Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

doi: 10.1073/pnas.2320591121

Figure Lengend Snippet: Fig. 2. DNA fragments enhance cGAS–LAMTOR1 interaction. (A) Silver staining of cGAS interactors by immunoprecipitating with M2 beads in HEK 293T cells treated with 5-FU (100 μM) or DMSO. (B) Overlapped proteins of annotated Lysosome gene sets and cGAS interactors whose amounts increased upon 5-FU (100 μM) treatment (ratio > 1.5). (C) Endogenous coimmunoprecipitation of cGAS and LAMTOR1 in HeLa cells. (D) GST pulldown assay of cGAS-Flag and GST- LAMTOR1 with full-length or different truncates as indicated. (E) GST pulldown assay of LAMTOR1-Flag and GST-cGAS with full-length or different truncates as indicated. (F and G) Representative images (F) and quantification (G) of colocalization for cGAS and HA (LAMTOR1) in HeLa cells treated with DMSO or 5-FU (100 μM) for indicated days. Scale bars, 5 μm. n = 4; one-way ANOVA was used. (H and I) Representative images (H) and quantification (I) of immunofluorescent imaging for cGAS and Flag (LAMTOR1) in HeLa cells transfected with HT-DNA (1 μg/mL) for 8 h. Scale bars, 10 μm. n = 4; a two-tailed unpaired t test was used. (J) Coimmunoprecipitation of cGAS-HA and LAMTOR1-Flag in HEK 293T upon treatment with 5-FU (100 μM), etoposide (20 μM), or DMSO for indicated days. NS, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001. Error bars represent SEM.

Article Snippet: His- tagged cGAS was purchased from MedChemExpress (HY- P72337) and His- tagged LAMTOR1 was purchased from CUSABIO (CSB- EP757083HU).

Techniques: Silver Staining, GST Pulldown Assay, Imaging, Transfection, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

doi: 10.1073/pnas.2320591121

Figure Lengend Snippet: Fig. 3. LAMTOR1 mediates DNA fragments-induced cGAS degradation. (A) Immunoblot analysis of cGAS in LAMTOR1 knockdown 4T1 tumors treated with 5-FU (100 mg/kg) or vehicle. (B) Immunoblot analysis of cGAS in LAMTOR1 knockdown HeLa cells exposed to 5-FU (100 μM) for indicated days. (C) Immunoblot analysis of cGAS in LAMTOR1 knockdown and control MC38 (above) or HeLa (below) cells. (D) Immunoblot analysis of cGAS in HeLa (Left) and MCF-7 (Right) cells transfected with gradient amounts of LAMTOR1-Flag plasmids. (E and F) Immunoblot analysis (E) and quantification (F) of cGAS in LAMTOR1 knockdown MC38 cells treated with cycloheximide (CHX) (25 μg/mL) for indicated hours. n = 3; one-way ANOVA was used. (G) Lysosome isolation and immunoblot analysis of cGAS and LAMTOR1 in TMEM192-3HA stably expressed MC38 tumors treated with 5-FU (100 mg/kg) or vehicle for three times and released for indicated days. (H) qPCR analysis of LAMTOR1 mRNA in 4T1 tumors treated with 5-FU (100 mg/kg) or vehicle. n = 6; a two-tailed unpaired t test was used. (I) qPCR analysis of LAMTOR1 mRNA in MC38 tumors treated with 5-FU (100 mg/kg) or vehicle. n = 6; a two-tailed unpaired t test was used. (J) qPCR analysis of cGAS mRNA in rectal tumors before or after chemotherapy. n = 6; a two-tailed unpaired t test was used. (K) Immunoblot analysis of cGAS and LAMTOR1 in B16F10 (Left) and HeLa (Right) cells treated with 5-FU (100 μM) for indicated days. (L and M) Representative images (L) and statistical analysis (M) of immunohistochemical staining for LAMTOR1 and cGAS in the same clinical colorectal cancer samples. Scale bars, 5×, 600 μm and 40×, 75 μm. n = 55. (N) Immunoblot analysis of cGAS and LAMTOR1 in clinical colorectal tumors. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

Article Snippet: His- tagged cGAS was purchased from MedChemExpress (HY- P72337) and His- tagged LAMTOR1 was purchased from CUSABIO (CSB- EP757083HU).

Techniques: Western Blot, Knockdown, Control, Transfection, Isolation, Stable Transfection, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

doi: 10.1073/pnas.2320591121

Figure Lengend Snippet: Fig. 4. LAMTOR1 targets cGAS for selective autophagy via p62. (A) Immunoblot analysis of cGAS in HeLa cells transfected with gradient amounts of LAMTOR1- Flag and after 24 h cells were treated with Bafilomycin A1 (Baf A1, 200 nM) for another 12 h. (B and C) Representative images (B) and quantification (C) of immunofluorescent imaging for cGAS and lysosome (Lysotracker) in living HeLa cells transfected with LAMTOR1-HA or vector for 36 h. Scale bars, 5 μm. n = 4; a two-tailed unpaired t test was used. (D) Lysosome isolation and immunoblot analysis in TMEM192-3HA stably expressed HeLa cells transfected with LAMTOR1- Flag or vector plasmids. (E) Overlapped proteins of LAMTOR1 interactors and cGAS interactors. (F) Immunoprecipitation of Flag-p62 and immunoblot analysis of cGAS-HA in HEK 293T cells transfected with cGAS-HA and Flag-p62 together with gradient amount LAMTOR1-HA. (G and H) Representative images (G) and quantification (H) of immunofluorescent imaging for cGAS and p62 in HeLa cells transfected with LAMTOR1-HA or vector for 36 h. Scale bars, 5 μm. n = 4; a two-tailed unpaired t test was used. (I) Immunoprecipitation of Flag-p62 and immunoblot analysis of endogenous cGAS in LAMTOR1 knockdown HeLa cells transfected with Flag-p62. (J) Immunoblot analysis of cGAS in p62 knockdown HeLa cells transfected with gradient amounts of LAMTOR1-Flag. (K) Immunoblot analysis of cGAS in p62 knockdown MC38 tumors treated with 5-FU (100 mg/kg) or vehicle.

Article Snippet: His- tagged cGAS was purchased from MedChemExpress (HY- P72337) and His- tagged LAMTOR1 was purchased from CUSABIO (CSB- EP757083HU).

Techniques: Western Blot, Transfection, Imaging, Plasmid Preparation, Two Tailed Test, Isolation, Stable Transfection, Immunoprecipitation, Knockdown

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

doi: 10.1073/pnas.2320591121

Figure Lengend Snippet: Fig. 5. Loss of LAMTOR1 enhances the cGAS–STING–IFN-β pathway. (A) qPCR analysis of IFN-β mRNA by in HeLa cells transfected with LAMTOR1-Flag or vector for 24 h followed by transfection with HT-DNA (1 μg/mL) for another 12 h. n = 3; a two-tailed unpaired t test was used. (B) Immunoblot analysis of cGAS signaling in HeLa cells transfected with LAMTOR1-Flag or vector for 24 h followed by transfection with HT-DNA (1 μg/mL) for indicated hours. (C) qPCR analysis of IFN-β (Left) and CXCL10 (Right) mRNA in LAMTOR1 knockdown MC38 cells transfected with HT-DNA (1 μg/mL) for 12 h. n = 3; one-way ANOVA was used. (D) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown HeLa cells transfected with HT-DNA (1 μg/mL) for 6 h. (E) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown B16F10 cells transfected with HT-DNA (1 μg/mL) for indicated hours. (F) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown HeLa cells treated with 5-FU (100 μM) for 24 h and released 24 h. (G) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown B16F10 cells treated with 5-FU (100 μM) for 24 h and released 24 h. (H) qPCR analysis of IFN-β mRNA in LAMTOR1 knockdown HeLa cells treated with 5-FU (100 μM), etoposide (20 μM), or cDDP (4 μM) for 24 h. n = 3; one-way ANOVA was used. (I) qPCR analysis of IFN-β mRNA in LAMTOR1 knockdown B16F10 cells treated with 5-FU (100 μM), etoposide (20 μM), or cDDP (4 μM) for 24 h. n = 3; one-way ANOVA was used. (J and K) Representative images (J) and statistical analysis (K) of immunofluorescent imaging of cGAS foci in LAMTOR1 knockdown HeLa cells transfected with Cy5-ISD (2 μg/mL) for 8 h. Scale bars, 20 μm. n = 4; one-way ANOVA was used. (L) Immunoblot analysis of cGAS signaling in cGAS KO together with LAMTOR1 knockdown HeLa cells transfected with HT-DNA (1 μg/mL) for 6 h. (M) Quantitative PCR assay of IFN-β (Left) and CXCL10 (Right) mRNA in HeLa cells were transfected with HT-DNA (1 μg/mL) for 12 h and treated with chloroquine (CQ, 100 μM) or Bafilomycin A1 (Baf A1, 200 nM) for another 8 h. n = 3; one-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent SEM.

Article Snippet: His- tagged cGAS was purchased from MedChemExpress (HY- P72337) and His- tagged LAMTOR1 was purchased from CUSABIO (CSB- EP757083HU).

Techniques: Transfection, Plasmid Preparation, Two Tailed Test, Western Blot, Knockdown, Imaging, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

doi: 10.1073/pnas.2320591121

Figure Lengend Snippet: Fig. 6. LAMTOR1 promotes tumor growth via impeding antitumor immunity. (A and B) Tumor volume (A) and tumor weight (B) of LAMTOR1 knockdown B16F10 tumors. n = 6; one-way ANOVA was used. (C) The survival time of C57BL/6J mice inoculated with LAMTOR1 knockdown B16F10 cells. n = 7, log-rank (Mantel-Cox) test was used. (D) ELISA of cGAMP in LAMTOR1 knockdown B16F10 tumors after 20 d post inoculation. n = 6; one-way ANOVA was used. (E and F) Tumor volume (E) and tumor weight (F) of LAMTOR1 knockdown B16F10 tumors together with cGAS knock-out. n = 6; one-way ANOVA was used. (G) LAMTOR1 expression level in normal and tumor tissue in COAD and READ by bioinformatics analysis of TCGA and GTEx database. A two-tailed unpaired t test was used. (H and I) Representative image (H) and statistical analysis (I) of immunohistochemical staining for LAMTOR1 in the paired cancer and paracancer tissues. Scale bars, 5×, 500 μm and 40×, 50 μm. n = 58. (J and K) Representative images (J) and quantification (K) of immunofluorescent imaging for markers of effective T lymphocytes, CD3, CD4, or CD8 in LAMTOR1 knockdown B16F10 tumors after 20 d post inoculation. Scale bars, 50 μm. n = 8-12; one-way ANOVA was used. (L) Quantification of infiltrating CD3+, CD4+, CD8+ T cells in LAMTOR1 knockdown B16F10 tumors via flow cytometry after 20 d post inoculation. n = 6; one-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent SEM.

Article Snippet: His- tagged cGAS was purchased from MedChemExpress (HY- P72337) and His- tagged LAMTOR1 was purchased from CUSABIO (CSB- EP757083HU).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Knock-Out, Expressing, Two Tailed Test, Immunohistochemical staining, Staining, Imaging, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet:

Article Snippet: cGAMP disodium , MCE , Cat# HY-110385.

Techniques: Virus, Recombinant, Negative Staining, Lysis, Control, Phospho-proteomics, Cell Isolation, DNA Purification, DNA Extraction, Bicinchoninic Acid Protein Assay, Protein Purification, Magnetic Beads, Software

Journal: iScience

Article Title: Global feather orientations changed by electric current

doi: 10.1016/j.isci.2021.102671

Figure Lengend Snippet: List of channel inhibitors

Article Snippet: Tetrodotoxin , Tocris , Compound CID: 5946; Cat. No. 1078.

Techniques: Blocking Assay, Inhibition

Journal: iScience

Article Title: Global feather orientations changed by electric current

doi: 10.1016/j.isci.2021.102671

Figure Lengend Snippet:

Article Snippet: Tetrodotoxin , Tocris , Compound CID: 5946; Cat. No. 1078.

Techniques: Recombinant, Software

Journal: bioRxiv

Article Title: Targeted Lipid Metabolism Screening Uncovers Regulatory Effects on the STING Immune Response in Mevalonate, Eicosanoid and Fatty Acid Pathways

doi: 10.64898/2026.01.05.697750

Figure Lengend Snippet: A) iBMDMs were pretreated for three hours either with vehicle (DMSO) or FATOSTATIN A (10µM) followed by stimulation with cGAMP (10µg/ml) added to culture media for an additional 3 hours. Total RNA was harvested for quantification of IFN-β and IP10 mRNA by qRT-PCR. B) iBMDMs were pretreated for three hours either with vehicle (DMSO) or FATOSTATIN A (10µM) followed by stimulation with synthetic TLR2/1 agonist Pam3CSK4 (100 ng/ml) added to culture media for an additional 3 hours. Total RNA was harvested for quantification of IL-6 and TNF mRNA by qRT-PCR. C) iBMDMs were pretreated for three hours either with vehicle (DMSO) or FATOSTATIN A (10µM) followed by stimulation with recombinant TNF (100 ng/ml) added to culture media for an additional 3 hours. Total RNA was harvested for quantification of IL-6 and TNF mRNA by qRT-PCR. N = 3. *p<.05.

Article Snippet: 3’,2’-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (cGAMP) was purchased from Tocris (cat no. 7718).

Techniques: Quantitative RT-PCR, Recombinant