Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the largest extracellular domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either alive (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), ECD-NaPi2b–mAbs L2 (20/3) directed to ECD of NaPi2b; (D,H) Merged images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each picture, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Mouse mAbs against ECD of NaPi2b L2 (20/3) , N-terminal domain of NaPi2b N-NaPi2b (15/1) , and rabbit mAbs against C-terminal domain of NaPi2b (D3V3I, Cell Signaling, United States) were used as the primary antibodies.

Techniques: Confocal Microscopy, Membrane

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the N-terminal domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either intact (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), N-NaPi2b–mAbs N-NaPi2b (15/1) directed to the N-terminal domain of NaPi2b; (D,H) Merged images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each picture, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Mouse mAbs against ECD of NaPi2b L2 (20/3) , N-terminal domain of NaPi2b N-NaPi2b (15/1) , and rabbit mAbs against C-terminal domain of NaPi2b (D3V3I, Cell Signaling, United States) were used as the primary antibodies.

Techniques: Confocal Microscopy, Membrane

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the C-terminal domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either alive (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), C-NaPi2b–mAbs D3V3I (Cell Signaling, United States) directed to the C-terminal domain of NaPi2b; (D,H) Merged Images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each image, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Mouse mAbs against ECD of NaPi2b L2 (20/3) , N-terminal domain of NaPi2b N-NaPi2b (15/1) , and rabbit mAbs against C-terminal domain of NaPi2b (D3V3I, Cell Signaling, United States) were used as the primary antibodies.

Techniques: Confocal Microscopy, Membrane

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Topological model of NaPi2b with color-coded antigenic regions recognized by available monoclonal analytical and therapeutic antibodies targeting different extramembrane domains. The epitope’s region in ECD of NaPi2b for mAbs, generated by: , 188–300 aa, marked in light purple; , 320–361 aa, marked in green; , 323–330 aa, marked in purple; , , Kiyamova et al. (2008), , , , , 324–338 aa, marked in red; Epitope region in N-termini and C-termini of NaPi2b for mAbs, generated by: Gryshkova et al. (2011), Cell Signaling, Abcam, and others against N-terminal domain of NaPi2b, 1–100 aa, marked in orange; Cell Signaling, Abcam, and others against С-terminal domain of NaPi2b, the region is unknown, marked in lavender. Positions of cysteine and asparagine amino acid residues potentially contributing to the oxidative folding and formation of disulfide bonds and N-linked glycosylation of the largest ECD, and palmytoilation site in C-terminal domain are also shown.

Article Snippet: Mouse mAbs against ECD of NaPi2b L2 (20/3) , N-terminal domain of NaPi2b N-NaPi2b (15/1) , and rabbit mAbs against C-terminal domain of NaPi2b (D3V3I, Cell Signaling, United States) were used as the primary antibodies.

Techniques: Generated, Glycoproteomics

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