crispr cas9 plasmid map  (TaKaRa)

Journal: BMC infectious diseases

Article Title: Exploring Candesartan, an angiotensin II receptor antagonist, as a novel inhibitor of NLRP3 inflammasome: alleviating inflammation in Neisseria gonorrhoeae infection.

doi: 10.1186/s12879-024-10208-3

Figure Lengend Snippet: Fig. 6 Effect of autophagy on CS-mediated NLRP3 Inflammasome inhibition in N. gonorrhoeae-infected macrophages. In panel A, J774A.1 macrophages were pre-incubated with CS or a vehicle in the presence or absence of 5 mM 3-MA for 0.5 h before N. gonorrhoeae infection, followed by an additional 24- hour incubation period. Levels of IL-1β in the supernatants were measured through ELISA. In panel B, wild-type or LC3-knockdown J774A.1 macrophages were incubated with 100 nM rapamycin or a vehicle for 4 h, and the levels of LC3 in the cell lysates were assessed via Western blotting. In panels C-E, wild- type or LC3-knockdown J774A.1 macrophages were pre-incubated with either CS or a control vehicle for 0.5 h before N. gonorrhoeae infection, followed by an additional 24-hour incubation period. The concentrations of IL-1β (C) and IL-6 (E) in the supernatants were measured through ELISA, while the levels of caspase-1 in the supernatants were assessed through Western blotting (D). The ELISA data are presented as the mean ± SD of three independent experiments. The presented Western blotting images represent individual experiments. Statistical significance is indicated as ***p < 0.001 as indicated

Article Snippet: Antibodies for ASC (SC-22514-R), inducible nitric oxide synthase (iNOS) (sc-7271), cyclooxygenase-2 (COX2) (sc-19999), actin (SC-47778) and LC3 CRISPR/Cas9 knockout plasmids (sc-426563 and sc-417828-HDR) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Control

Journal: Journal of Inflammation (London, England)

Article Title: Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei -infected macrophages

doi: 10.1186/s12950-024-00395-w

Figure Lengend Snippet: Role of autophagy in CA-mediated IL-1β inhibition in S. sonnei -infected macrophages. ( A-C ) J774A.1 macrophages were incubated with 40 µM CA for 4–20 h or 1 µM rapamycin for 4 h. The expression levels of LC3 ( A ), ATG5 ( B ) and p62 ( C ) in the cell lysates were analysed by Western blotting. ( D ) J774A.1 macrophages were primed for 4 h with LPS in the presence or absence of 1 mM 3-MA, followed by incubation with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. ( E ) Wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 20 h. The expression levels of LC3 in the cell lysates were analysed by Western blotting. ( F ) LPS-primed wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. The Western blotting images are representative of separate experiments. The ELISA data are expressed as the means ± SD of three separate experiments. * p < 0.05 and *** p < 0.001 as indicated

Article Snippet: Mouse LC3 CRISPR/Cas9 knockout plasmids (sc-426,563 and sc-417,828-HDR) and antibodies against IL-18 (SC-6177), ASC (SC-22,514-R), cathepsin B (SC-365,558) and actin (SC-47,778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Infection, Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cell reports

Article Title: Endothelial p130cas confers resistance to anti-angiogenesis therapy

doi: 10.1016/j.celrep.2022.110301

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MAP-LC3β2 GRISPR/Gas9 knockout plasmids (Human) , Santa Cruz Biotechnology , Cat# sc-417828, sc-417828-HDR.

Techniques: Recombinant, Plasmid Preparation, Knock-Out, Gene Knockout, CRISPR, Control, esiRNA, shRNA, Construct, Software

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B induces autophagy. (A, B) J774A.1 macrophages were incubated for 3–24 h with 0.3 µM F240B or for 4 h with 0.1 µM rapamycin. The levels of LC3 (A) and p62 (B) in the lysates were measured by Western blotting. (C) J774A.1 macrophages were incubated for 3 h with 0.1–1 µM F240B or for 4 h with 0.1 µM rapamycin. The levels of p62 in the lysates were measured by Western blotting. (D) GFP-LC3 expressed J774A.1 macrophages were incubated for 3 h with 1 µM F240B or for 4 h with 0.1 µM rapamycin. The LC3-GFP speck formation was measured by confocal microscopy. (E) J774A.1 macrophages were incubated for 3 h with 1 µM F240B. The cells were stained with 50 nM MDC or 1 µg/ml AO and the fluorescent signals were acquired by confocal microscopy.

Article Snippet: Antibodies against Actin (sc-47778), ASC (sc-25514-R), cyclooxygenase-2 (COX-2) (sc-376861), phospho-IκB-α (sc-8404) and CRISPR/Cas9 knockout plasmids targeting LC3 (sc-426563 and sc-417828-HDR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Western Blot, Confocal Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits the NLRP3 inflammasome through autophagy induction. (A, B) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The levels of IL-1β and caspase-1 (A) or NLRP3 and ASC (B) in the supernatants were measured by Western blotting. (C) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B in the presence or absence of 5 mM 3-MA for 3 h. Cells then were incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1β in the supernatants were measured by ELISA. (D) Wild-type and LC3-knockout J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1β in the supernatants were measured by ELISA. The data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Article Snippet: Antibodies against Actin (sc-47778), ASC (sc-25514-R), cyclooxygenase-2 (COX-2) (sc-376861), phospho-IκB-α (sc-8404) and CRISPR/Cas9 knockout plasmids targeting LC3 (sc-426563 and sc-417828-HDR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Activation of autophagy by F240B inhibits NF-κB activation and proIL-1β expression. (A) J774A.1 macrophages were incubated with F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 6 h. The levels of proIL-1β and NLRP3 in the cell lysates were measured by Western blotting. (B) J-Blue cells were incubated with F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 24 h. The NF-κB transcriptional activity was measured by NF-κB reporter assay. (C, D) J774A.1 macrophages were incubated with F240B (1 µM for ROS assay) for 0.5 h followed by incubated with 1 µg/ml LPS for 10 min. The phosphorylation levels of IκBα in the cell lysates were measured by Western blotting (C) , and the intracellular ROS production was analysed by H 2 DCFDA staining (D) . (E) J774A.1 macrophages were incubated with 1 µM F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 10-30 min. The phosphorylation levels of ERK1/2, JNK1/2 and p38 in the cell lysates were measured by Western blotting. (F) Will-type or LC3-knockout J774A.1 macrophages were incubated with1 µM F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 6 h. The levels of proIL-1β in the cell lysates were measured by Western blotting. ** and *** indicate a significant difference at the level of p < 0.01 and p < 0.001, respectively compared to LPS.

Article Snippet: Antibodies against Actin (sc-47778), ASC (sc-25514-R), cyclooxygenase-2 (COX-2) (sc-376861), phospho-IκB-α (sc-8404) and CRISPR/Cas9 knockout plasmids targeting LC3 (sc-426563 and sc-417828-HDR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Activity Assay, Reporter Assay, ROS Assay, Phospho-proteomics, Staining, Knock-Out

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Activation of autophagy by F240B limits mitochondrial integrity loss. (A) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B in the presence or absence of 5 mM 3-MA for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. (B) Wild-type or LC3-knockout J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The mitochondrial membrane integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green. The percentage of cells with mitochondrial membrane integrity loss are expressed as the mean ± SD of three separate experiments. * and ** indicate a significant difference at the level of p < 0.05 and p < 0.01, respectively.

Article Snippet: Antibodies against Actin (sc-47778), ASC (sc-25514-R), cyclooxygenase-2 (COX-2) (sc-376861), phospho-IκB-α (sc-8404) and CRISPR/Cas9 knockout plasmids targeting LC3 (sc-426563 and sc-417828-HDR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Knock-Out, Membrane, Staining