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: Advanced Healthcare Materials

Article Title: Generation of an Induced Pluripotent Stem Cell‐Derived Alveolar Type II In Vitro Model to Study Influenza A Virus Infection and Drug Treatments

doi: 10.1002/adhm.202405141

Figure Lengend Snippet: Differentiation, cultivation, and characterization of iAT2 cells. (a) Overview of the differentiation, cultivation, and plating procedure of iAT2s as described in the methods. iPSCs: induced pluripotent stem cells; iAT2s: iPSC‐derived alveolar type II cells; ALI: air–liquid interface; NKX2.1: NK2 homeobox 1; SP‐C: surface active agent protein C; for StemDiff, DS/SB, CBRa, CK/DCI, CK/DCI+Y: see methods. (b) Representative flow cytometry plots of the lung progenitor markers NKX2.1 coupled to GFP on day 14 (left) and SP‐C coupled to tdTomato on day 30 (right). (c) Representative transmission light image of alveolospheres within matrigel before passaging on day 35. Scale bar = 30 µm. (d) Representative transmission light images of iAT2s seeded on transwell inserts directly after seeding (day X) and 4 or 6 days after seeding (day X + 4 and X + 6). Scale bars = 50 µm. (e) Immunofluorescence images of iAT2s showing expression of antigen Kiel 67 (Ki67; green, left), SP‐C (magenta, left), lysophosphatidylcholine acyltransferase 1 (LPCAT1; yellow, middle), and NKX2.1 (green, right). Nuclei are displayed in blue. Single z‐planes are shown. Scale bars = 20 µm (left, middle) or 50 µm (right).

Article Snippet: Transwell inserts (Greiner Bio‐one, 662 641) were placed in 24‐well plates and coated with 100 μL of Corning Matrigel Human Embryonic Stem Cell‐qualified matrix (Corning, 354 277) according to the manufacturer's instructions.

Techniques: Derivative Assay, Flow Cytometry, Transmission Assay, Passaging, Immunofluorescence, Expressing