Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

doi: 10.3389/fcimb.2015.00067

Figure Lengend Snippet: Flow cytometry analyses for GAPDH expression on the surface of P. blaschkeae (SAG 2064), P. zopfii GT2 (SAG 2021), and P. zopfii GT1 (SAG 2063) . A representative image is shown. Cells in suspension were incubated with anti-GAPDH polyclonal antibodies, followed by incubation with Alexa-Fluor 488-labeled goat anti-rabbit IgG. As controls, cells were incubated only with the Alexa-Fluor 488-labeled goat anti-rabbit IgG to exclude any background. The experiment was repeated three independent times and results of one representative experiment are shown.

Article Snippet: Cells were incubated with 5–10 μg/ml polyclonal anti-GAPDH (Rockland, Limerick, USA) for 1 h at 37°C.

Techniques: Flow Cytometry, Expressing, Incubation, Labeling

Journal: Cancer Prevention Research

Article Title: Curcumin: A Double Hit on Malignant Mesothelioma

doi: 10.1158/1940-6207.capr-13-0259

Figure Lengend Snippet: Figure 1. Curcumin induced NLRP3 priming and caspase-1 activation in mouse malignant mesothelioma cells without proinflammatory cytokine processing. A, MTS assays revealed that curcumin inhibited mouse malignant mesothelioma cell growth in a manner that was dose and time dependent. Mouse malignant mesothelioma cells treated with curcumin (40 mmol/L for 48 hours) resulted in increased mRNA levels of NLRP3 (B) compared with control malignant mesothelioma cells, which was inhibited by actinomycin D pretreatment for 1 hour (C). Pro-IL-1b mRNA levels were not significantly altered by curcumin (D), whereas ASC mRNA levels were significantly decreased (E). Caspase-1 activation (F) and HMGB-1 release (G) significantly increased in mouse malignant mesothelioma cells in response to curcumin (20 and 40 mmol/L for 48 hours). , P 0.05 when compared with control; †, P 0.05 when compared with curcumin-only treatment group.

Article Snippet: The amount of proteinwas determined using the RCDCProtein Assay (Bio-Rad).Western blotswere performed as described previously (23), using monoclonal rabbit NLRP3 antibodies (Novus Biologicals) at a dilution of 1:500 and amouse b-Actin antibody (Abcam) at a dilution of 1:2,000.Quantity One was used to quantify band density.

Techniques: Activation Assay, Control

Journal: Cancer Prevention Research

Article Title: Curcumin: A Double Hit on Malignant Mesothelioma

doi: 10.1158/1940-6207.capr-13-0259

Figure Lengend Snippet: Figure 2. NLRP3 and ASC inhibition did not attenuate curcumin- induced caspase-1 activation. Mouse malignant mesothelioma cells transfected with siNLRP3 or siASC suppressed NLRP3 (A) and ASC (C) mRNA levels, however did not inhibit caspase-1 activity (B and D) when treated with curcumin (40 mmol/L for 24 hours). , P 0.05 when compared with control; †, P 0.05 when compared with siCon þ curcumin.

Article Snippet: The amount of proteinwas determined using the RCDCProtein Assay (Bio-Rad).Western blotswere performed as described previously (23), using monoclonal rabbit NLRP3 antibodies (Novus Biologicals) at a dilution of 1:500 and amouse b-Actin antibody (Abcam) at a dilution of 1:2,000.Quantity One was used to quantify band density.

Techniques: Inhibition, Activation Assay, Transfection, Activity Assay, Control

Journal: Cancer Prevention Research

Article Title: Curcumin: A Double Hit on Malignant Mesothelioma

doi: 10.1158/1940-6207.capr-13-0259

Figure Lengend Snippet: Figure 3. Curcumin mediates NLRP3 priming and caspase-1 activation without proinflammatory cytokine secretion in human malignant mesothelioma cells. MTS assays revealed that curcumin inhibited HMESO (A), H2461 (B), and H2595 (C) cell growth in a manner that was dose and time dependent. HMESO cells treated with curcumin (40 mmol/L for 48 hours) resulted in increased mRNA levels (D) and protein levels (I) of NLRP3 compared with control malignant mesothelioma cells. Pretreatment with actinomycin D 1 hour before curcumin treatment inhibited curcumin-induced increased mRNA levels of NLRP3 (E). Caspase-1 activation was significantly increased in response to curcumin (40 mmol/L for 48 hours) when measured by caspase-1 activity assay (F) and Western blot analysis of p20 (J) without significant change in secretion of IL-1b (G) and IL-18 (H). Curcumin treatment (40 mmol/L for 48 hours) resulted in increased HMBG1 release in HMESO cells compared with control cells (K). , P value 0.05 when compared with control. †, P < 0.05 when compared with curcumin-only treatment group.

Article Snippet: The amount of proteinwas determined using the RCDCProtein Assay (Bio-Rad).Western blotswere performed as described previously (23), using monoclonal rabbit NLRP3 antibodies (Novus Biologicals) at a dilution of 1:500 and amouse b-Actin antibody (Abcam) at a dilution of 1:2,000.Quantity One was used to quantify band density.

Techniques: Activation Assay, Control, Activity Assay, Western Blot

Journal: Cancer Prevention Research

Article Title: Curcumin: A Double Hit on Malignant Mesothelioma

doi: 10.1158/1940-6207.capr-13-0259

Figure Lengend Snippet: Figure 5. Curcumin-induced pyroptosis is ROS dependent. Curcumin treatment (40 mmol/L for 6 hours) resulted in significantly increased ROS in HMESO cells (A) compared with control cells, which was attenuated by NAC pretreatment (40 mmol/L for 18 hours; B). Pretreatment with NAC (40 mmol/L for 18 hours) blocked increased NLRP3 mRNA levels (C), caspase-1 activity (D), cleavage of p20 (E), and HMGB1 release (F) by curcumin (40 mmol/L for 48 hours) in HMESO cells. Expression of NLPR3 protein (G) was not altered with NAC pretreatment. H, schema showing regulation of pyroptosis by curcumin. , P 0.05 when compared with control; †, P < 0.05 when compared with curcumin-only treatment group.

Article Snippet: The amount of proteinwas determined using the RCDCProtein Assay (Bio-Rad).Western blotswere performed as described previously (23), using monoclonal rabbit NLRP3 antibodies (Novus Biologicals) at a dilution of 1:500 and amouse b-Actin antibody (Abcam) at a dilution of 1:2,000.Quantity One was used to quantify band density.

Techniques: Control, Activity Assay, Expressing