMTOR1 WT or the indicated variants in RAW cells, as assessed by FLAG immunoprecipitation. ( F ) Ifnb1 mRNA expression in Lamtor1 KO cells reconstituted with FLAG-tagged human WT, NIV, or LVV mutant LAMTOR1 after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( G ) IFN-β secretion from LAMTOR1 WT, NIV, and LVV cells after PRR stimulation, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( H ) Measurement of endolysosomal pH in parental, Lamtor1 KO, Rraga KO, and Rragc KO cells using LysoSensor TM Yellow/Blue DND-160. Data were mean ± SD, n = 3 biological replicates per group; one-way ANOVA followed by Dunnett’s test, adjusted P value as indicated. Representative results for ( D – H ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Mutagenesis, Modification, Immunofluorescence, Stable Transfection, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Ifnb1 mRNA expression in parental, Lamtor1 KO, Rraga KO, and Rragc KO cells after stimulation with PRR agonists. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( B ) IFN-β secretion from parental, Lamtor1 KO, Rraga KO, and Rragc KO cells after stimulation with PRR agonists, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls in ( A , B ) were shared across PRR stimulations, except for cGAMP stimulation, which used separate mock controls to account for reagent lot variability. ( C – E ), Immunoblot analysis of the indicated ISGs in parental, Lamtor1 KO ( C ), Rraga KO ( D ), and Rragc KO ( E ) cells in response to PRR stimulation. ( F ) Schematic diagram of the Rragc -floxed allele and generation of Rragc Lyz2 mice. Exons 2–3 of Rragc are flanked by two loxP sites. ( G ) Genotyping (top) and immunoblotting (bottom) showing the myeloid-specific deletion of RagC in Rragc Lyz2 mice. Brain tissue was used as a negative control. BMDM, bone marrow-derived macrophage. ( H ) Survival rate (top) and body weight change (bottom) were monitored following PR8 infection in Rragc f/f and Rragc Lyz2 mice. Data were mean ± SD of body weight measurements (bottom), n = 10 mice per group. ( I ) Viral titers in bronchoalveolar lavage fluid were quantified using the TCID 50 assay 3 d after infection. Data were mean ± SD, n = 5 mice per group. ( J ) IFN-β levels in bronchoalveolar lavage fluid were measured 3 d after infection. Data were mean ± SD, n = 5 mice per group. ( K ) Representative hematoxylin and eosin staining of lung tissue sections from PR8-infected mice. n = 5 mice per group. Representative results for A – E , G – K from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control, Derivative Assay, Infection, Staining

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Immunoblot analysis of resting-state mTORC1 signaling in parental, Lamtor1 KO, Rraga KO, and Rragc KO cells. Cells were lysed and analyzed for the levels of the indicated proteins and phosphorylation status of TFEB (S122), S6K1 (T389), 4E-BP1 (S65), and S6 (S235/236). ( B ) Ifnb1 mRNA expression in RAW cells after stimulation with PRR agonists with or without rapamycin (100 nM) or Torin 1 (250 nM). The drugs were added 30 min before PRR stimulation and remained throughout. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( C ) IFN-β secretion from RAW cells as treated in ( B ). Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( D ) Immunoblot analysis of mTORC1 signaling in cells treated with mTORC1 inhibitors. Cells were lysed and analyzed for the levels of the indicated proteins and phosphorylation status of TFEB (S122), S6K1 (T389), 4E-BP1 (S65), and S6 (S235/236). ( E ) Immunoblot analysis of S6K1 phosphorylation in parental and Rptor KO cells following PRR stimulation. ( F ) Ifnb1 mRNA expression in parental, Lamtor1 KO, and Rptor KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( G ) IFN-β secretion from parental, Lamtor1 KO, and Rptor KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( H ) Immunoblot analysis of the indicated ISGs in parental and Rptor KO cells in response to PRR stimulation. ( I ) Immunoblot analysis of TFEB and S6K1 phosphorylation status in parental, Lamtor1 KO, and Lamtor1 KO cells stably expressing Raptor-Rheb15. ( J ) Ifnb1 mRNA expression in parental, Lamtor1 KO, and Lamtor1 KO cells expressing Raptor-Rheb15 after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( K ) IFN-β secretion from parental, Lamtor1 KO, and Lamtor1 KO cells expressing Raptor-Rheb15 after PRR stimulation, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Representative results for ( A – K ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Phospho-proteomics, Expressing, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of TFEB phosphorylation in parental, Lamtor1 KO ( A ), Rraga KO ( B ), and Rragc KO cells ( C ) following PRR stimulation. ( D ) Validation of TFE3 expression in Tfe3 KO cells. ( E , F ) Ifnb1 transcription ( E ) and IFN-β release ( F ) from Tfe3 KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( G ) Validation of TFEB expression in parental, Lamtor1 KO, Tfeb KO, and Lamtor1/Tfeb DKO cells. ( H , I ) Ifnb1 transcription ( H ) and IFN-β release ( I ) from parental, Lamtor1 KO, Tfeb KO, and Lamtor1/Tfeb DKO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Representative results for ( A – I ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Phospho-proteomics, Biomarker Discovery, Expressing

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Schematic depicting where MyD88 and UNC93B1 act in the innate immune signaling pathway. ( B ) Ifnb1 mRNA expression in parental, Lamtor1 KO, Myd88 KO, and Unc93b1 KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( C ) IFN-β secretion from parental, Lamtor1 KO, Myd88 KO, and Unc93b1 KO cells after PRR stimulation, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( D , E ) Immunoblot analysis of ISGs in parental, Myd88 KO ( D ), and Unc93b1 KO ( E ) cells in response to PRR stimulation. qRT-PCR ( E , right) confirmed the knockout of Unc93b1 owing to the lack of validated antibodies against UNC93B1. ( F ) Immunoblot analysis of Lamtor1 protein levels in THP-1 cells depleted of LAMTOR1 , using three different shRNAs via lentiviral transduction. Luciferase was used as a non-targeting control. ( G – J ) mRNA expression levels of IFNB1 ( G ), ISG15 ( H ), RSAD2 ( I ), and IFITM ( J ) in THP-1 cells after 14 h of stimulation with either R848 (5 µg ml −1 ) or LPS (20 µg ml −1 ). Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( K ) IFN-β secretion from the indicated THP-1 cells after stimulation with either R848 (top) or LPS (bottom), as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Representative results for ( B – K ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Knock-Out, Transduction, Luciferase, Control

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Schematic illustrating the activation of the MAVS signaling cascade to trigger IFN induction. ( B ) Time-course analysis of IFN-β secretion from the indicated RAW macrophages after stimulation with Poly(I:C) (0.5 µg ml −1 ) via FuGENE transfection, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group at each time point; two-way repeated measures ANOVA followed by Dunnett’s test, adjusted P value as indicated. ( C ) Immunoblot analysis of the indicated ISGs in LAMTOR1 WT and 3A cells in response to 14 h of stimulation with transfected Poly(I:C). ( D ) Ifnb1 mRNA expression in parental, Lamtor1 KO, Rraga KO, and Rragc KO RAW cells after stimulation with transfected Poly(I:C). Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( E ) IFN-β secretion from the indicated RAW macrophages after stimulation with transfected Poly(I:C), as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( F ) Immunoblot analysis of Lamtor1 protein levels in HEK293T cells expressing two different shRNAs via lentiviral transduction. Luciferase was used as a non-targeting control. ( G ) ISRE luciferase reporter activity in Lamtor1-depleted HEK293T cells. Cells were transfected with ISRE Firefly luciferase and Renilla luciferase constructs and stimulated with Poly(I:C) (0.8 µg ml −1 ) via FuGENE transfection for 24 h, followed by the measurement of reporter activity. Normalized data were expressed as luciferase fold change over the mock-treated shLuc group. ( H – J ) mRNA expression of IFNB1 ( H ), ISG15 ( I ), and RSAD2 ( J ) in the indicated HEK293T cells transfected with Poly(I:C) (1 µg ml −1 ) for 24 h. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( K ) IFN-β secretion from the indicated HEK293T cells after 24 h stimulation with transfected Poly(I:C), as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( L ) Representative micrographs of parental and LAMTOR1 KO HEK293T cells infected with IAV PR8-NS1-GFP at an MOI of 0.001, taken at 24 h post-infection (hpi). Scale bars: 100 µm. ( M ) Histograms showing the percentage of GFP-positive cells upon IAV PR8-GFP infection, as in ( L ), quantified by flow cytometry. Data were mean ± SD, n = 5 biological replicates per group; two-tailed unpaired Student’s t -test. Representative results for ( B – M) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transduction, Luciferase, Control, Activity Assay, Construct, Infection, Flow Cytometry, Two Tailed Test

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Ifnb1 mRNA expression in Rraga KO cells reconstituted with FLAG-tagged WT, GTP-bound (Q66L), or GDP-bound (T21L) forms of mouse RagA after stimulation with PRR agonists. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls were shared across PRR stimulations, except for cGAMP. ( B ) IFN-β secretion from cells reconstituted with the WT, GTP-bound, and GDP-bound forms of mouse RagA after stimulation with PRR agonists, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls were shared across PRR stimulation conditions. ( C ) Ifnb1 mRNA expression in Rragc KO cells reconstituted with FLAG-tagged WT, GTP-bound (Q119L), or GDP-bound (S74N) forms of mouse RagC after stimulation with PRR agonists. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( D ) IFN-β secretion from cells reconstituted with the WT, GTP-bound, and GDP-bound forms of mouse RagC after stimulation with PRR agonists, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls in ( C , D ) were shared across PRR stimulations, except for cGAMP stimulation, which used separate mock controls to account for reagent lot variability. ( E ) Heatmap comparing the gene expression profiles of IRF family members between LAMTOR1 WT and 3A cells in response to PRR stimulation. ( F ) Immunoblot analysis of the indicated TFs in parental, Lamtor1 KO, WT, and 3A cells. ( G ) Immunoblot analysis of the indicated TFs in parental, Lamtor1 KO, Rraga KO, and Rragc KO cells. ( H , I ) Immunoblot analysis of the indicated TFs in Rraga KO ( H ) or Rragc KO cells ( I ) reconstituted with recombinant cDNAs encoding the corresponding WT, GTP-bound, or GDP-bound variants. Representative results for ( A – D , F – I ) from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot, Recombinant

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A , B ) Immunoblot analysis of ISGs in cells reconstituted with WT, GTP-bound ( A ), and GDP-bound ( B ) forms of mouse RagA in response to PRR stimulation. ( C , D ) Immunoblot analysis of ISGs in cells reconstituted with WT, GTP-bound ( C ), and GDP-bound ( D ) forms of mouse RagC in response to PRR stimulation. ( E – H ) Immunoblot analysis of TFEB phosphorylation in Rraga/c KO cells reconstituted with WT, GTP-bound ( E , G ), and GDP-bound ( F , H ) forms of mouse RagA/C in response to PRR stimulation. ( I ) Transcription factor (TF) expression in WT versus 3A cells. Volcano plots depict TF transcripts differentially expressed in 3A cells relative to WT, with or without PRR stimulation. Significant DEGs (FDR-adjusted P < 0.05, |fold change| ≥2; DESeq2) are indicated. n = 3 biological replicates per condition. ( J ) Immunoblot analysis of IFN signaling in parental, Lamtor1 KO, Rraga KO, and Rragc KO cells treated with 20 ng ml −1 recombinant IFN-β for 4 h. Cells were lysed and analyzed for the levels of the indicated proteins and phosphorylation status of Stat1 (Y701) and Stat1 (S727). ( K ) Immunoblot analysis of nutrient-dependent mTORC1 signaling in parental cells, as well as Lamtor1 KO, Rraga KO, and Rragc KO cells. The cells were deprived of amino acids for 120 min, or they were deprived for 100 min followed by amino acid refeeding for 20 min prior to harvest. ( L ) Ifnb1 mRNA expression in parental RAW cells after PRR stimulation in the absence or presence of amino acids. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( M ) Immunoblot analysis of innate immune signaling in cells starved for amino acids in response to PRR stimulation. ( N ) IFN-β secretion from parental RAW cells after PRR stimulation in the absence or presence of amino acids as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( O ) Immunoblot analysis of ISGs in amino acid-deprived cells in response to PRR stimulation. Representative results for ( A – O ) from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Phospho-proteomics, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Immunoblot analysis of IRF-3 activation in Lamtor1 KO cells transfected with Poly(I:C). Cells were lysed and analyzed for levels of the indicated proteins, phosphorylation status of IRF-3 (S396), and dimerization of IRF-3. ( B ) Nuclear translocation of IRF-3 in Lamtor1 KO cells transfected with Poly(I:C) as analyzed by nuclear-cytoplasmic fractionation. Tubulin was used as a cytosolic control, whereas PARP-1 was used as a nuclear control. ( C ) Ectopic expression of IRF5 and IRF7 in Lamtor1 KO cells. ( D , E ) Induction of Ifnb1 transcript ( D ) and IFN-β release ( E ) from parental, Lamtor1 KO, and Lamtor1 KO cells stably expressing IRF-5 and IRF-7 after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls in ( D , E ) were shared across PRR stimulations. ( F ) Schematic overview of the ATAC-seq workflow for chromatin accessibility analysis in WT and 3A cells in response to cGAMP stimulation, with n = 3 biological replicates per group. ( G ) Chromatin accessibility and functional enrichment of differentially accessible regions. Heatmaps display normalized ATAC-seq signal intensity across a 10 kb region (±5 kb) centered on genomic loci within the indicated clusters (left). GO term analysis of biological processes significantly associated with the ‘up’ and ‘stable’ clusters in 3A cells (right). Enrichment was calculated using rGREAT with a hypergeometric test (FDR <0.05). The top ten enriched terms are shown. ( H ) Coordinated transcriptional and chromatin remodeling upon LAMTOR loss. Genes concurrently identified by RNA-seq and ATAC-seq are shown in the Venn diagram (left). The biological processes most significantly associated with the upregulated 3A-specific gene cluster are listed (right). GO enrichment was calculated via hypergeometric distribution using Clusterfiler (FDR <0.05). ( I ) HOMER motif TF analysis of the indicated gene clusters. ( J ) ATAC-seq analysis highlighting accessible chromatin regions of Ifnb1 in WT and 3A cells under mock- and cGAMP-treated conditions. n = 3 biological replicates per group. ( K ) Schematic representation illustrating communal changes in chromatin accessibility of the indicated genes in the absence of LAMTOR. ( L ) Immunoblot analysis of phosphorylation status of ERK1/2 (T202/Y204), JNK (T183/Y185), and p38 (T180/Y182) in Lamtor1 KO, Rraga KO, and Rragc KO cells. ( M ) Immunoblot analysis of the phosphorylation status of the indicated MAPKs, ATF-2 (T71), and c-Jun (S73) in Rraga KO cells and Rragc KO cells reconstituted with WT or its variants. Representative results for ( A – E and L , M) from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Activation Assay, Transfection, Phospho-proteomics, Translocation Assay, Fractionation, Control, Expressing, Stable Transfection, Functional Assay, RNA Sequencing

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A , B ) Ifnb1 transcription ( A ) and IFN-β release ( B ) from parental, Mapk14 KO, and Mapk11 KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( C ) qRT-PCR analysis of Mapk11 transcript in parental, Mapk14 KO, and Mapk11 KO cells. ( D ) Immunoblot analysis of IFN signaling in parental, Mapk14 KO, and Mapk11 KO cells treated with 20 ng ml −1 recombinant IFN-β for 4 h. ( E ) Immunoblot analysis of p38 MAPK phosphorylation in parental and Mapk14/11 DKO cells following 4 h of stimulation with sorbitol (0.5 M) or sodium arsenite (250 μM). ( F ) Ifnb1 transcription (left) and IFN-β release (right) from parental, Mapk14/11 DKO, and Lamtor1 KO cells after 4 h of treatment with sorbitol, sodium arsenite, and LPS. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( G ) Ectopic expression of MAPK14 WT and its variants in parental RAW cells. ( H , I ) Ifnb1 transcription ( H ) and IFN-β release ( I ) from parental cells or cells expressing MAPK14 WT, D168A, or D176A/F327S after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( J ) Immunoblot analysis of phagolysosomes isolated from parental cells after PRR stimulation. ( K ) Representative micrographs showing abolished co-localization of mBaoJin-MAPK14 with LysoView-positive lysosomes in response to amino acid starvation. Scale bars: 10 µm. Representative results for ( A – K ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Quantitative RT-PCR, Western Blot, Recombinant, Phospho-proteomics, Expressing, Isolation

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Immunoblot analysis of p38 MAPK phosphorylation in Rragc KO, Flcn KO, and Depdc5 KO cells compared with parental cells. ( B ) Ifnb1 mRNA expression in parental, Flcn KO, and Depdc5 KO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls were shared across PRR stimulations, except for cGAMP. ( C ) IFN-β secretion from parental, Flcn KO, and Depdc5 KO cells after PRR stimulation, as measured by ELISA. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls were shared across PRR stimulations, except for cGAMP and Poly(I:C)/FuGENE. ( D ) Ectopic expression of FLAG-tagged RagA-GTP (Q66L) or RagC-GDP (S74N) in Mapk14/11 DKO cells. ( E ) Ifnb1 mRNA expression in Mapk14/11 DKO cells expressing FLAG-tagged RagA-GTP or RagC-GDP after PRR stimulation. Data are mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( F ) IFN-β secretion from Mapk14/11 DKO cells expressing FLAG-tagged RagA-GTP or RagC-GDP after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Mock-treatment controls in ( E , F ) were shared across PRR stimulations, except for cGAMP. ( G ) Schematic showing epistasis across the GATOR1 and FLCN-FNIP1/2 complex, RagA/C GTPases, and p38 MAPK. Representative results for ( A – F ) from two independent experiments. .

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: The lysosomal LAMTOR-Rag complex functions as a checkpoint for antiviral interferon production

doi: 10.1038/s44318-026-00695-2

Figure Lengend Snippet: ( A ) Immunoblot analysis of the phosphorylation status of S6K1, TFEB, and p38 in parental, Mapk14/11 DKO, Depdc5 KO, and Depdc5/Mapk14/11 TKO cells. ( B , C ) Ifnb1 transcription ( B ) and IFN-β release ( C ) from parental, Mapk14/11 DKO, Depdc5 KO, and Depdc5/Mapk14/11 TKO cells after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. ( D ) Ectopic expression of the constitutively active p38 (D176A/F327S) in Lamtor1 KO, Rraga KO, and Rragc KO cells. ( E , F ) Ifnb1 transcription ( E ) and IFN-β release ( F ) from parental, LAMTOR-Rag deficient cells or LAMTOR-Rag deficient cells stably expressing the constitutively active p38 after PRR stimulation. Data were mean ± SD, n = 3 biological replicates per group; two-way ANOVA followed by Tukey’s test, adjusted P value as indicated. Representative results for ( A – F ) from two independent experiments.

Article Snippet: Human IFN-β ELISA kit , Multi Sciences , EK1236.

Techniques: Western Blot, Phospho-proteomics, Expressing, Stable Transfection