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. 2022 Jan 25;14(5):477–492. doi: 10.1159/000521602

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Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel

This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC). Usage and distribution for commercial purposes requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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Fig. 4 — HO53 treatment leads to nuclear translocation of TFEB and augments expression of autophagy-related genes in differentiated BCi cells. aAnalysis of TFEB nuclear translocation upon treatment with 75 μM HO53 after 24 h by confocal imaging of nuclei (blue, DAPI), TFEB (red), and extracted signal from co-localized TFEB/nuclei (pink) from merged channels with scale bar 50 μm. Representative single slices from z-stacked images (15/39) were placed for each channel of HO53-treated samples with scale bar 10 μm. The representative images from at least 3 different areas of the ALI filter from n = 3 independent experiments with quantification of pixel intensity for a signal from co-localized TFEB/nuclei (pink) extracted from merged DAPI/TFEB images ± SEM. Statistical analysis was performed by using an unpaired t-test with Welch's correction, *p < 0.05. Changes in autophagy-related genes expression in differentiated BCi cells in ALI at 4 h, 8 h, and 24 h of treatment with HO53. bA heatmap showing the expression level represented as a log2FC of significantly differentially expressed genes in comparison to control cells collected at the same time points (p < 0.05). Genes selected for further analysis were marked in red. Nonsignificant differentially expressed genes have arbitrary value 0. Expression level of selected autophagy-related genes: MAP1LC3A (c), MAP1LC3C (d), ATG16L1 (e), and ATG16L2 (f) analyzed by qRT-PCR. Gene expression was represented as a fold change in comparison to control cells (value 1, dashed line) collected at the same time point as treated cells and normalized to the EEF2 reference gene. n = 3 trans-well inserts from 3 independent experiments ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 versus control cells analyzed by one-way ANOVA with the Dunnett post hoc test. gEnrichment plot for autophagy-lysosomal genes obtained from GSEA of the expression data from differentiated BCi cells in the ALI culture after 24 h of treatment with HO53. NES and NOM p are indicated on the plot. log2FC, log₂ fold change; NES, normalized enrichment score; NOM p, nominal p value; SEM, standard error of mean.