Journal: Journal of Innate Immunity

Article Title: The Novel Inducer of Innate Immunity HO53 Stimulates Autophagy in Human Airway Epithelial Cells

doi: 10.1159/000521602

Figure Lengend Snippet: HO53 treatment induces autophagy in human airway epithelial cells. BCi cells were stimulated for 24 h with different doses of HO53, and 250 nM Rapa was used as a positive control for autophagy induction, and DMSO (final concentration of 0.3%) was used as a solv, all in combination with (+Baf.A1) or without (−Baf.A1) Bafilomycin A1 (100 nM). Treatment of differentiated BCi cells was performed by addition of the compound to the lower chamber of the trans-well insert. Induction of autophagy in the ALI culture ( a ) and undifferentiated BCi ( b ) was evaluated by analysis of LC3B processing on Western blotting. The processing of LC3B-I to LC3B-II was quantified by measurement of the LC3B-II band intensity versus GAPDH loading control and presented as the LC3B-II/GAPDH ratio. Data present average ± SEM from n = 3 independent experiments analyzed by one-way ANOVA with Sidak post hoc test, where * p < 0.05, ** p < 0.01 and ns versus solvent control, & p < 0.05 versus 50 μM HO53, && p < 0.01 versus 12.5 μM HO53. Samples were run in one experiment on separate gels/blots processed in parallel, and full-length blots are presented in Supplementary Figure S7. c Analysis of the autophagy induction by HO53 (75 μM) in ALI-cultured BCi cells by immunostaining of LC3B puncta (green), nuclei (blue), and occludin (red), a tight-junction protein characteristic for the differentiated BCi cells in the ALI culture. The scale bar is 10 μm. Autophagy flux in ALI-cultured BCi was calculated based on number of LC3B+ (positive) puncta using the formula: ( sample + Baf.A1 )/ sample presented as autophagy flux LC3B + puncta. Data present average ± SEM from n = 5 independent experiments analyzed by an unpaired t -test, where * p < 0.05. d TEM analysis of differentiated BCi cells treated with HO53 (75 μM) and Bafilomycin A1 (100 nm) for 24 h. Autophagosomes are indicated by red arrows. TF indicates the trans-well filter/insert; red squares indicate a magnified area with scale bars for images as indicated (from left to right) 2 μm, 1 μm and 200 nm. Representative images of n = 4 trans-well inserts from 2 independent experiments. ns, nonsignificant; solv, solvent control; Rapa, rapamycin; SEM, standard error of mean.

Article Snippet: UltroserG (15950-017) was obtained from Pall Life Sciences and DMSO (sc-358801), Bafilomycin A1 (sc-201550), and rapamycin (sc-3504) from Santa Cruz.

Techniques: Positive Control, Concentration Assay, Western Blot, Control, Solvent, Cell Culture, Immunostaining

Journal: ACS Infectious Diseases

Article Title: Using Next Generation Chemiluminescent Probes to Improve the Plasmodium falciparum in vitro Parasite Reduction Ratio (PRR) Assay

doi: 10.1021/acsinfecdis.5c00924

Figure Lengend Snippet: Comparison of the 14-day vs 7-day lacZ /β-gal SENSOR probe PRR assay in comparison to the published PRR v2 assay. The four reference drugs artemisinin (58 nM), atovaquone (100 nM), chloroquine (112 nM) and pyrimethamine (170 nM) were tested in at least three independent experiments. Time-killing profiles were normalized with error bars representing the standard error of the mean (SEM) of the independent experiments in technical quadruplicates.

Article Snippet: All wells of the 7- and 14-day lacZ /β-gal SENSOR probe PRR assay were treated with β-gal SENSOR probe (AquaSpark beta-D-galactoside, Biosynth AG, cat. #A-8169_P00) diluted in purified, sterile-filtered water containing MgCl 2 to achieve final concentrations of 10 μM β-gal SENSOR probe and 200 μM MgCl 2 per well.

Techniques: Comparison