Journal: Frontiers in Microbiology

Article Title: Antimycobacterial and Anti-inflammatory Mechanisms of Baicalin via Induced Autophagy in Macrophages Infected with Mycobacterium tuberculosis

doi: 10.3389/fmicb.2017.02142

Figure Lengend Snippet: Baicalin can promote the activation of autophagy in Mtb-infected macrophages. (A) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected Raw264.7 cells after treatment with different concentrations of baicalin (0, 12.5, 25, 50, 100 μM) or rapamycin (1 μg/ml) for 12 h. The right bar graphs show the statistical results for the relative quantitative expression of LC3 II and p62. (B) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected Raw264.7 cells. After different times of Mtb infection (0, 4, 12, 24 h), cells were treatment with or without baicalin (100 μM). The right bar graphs showed the statistical results for the relative quantitative expression of LC3 II and p62. ( C) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected Raw264.7 cells after treatment with baicalin (100 μM) or CQ (10, 20 μM) for 12 h. The right bar graphs showed the statistical results for the relative quantitative expression of LC3 II and p62 expression. Data are shown with the means ± SD of at least three independent experiments. * p < 0.05.

Article Snippet: The following antibodies were used: anti-NLRP3 (cat. #15101), anti-IL-1β (cat. #12507), anti-ASC (cat. #67824), anti-LC3 (cat. #2775), anti-p62 (cat. #5114), anti-Akt (cat. #4691), anti-phosphorylated Akt (Ser473) (cat. #4060), anti-mTOR (cat. #2972), anti-phosphorylated mTOR (Ser2448) (cat. #5536), anti-phosphorylated p38 (cat. #4511), anti-phosphorylated JNK (cat. #4668), and anti-phosphorylated ERK1/2 (cat. #4370) were purchased from Cell Signaling Technology, Inc. (CST, Danvers, MA, USA); rabbit anti-caspase-1 (cat. #sc-514), goat anti-rabbit (cat. #sc-2012), donkey anti-goat (cat. #sc-2094), and goat anti-mouse LC3 (cat. #sc-16755) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz CA, USA); donkey anti-rabbit IgG H&L antibody (conjugated with Alexa Fluor® 488; ab150073) and donkey anti- goat IgG H&L antibody (conjugated with Alexa Fluor® 647; ab150131) were acquired from Abcam (Cambridge, UK); anti-β-actin (cat. #66009-1-lg) monoclonal antibody was from ProteinTech Group (Chicago, IL).

Techniques: Activation Assay, Infection, Western Blot, Expressing

Journal: Frontiers in Microbiology

Article Title: Antimycobacterial and Anti-inflammatory Mechanisms of Baicalin via Induced Autophagy in Macrophages Infected with Mycobacterium tuberculosis

doi: 10.3389/fmicb.2017.02142

Figure Lengend Snippet: Baicalin inhibits the expression of Mtb-induced NLRP3 in primary peritoneal macrophage cells and chloroquine (CQ) prevented this effect. The expression of NLRP3, LC3 I/II and p62 in Mtb-infected primary peritoneal macrophage cells after treatment with different concentrations of baicalin or CQ (10, 20 μM) was detected by Western blot. The concentration of baicalin when used in combination with CQ was 100 μM.

Article Snippet: The following antibodies were used: anti-NLRP3 (cat. #15101), anti-IL-1β (cat. #12507), anti-ASC (cat. #67824), anti-LC3 (cat. #2775), anti-p62 (cat. #5114), anti-Akt (cat. #4691), anti-phosphorylated Akt (Ser473) (cat. #4060), anti-mTOR (cat. #2972), anti-phosphorylated mTOR (Ser2448) (cat. #5536), anti-phosphorylated p38 (cat. #4511), anti-phosphorylated JNK (cat. #4668), and anti-phosphorylated ERK1/2 (cat. #4370) were purchased from Cell Signaling Technology, Inc. (CST, Danvers, MA, USA); rabbit anti-caspase-1 (cat. #sc-514), goat anti-rabbit (cat. #sc-2012), donkey anti-goat (cat. #sc-2094), and goat anti-mouse LC3 (cat. #sc-16755) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz CA, USA); donkey anti-rabbit IgG H&L antibody (conjugated with Alexa Fluor® 488; ab150073) and donkey anti- goat IgG H&L antibody (conjugated with Alexa Fluor® 647; ab150131) were acquired from Abcam (Cambridge, UK); anti-β-actin (cat. #66009-1-lg) monoclonal antibody was from ProteinTech Group (Chicago, IL).

Techniques: Expressing, Infection, Western Blot, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Antimycobacterial and Anti-inflammatory Mechanisms of Baicalin via Induced Autophagy in Macrophages Infected with Mycobacterium tuberculosis

doi: 10.3389/fmicb.2017.02142

Figure Lengend Snippet: Baicalin promotes co-localization of inflammasome with autophagosome. Confocal microscopy of Raw264.7 cells with different treatments immunostained with anti-LC3 antibody (pink), anti-ASC antibody (green), and DAPI (blue). Scale bars shown are 5 μm.

Article Snippet: The following antibodies were used: anti-NLRP3 (cat. #15101), anti-IL-1β (cat. #12507), anti-ASC (cat. #67824), anti-LC3 (cat. #2775), anti-p62 (cat. #5114), anti-Akt (cat. #4691), anti-phosphorylated Akt (Ser473) (cat. #4060), anti-mTOR (cat. #2972), anti-phosphorylated mTOR (Ser2448) (cat. #5536), anti-phosphorylated p38 (cat. #4511), anti-phosphorylated JNK (cat. #4668), and anti-phosphorylated ERK1/2 (cat. #4370) were purchased from Cell Signaling Technology, Inc. (CST, Danvers, MA, USA); rabbit anti-caspase-1 (cat. #sc-514), goat anti-rabbit (cat. #sc-2012), donkey anti-goat (cat. #sc-2094), and goat anti-mouse LC3 (cat. #sc-16755) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz CA, USA); donkey anti-rabbit IgG H&L antibody (conjugated with Alexa Fluor® 488; ab150073) and donkey anti- goat IgG H&L antibody (conjugated with Alexa Fluor® 647; ab150131) were acquired from Abcam (Cambridge, UK); anti-β-actin (cat. #66009-1-lg) monoclonal antibody was from ProteinTech Group (Chicago, IL).

Techniques: Confocal Microscopy

Journal: Oncotarget

Article Title: Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells

doi: 10.18632/oncotarget.17069

Figure Lengend Snippet: (A) PON1 protein expression in different lung normal (L132) or cancer cell lines (A549, H460, H1299, H358) including another normal cell line (HEK-293). (B) Western blot analysis of nuclear and cytosolic cell extracts (50 μg of each fraction). (C) Live A549 and A549-shPON1 cells stained with reduced FITC (pseudocolored in green) and DAPI. Cells were analyzed by confocal microscopy. Scale bars = 40 μm or 2 μm as indicated. (D, E) Stable overexpression and knockdown of PON1 by lentiviral-induced systems were examined by Western blotting. In PON1 overexpression system (H460- and H1299-PON1), two isoforms of stable PON1 overexpression (#1 and #2, respectively) differ in lentiviral particles used for transfection. (F) Immunoprecipitation of cells with anti-human PON1 antibody. Representative examples of three independent experiments are shown. All experiments were conducted at least in triplicate and biologically repeated at least twice. Statistical analysis was performed using unpaired Student's t test.

Article Snippet: A549, H358, H460, and H1299 cells (ATCC CCL-185, CRL-5807, HTB-177, and CRL-5803 respectively; obtained in 2012; passage 2 to 15 after obtaining except for cell senescence experiment) and Lewis lung cancer (LLC) cells (ATCC CLR-1642; obtained in 2011; passage 4 to 12) were all grown in RPMI (Invitrogen) with the same supplementation and cell culture procedure as mentioned above.

Techniques: Expressing, Western Blot, Staining, Confocal Microscopy, Over Expression, Knockdown, Transfection, Immunoprecipitation

Journal: Oncotarget

Article Title: Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells

doi: 10.18632/oncotarget.17069

Figure Lengend Snippet: (A, B) Invasion and migration counts per field of PON1-overexpressing H460-PON1 and H1299-PON1 cells versus vector control cells for 24 h. (C, D) Invasion and migration counts per field of PON1-knocked down A549-shPON1 cells versus shControl cells for 24 h. (E) Scratch-wound healing ability of cells after pre-exposure to 0.01% DMSO, 10 mM NAC, or 10 mM NAC in combination with 10 μM Akt-inhibitor LY294002. Representative bar graphs show the width in μm of scratch recorded. Representative migration micrographs are shown taken using phase-contrast microscope. (F, G) Western blot analysis of whole cell lysates. Cells were treated with 0.01% DMSO, 10 mM ROS-inhibitor NAC, 1 mM ROS-stimulators H 2 O 2 and 7 mM FeSO 4 . Cells were treated with or without combination of NAC and Akt-inhibitor LY294002 for G. All experiments were conducted at least in triplicate and biologically repeated at least twice. Statistical analysis was performed using unpaired Student's t test.

Article Snippet: A549, H358, H460, and H1299 cells (ATCC CCL-185, CRL-5807, HTB-177, and CRL-5803 respectively; obtained in 2012; passage 2 to 15 after obtaining except for cell senescence experiment) and Lewis lung cancer (LLC) cells (ATCC CLR-1642; obtained in 2011; passage 4 to 12) were all grown in RPMI (Invitrogen) with the same supplementation and cell culture procedure as mentioned above.

Techniques: Migration, Plasmid Preparation, Control, Microscopy, Western Blot

Journal: Oncotarget

Article Title: Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells

doi: 10.18632/oncotarget.17069

Figure Lengend Snippet: (A) Immunoblot detection of PON1 from whole cell lysates after glucose-enrichment in the media with indicated concentrations for 24 h. (B) Cells were serum-starved, glucose-starved, or glucose-enriched prior to Western blotting with PON1 antibody. (C) Homogenous detection of oxidized and reduced NAD+ and NADPH in H460-PON1, H1299-PON1, A549-shPON1, and their respective controls as indicated after the transfected cells have been exposed to the indicated glucose-deprivation periods. (D) ROS production after cells were either enriched with glucose or glucose-deprived. (E) Immunoblotting and immunoprecipitation of cells after glucose-enrichment or glucose-deprivation. Three independent experiments were summarized. Symbols represent mean ± S.E.M. n = 3~7; n.s., not significant; * P <0.05; ** P <0.01.

Article Snippet: A549, H358, H460, and H1299 cells (ATCC CCL-185, CRL-5807, HTB-177, and CRL-5803 respectively; obtained in 2012; passage 2 to 15 after obtaining except for cell senescence experiment) and Lewis lung cancer (LLC) cells (ATCC CLR-1642; obtained in 2011; passage 4 to 12) were all grown in RPMI (Invitrogen) with the same supplementation and cell culture procedure as mentioned above.

Techniques: Western Blot, Transfection, Immunoprecipitation