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