Journal: Nature Communications

Article Title: Human iPSC-based Modeling of Pulmonary Fibrosis Reveals p300/CBP Inhibition Suppresses Alveolar Transitional Cell State

doi: 10.1038/s41467-026-68909-z

Figure Lengend Snippet: a Gene expression of ATCS markers in the micro-patterned culture. Each well was treated with either DMSO or p300/CBP inhibitors (10 μM) from days 11 to 14. Data are presented as mean ± SEM (n = 3 biologically independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. b Dot plots displaying the gene expression of iATCs-specific markers in representative cell populations from the micro-patterned culture, corresponding to those shown in Fig. . c Immunostaining of CD54 (ICAM1), lineage markers for ATCS (KRT19), AT1 cells (HT1-56), AT2 cells (NaPi2b), and nuclei (Hoechst) in micro-patterned cultures. Representative images from three biologically independent experiments with similar results are shown. Scale bar: 100 μm. d, e Flow cytometry analysis assessing the CD54 + cell ratio in the micro-patterned culture. Each well was treated with either DMSO or p300/CBP inhibitors (10 μM) from days 11 to 14. Data are presented as mean ± SEM (n = 3 biologically independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test; ** p < 0.01. f Schematic outline for sorting CD54 + iATCs from day 14 of the micro-patterned culture. Created in BioRender. Tsutsui, Y. (2026) https://BioRender.com/v51i925 g Gene expression data of ATCS markers in the micro-patterned culture. Data are presented as mean ± SEM ( n = 3 biologically independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. h Schematic outline of the co-culture experiment involving isolated CD54 + iATCs and NHLFs. Created in BioRender. Tsutsui, Y. (2026) https://BioRender.com/nbi6c0b i Gene expression analysis of ATCS markers in isolated CD54 + iATCs. Data are presented as mean ± SEM ( n = 3 biologically independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test; ** p < 0.01. ns; not significant ( p > 0.05). j Gene expression analysis of fibroblast activation markers in NHLFs cocultured with or without iATCs. Data are presented as mean ± SEM ( n = 3 biologically independent experiments). Unpaired two-tailed Student’s t test: ∗ p < 0.05.

Article Snippet: Primary antibodies used in this study included GFP (1:500, Aves Labs, GFP-1020), SFN (1:200, Abcam, ab77187), act-p300 (1:200, biorbyt, ORB6262), EpCAM (1:200, Santa Cruz Biotechnology, sc-66020), KRT19 (1:200, Merck, MABT913), KRT17 (1:100, Abcam, ab109725), COL1A1 (1:200, Abcam, ab138492), CD54 (1:200, BioLegend, 353102), CD54 (1:200, Atlas, HPA002126), HT1-56 (1:200, Terrace Biotech, TB29AHT1-56), NaPi2b (1:200, kindly provided by Dr. Gerd Ritter (MX35)), AGER (R&D systems, AF1145), H3K27ac (1:200, Cell Signaling technology, 8173), FLAG (1:500, Cell Signaling technology, 14793), alpha smooth muscle Actin (1:100, Abcam ab5694), and Fluorescin (1:1000, Vector Laboratories, FL-1171).

Techniques: Gene Expression, Immunostaining, Flow Cytometry, Co-Culture Assay, Isolation, Activation Assay, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Involvement of the coupled RAGE ligand/receptor in human corneal epithelial (HCE) cell wound healing. Representative images of scratch assays performed on HCE cells (left panel) treated with HMGB1 (high mobility group box 1, 100 ng/mL) ( A ) or AGEs (advanced glycation end products) (10/100/200 µg/mL) (B , C ). Percentage of residual wound area (right panel) after treatment with HMGB1 (100 ng/mL) ( A ) or AGEs (10/100/200 µg/mL) ( B , C ), compared with 0 hours and standardized to the untreated condition (100%) (n = 5 experiments, each conducted in duplicate). ( D ) Representative images of scratch assays performed on HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) or siRNA control (Scramble) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) (left panel). Percentage of the residual wound area of HCE cells transfected with siRNA against RAGE (100 nM) for 36 hours and then treated with AGEs (100 µg/mL), compared with 0 h (right panel) (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney test after a nonparametric ANOVA analysis; * P < 0.05; ** P < 0.01; *** P < 0.005; ns: not significant.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Transfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Functionality of the RAGE pathway. Characterization of the RAGE ( A ) mRNA and ( B ) protein expression in human cornea, primary human epithelial cells (mRNA only), and the HCE cell line (mRNA and protein) evaluated by ( A ) RT-PCR, ( B ) immunofluorescence, and ( B ) western blotting. For RT-PCR, negative controls (NC) were performed ( A ) without cDNA. ( B , left panel) Representative images of RAGE expression ( green ) in human corneas (top panel) and HCE cells (bottom panel). Nuclei were stained with Hoechst ( blue ); NC (left) were obtained by incubating HCE cells without primary antibody. ( B , right panel) Western blot experiments identified the RAGE protein at the described molecular weight (46 kDa). ( C ) Functionality of the NF-κB pathway (by luciferase reporter gene activity) was measured after treatment of HCE cells with AGEs (100 µg/mL) for 45 minutes (n = 5 experiments, each conducted in duplicate) (right panel). Positive controls (T+) were obtained by co-transfection with pMEKK (n = 5 experiments, each conducted in duplicate) (left panel). Each bar graph shows mean ± SEM. Mann-Whitney; * P < 0.05.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Staining, Molecular Weight, Luciferase, Activity Assay, Cotransfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Cx43 expression is induced by the AGEs/RAGE axis in HCE cells. Characterization of Cx43 protein expression ( arrow ) by immunostaining (bottom panel). Cells incubated without primary antibody served as a negative control (NC). ( B ) Relative quantification of Cx43 mRNA expression in untreated HCE cells 0, 6, and 24 hours after scratch wounding. Results were expressed as a ratio of the 0-hour condition (n = 5 experiments, each conducted in duplicate). ( C ) Quantification of Cx43 mRNA expression in HCE cells treated with AGEs (100 µg/mL) for 6 hours or 24 hours without scratch wounding (left panel) or with scratch wounding (right panel). Cells not treated with AGEs served as a control. Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate). (Left panel) Quantification of Cx43 mRNA expression in HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) for 6 hours. Results were expressed as a ratio of the scrambled siRNA condition (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney after a nonparametric ANOVA analysis; * P < 0.05; ns: not significant. ( D ) Relative quantification of Cx43 protein expression in HCE cells following treatment with AGEs (100 µg/mL) after scratch wounding. Untreated cells served as a control. Results were expressed as a ratio of the 0-hour, unwounded condition (n = 5 experiments) (top panel). Quantification of Cx43 protein expression in HCE cells treated with AGEs (100 µg/mL) for 12 and 24 hours after scratch wounding (bottom panel). Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate) (** P < 0.01). (E) Characterization by immunostaining of Cx43 protein expression according to the distance from the wound, time, and treatment with AGEs (100 µg/mL). Staining of Cx43 protein expression in HCE cells treated near the wound margins (left panel) and behind the wound (right panel). Cells not treated with AGEs (100 µg/mL) served as a control. Cells incubated without primary antibody served as a negative control (NC).

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Immunostaining, Incubation, Negative Control, Transfection, MANN-WHITNEY, Staining

Journal: Brain Pathology

Article Title: Environmental Enrichment Counteracts Alzheimer’s Neurovascular Dysfunction in TgCRND8 Mice

doi: 10.1111/j.1750-3639.2007.00094.x

Figure Lengend Snippet: Differential regulation of Aβ receptor/transporter molecules following environmental enrichment. Western blot analysis revealed a decrease in cerebral RAGE levels (A) and an increase in LRP1 protein (B) following enrichment (cropped images). C. Densitometry analysis of immunoblots identified a significant decrease of cerebral RAGE by 60% (P = 0.004). D. Densitometry analysis of immunoblots identified a significant increase of LRP1 protein by 384% (P = 0.04). E. TaqMan quantitative real‐time PCR revealed enhanced gene expression of the Aβ binding proteins Apoe by 42% (P = 0.006) and A2m by 50% (P = 0.02) in enriched housed mice, whereas gene expression levels of Rap (P = 0.31) and Apoj (P = 0.25) remained unaltered. Data are given as mean ± SD, statistics unpaired Student’s t‐test. RAGE = receptor for advanced glycation end products; LRP1 = low‐density lipoprotein receptor‐related protein 1; PCR = polymerase chain reaction; EH = enriched housing; SH = standard housing.

Article Snippet: Electrophoresis and wet‐blotting preceded membrane blocking with 5% non‐fat milk in TST buffer (10 mM Tris‐HCl pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature and subsequent incubation with RAGE antibody (SP5151P, 1:500, Acris Antibodies, Hiddenhausen, Germany), LRP1 antibody (5A6, 1:1000, Merck KGaA, Darmstadt, Germany), EFNB2 antibody (AF496, 1:500, R&D Systems, Minneapolis, MN, USA) or anti‐FLT1 (1303‐1, 1:5000, Epitomics) overnight at 4°C followed by secondary antibody (A2074, 1:2000, Sigma‐Aldrich, for RAGE; A0168, 1:10 000, Sigma‐Aldrich, for LRP1; A9452, 1:5000, Sigma‐Aldrich, for EFNB2; 111‐035‐003, 1:5000, Jackson ImmunoResearch, West Grove, PA, USA for FLT1) and peroxidase‐catalyzed enhanced chemiluminescence (ECL‐Plus, Amersham Biosciences, Freiburg, Germany).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Gene Expression, Binding Assay, Polymerase Chain Reaction