Journal: PloS one

Article Title: HMGB1 accelerates alveolar epithelial repair via an IL-1β- and αvβ6 integrin-dependent activation of TGF-β1.

doi: 10.1371/journal.pone.0063907

Figure Lengend Snippet: Figure 1. Endogenous HMGB1 released by damaged primary rat ATII cells increases alveolar epithelial wound closure. (A) HMGB1 was elevated in cell supernatant from rat ATII monolayers that underwent scratch wounds (MS Cell Sup) compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds (condition media). (B) MS Cell Sup increases the rate of wound closure of primary rat ATII cell monolayers compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds. HMGB1 was depleted from MS Cell Sup by immunoprecipitation using 30 mg/ml of HMGB1 specific Ab (MS Cell Sup IP w/HMGB1 Ab). Controls were MS Cell Sup immunoprecipitated with a control IgG (MS Cell Sup IP w/Cont Ab). (C) HMGB1 is secreted by primary rat ATII cell monolayers after scratch wounds. Multiple scratches (MS) were performed on primary rat ATII cell monolayers. Fresh cell media were added for 6 hours to the monolayers after extensive washes. Cell supernatants were then centrifuged to remove dead cells and cell debris, then analyzed by western blot (40 ml loaded per lanes from a 1 ml MS Cell Sup sample). (D) MS Cell Sup increases the rate of wound closure of a primary rat ATII cell monolayers via RAGE- and TLR4-dependent pathways, but not via a CXCR4-dependent mechanism. MS Cell Sup, and either 30 mg/ml of blocking RAGE or TLR4 antibodies or their isotype control IgG, or 1 mM of AMD3100, a CXCR4 inhibitor, were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *p,0.05 from monolayers exposed to control cell media; **p,0.05 from monolayers exposed to MS Cell Sup. For western blot experiments, one representative experiment is shown, three additional experiments gave comparable results; *p,0.05 from monolayers exposed to condition media. doi:10.1371/journal.pone.0063907.g001

Article Snippet: Rat RAGE blocking antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Control, Western Blot, Blocking Assay

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: Journal of Clinical Investigation

Article Title: A multimodal RAGE-specific inhibitor reduces amyloid β–mediated brain disorder in a mouse model of Alzheimer disease

doi: 10.1172/jci58642

Figure Lengend Snippet: Figure 2 FPS-ZM1 and FPS2 inhibit Aβ/RAGE binding in cell-free and cell-based assays. (A) 125I-Aβ40 (5 nM) binding to immobilized human sRAGE in the presence of FPS2 or FPS-ZM1 (10–500 nM). (B) 125I-HMGB1 (5 nM) or 125I-S100B (5 nM) binding to sRAGE in the presence of FPS-ZM1 (10–1,000 nM). In A and B, Ki represents inhibitory constant. (C) 125I-Aβ40 (5 nM) binding to immobilized human recombinant RAGE V domain (Vd) or C1C2 domain (C1C2d) with and without FPS-ZM1 (200 nM) and to sRAGE with and without RAGE-specific C1 domain (anti-C1d), C2 domain (anti-C2d), or V domain (anti-Vd) antibodies (20 μg/ml), NI-IgG, and FPS-ZM1 (100 nM). (D) Aβ40 and Aβ42 binding to immobilized human sLRP with and without FPS-ZM1 (1 μM) or RAP (1 μM). (E) Aβ40-induced (1 μM) TBARS in RAGE-CHO cells in the presence of vehicle (closed triangle) or various concentrations of FPS2 (white circles) and FPS-ZM1 (black circles). (F) Aβ40-induced (1 μM) NF-κB activation in RAGE-CHO cells with and without FPS2 and FPS-ZM1. (G–I) BACE1 mRNA (G), BACE1 protein (H), and secreted sAPPβ (I) levels determined by qRT-PCR, immunoblotting, and ELISA, respectively, in SH-SY5Y cultures treated with vehicle or Aβ40 (1 μM) with or without FPS2 or FPS- ZM1 (50 nM) after transduction with Ad. GFP or a mutant Ad. IκB-α (S32, 36A), and/or transfection with scrambled siRNA or RAGE-siRNA. All values are means ± SEM. n = 3–5 independent experiments. β-actin was used as a loading control in H.

Article Snippet: The effect of a RAGE antibody (RAGE antibody AF1179, 40 μg/ml; R&D Systems), NI IgG (40 μg/ml), or FPS2 or FPS-ZM1 (200 nM) was studied over 5 minutes of infusion, which was within the linear range of Aβ transport (influx) into the brain, as described (10).

Techniques: Binding Assay, Recombinant, Activation Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transduction, Mutagenesis, Transfection, Control