Fig 1: Effects of A02 and Octa on viability, proliferation, and differentiation of A431 cells. (A) Cell number analysis was performed in A431 treated with Octa or A02 (90 µM) for 72 h. The data are presented as the mean ± SD of three independent experiments and are expressed as the fold change with respect to untreated control cells (* p < 0.05 vs. untreated control). (B) Cell cycle distribution evaluated by flow cytometric analysis on A431 treated with Octa or A02 (90 µM) for 48 h. The bar graph shows the distribution of cells among the different phases of the cell cycle. The data are expressed as the mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (C) Western blot analysis of p21 and cyclin D1 protein expression in A431 treated with Octa or A02 (90 µM) for 24 h and 48 h, respectively. Representative blots are shown. (D) Real-time RT-PCR analysis of FLG, IVL, and LOR in A431 treated with Octa or A02 (90 µM) for 48 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control). (E) Western blot analysis of filaggrin, involucrin, and loricrin protein expression in A431 treated with Octa or A02 (90 µM) for 72 h. β-actin and GAPDH were used as endogenous loading control for Western blot analyses. Densitometric scanning of band intensities was performed to quantify the change in protein expression. Data represent the mean ± SD of three independent experiments and are expressed as fold change with respect to untreated control cells (control value taken as 1-fold in each case).
Fig 2: Recovery of IL-4/IL-13-induced epidermal and dermal impairment by dhAvD treatment. (A) Histological images of hematoxylin and eosin (H&E)-stained skin equivalents treated with IL-4/IL-13 with or without dhAvD (scale bar = 200 μm) (n = 3). (B) Immunostaining results of FLG, IVL, OCLN, ZO-1, MMP1, and COL1A1 expression in IL-4/IL-13-treated skin equivalents with or without dhAvD and their quantification (scale bar = 200 μm) (n = 3). * p < 0.05 and ** p < 0.01 vs. non-treated control; # p < 0.05 and ## p < 0.01 vs. IL-4/IL-13 treatment.
Fig 3: MAB21L4 up-regulates TGF-β target genes. (A) The effect of MAB21L4 siRNA on the TGF-β-induced expression of SERPINE1 and CDKN1A in HaCaT cells. The same samples shown in Fig. 4B were used for this evaluation. (B) MAB21L4 protein physically binds to Smad proteins; 293T cells were transfected with the expression plasmids as indicated and lysed for co-immunoprecipitation analysis. The experiment was repeated with the similar results and the representative data are shown. (C) Cytoplasmic localization of FLAG-MAB21L4 protein in transfected HaCaT cells. Cells were transfected with the plasmids encoding FLAG-tagged MAB21L4 or empty vector. Data were obtained from the two microscopic fields, and the representative images are shown. (D) Cell fractionation assay showing the localization of endogenous MAB21L4 protein. HaCaT cells were lysed with NE-PER (Thermo Fisher Scientific) to obtain the cytoplasmic and nuclear fractions. The experiment was repeated with similar results, and the representative data are shown. (E) Effect of knockdown of MAB21L4 on the Smad3 binding to the target genomic regions. HaCaT cells transfected with siRNA as indicated were stimulated with TGF-β for 1.5 h and harvested for ChIP-qPCR analysis. Data represents the mean ± standard deviation of two biological replicates. (F) Down-regulation of H3K27ac by MAB21L4 siRNAs. HaCaT cells were transfected with the siRNAs as indicated and were stimulated with TGF-β for 1.5 h. Data represents the mean ± standard deviation of two biological replicates. Note that the most enriched genomic positions of Smad3 and H3K27ac appeared different at the IVL locus and the different primers were used between Fig. 5E and F, as shown in Supplementary Table S2. NC, control siRNA; IP, immunoprecipitation; IB, immunoblotting; ALK5-TD, constitutively active ALK5; n.s.: not significant. *P < 0.05 by Dunnett test.
Fig 4: Downregulation of ELF3 recovered FOXP4‐induced inhibition of squamous differentiation in HaCaT cells. (A) In FOXP4‐shRNA‐treated HaCaT cells, mRNA expression of SPRR1, ELF3, GRHL3, HES1, and HES5 was significantly promoted. (B) Knockdown of ELF3 inhibited gene induction of the transcriptional factor and squamous differentiation markers TGM1, SPRR1, IVL, and GRHL3 as well as NOTCH3, but not NOTCH1. Three separate experiments were performed and bars represent SD. The data were analyzed using ANOVA followed by Dunnett test. *p < 0.05; **p < 0.01; N.S., not significant
Fig 5: Co-cultivated constructs show adherent cell junctions and multiple epithelial layers.Representative staining for E-cadherin staining of vocal folds (A), buccal mucosa (B) and co-cultivated construct (C); CK5/14 staining of vocal folds (D), buccal mucosa (E) and co-cultivated construct (F); p63 staining of vocal folds (G), buccal mucosa (H) and co-cultivated construct (I); and IVL staining of vocal folds (J), buccal mucosa (K) and co-cultivated construct (L). All formalin-fixed, paraffin-embedded sections, 20x magnification.
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