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: Gene therapy

Article Title: Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates.

doi: 10.1038/sj.gt.3300648

Figure Lengend Snippet: Figure 1 Expression of Epo following infection of HT1080 cells. Super- natant was harvested from cells 48 h after infection and analyzed by ELISA. Titers of rAAV-cm-Epo are indicated on X-axis. Bar marked lep- tin represents background levels of Epo secreted from cells infected with 5 × 109 particles of an irrelevant virus (rAAV-m-leptin).

Article Snippet: The levels of Epo were determined from monkey plasma or mouse serum using the R&D Systems ELISA Quantikine kit (R&D Systems).

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Virus

Journal: Gene therapy

Article Title: Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates.

doi: 10.1038/sj.gt.3300648

Figure Lengend Snippet: Figure 3 Titer of anti-Epo antibodies in C57BL/6 mice following rAAV- cm-Epo administration. Antibodies were measured as described in the experimental protocol. For saline-treated mice, sera were pooled before ELISA, sera from rAAV-treated mice were measured individually. Posi- tive control (+ control) represents sera from Cynomolgus monkey-Epo plasmid DNA injected BALB/c mice which had previously been shown to have anti-cm-Epo antibodies. Titer is defined as the dilution of serum required to reduce the signal to levels obtained in wells containing dilution buffer alone.

Article Snippet: The levels of Epo were determined from monkey plasma or mouse serum using the R&D Systems ELISA Quantikine kit (R&D Systems).

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Injection

Journal: Gene therapy

Article Title: Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates.

doi: 10.1038/sj.gt.3300648

Figure Lengend Snippet: Figure 2 Effect of rAAV-cm-Epo administration in C57BL/6 mice. (a) Serum Epo concentration (±s.e.m.), as measured by ELISA, following rAAV administration. At all time-points, the saline-injected mice had undetectable levels of serum Epo and are not included on this graph. (b) The average hematocrit of four mice injected with either rAAV-cm-Epo or saline. Although error bars are included, they are obscured by the plot symbols The 0 week time-points represent the average baseline hematocrit and serum Epo concentrations for untreated C57BL/6 mice.

Article Snippet: The levels of Epo were determined from monkey plasma or mouse serum using the R&D Systems ELISA Quantikine kit (R&D Systems).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Saline, Injection

Journal: Gene therapy

Article Title: Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates.

doi: 10.1038/sj.gt.3300648

Figure Lengend Snippet: Figure 4 Response of baboons to administration of rAAV-cm-Epo. (a) The plasma Epo levels as measured by ELISA. (b) The hematocrits of two baboons at times before injection (negative numbers) and after injection. To prevent stroke, 34 or 40 ml of blood was removed from baboon 2 at weeks 11 and 13.

Article Snippet: The levels of Epo were determined from monkey plasma or mouse serum using the R&D Systems ELISA Quantikine kit (R&D Systems).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A and B) Sestrin2 destabilizes physical interaction between GATOR1 and GATOR2 in HEK293 cells. HA-tagged Sestrin2 was co-transfected with GATOR1 and GATOR2 components as indicated. NPRL2, a GATOR1 component, or WDR24, a GATOR2 component, was IPed using Flag antibody in (A) and (B), respectively. Input (WCL) and IP complex were analyzed by IB. (C) Purification of Sestrin2, GATOR1 and GATOR2 proteins. Sestrin2 and HA-GATOR1/2 complexes were purified from transformed E. coli and transfected HEK293 cells, respectively. (D) Sestrin2 inhibits assembly of GATOR1-GATOR2 supercomplex in vitro . As indicated, the purified proteins were mixed and incubated together. GATOR1 was pulled down from the mixture using NPRL2 antibody. GATOR1 and co-purified GATOR2 was detected through IB with HA antibody. (E) Sestrin2 overexpression decreases GATOR1-GATOR2 association in mouse liver. 10 9 pfu of adenoviruses expressing GFP or Sestrin2 were injected into two-month-old WT mice through the tail vein. After 4 days, liver lysates were prepared, and endogenous GATOR2 was IPed with WDR24 antibody. WCL and IP complex were analyzed by IB. (F) Sestrin2 is required for the effect of tunicamycin (Tm) on GATOR1-GATOR2 interaction. Two-month-old WT or Sesn2 −/− mice were injected with Tm (500 mg/Kg i.p.) as described . After 24 hrs, liver lysates were prepared, and GATOR2 was IPed with WDR24 antibody. WCL and IP complex were analyzed by IB. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Transfection, Purification, Transformation Assay, In Vitro, Incubation, Over Expression, Expressing, Injection

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A) Inactivation of GST-RagB by Sestrin2. Flag-tagged Sestrin2 was co-transfected with GST-tagged RagB. Following metabolic labeling of the guanine nucleotide pools with 32 P, GST-RagB was pulled-down using Glutathione-Sepharose 4B beads. RagB-bound nucleotides were separated by thin layer chromatography (TLC) and visualized through autoradiography. The percentage of GDP-bound RagB protein is shown below each lane. Expression of Sestrin2 and GST-RagB were analyzed by IB of WCL. (B-D) Inactivation of endogenous RagB by Sestrin2-potentiated GATOR1. HEK293 cells were transduced with GFP (-) or Sestrin2-overexpressing (+) lentiviruses. After 48 h, RagB was IPed from cell lysates using RagB antibody (B,C) or NPRL2 antibody (D). 32 P-labeled nucleotides in the IP complex were separated by TLC and visualized through autoradiography (B). Input (WCL) and IP complex were analyzed by IB of endogenous proteins (C,D). (E) Sestrin2 promotes physical association between GATOR1 and RagB/RagC heterodimer. Sestrin2 was co-transfected with GATOR1, GATOR2 and GST-tagged RagB/RagC proteins as indicated. RagB/RagC proteins were pulled-down using Glutathione-Sepharose 4B beads. Input (WCL) and GST-purified complex (IP: GST) were analyzed by IB. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Transfection, Labeling, Thin Layer Chromatography, Autoradiography, Expressing, Transduction, Purification

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A) Sestrin2 inhibits phosphorylation of mTORC1 targets through GATOR. HEK293 cells stably transduced with indicated shRNA were infected with GFP or Sestrin2-overexpressing lentiviruses. After 48 h, cells were analyzed by IB with indicated antibodies against endogenous proteins. (B and C) Silencing Npr2, the Drosophila homologue of NPRL2, relieves Drosophila Sestrin (dSesn)-induced growth arrest. Anterior (B, upper panels) and dorsal (B, lower panels) views of wing blades with ap-GAL4 –driven expression of indicated transgenic elements were imaged under a dissection microscope and a transmitted light microscope, respectively. Dorsal wing area were quantified and presented as a bar graph (C, n = 7, means ± s.e.m.). P value was calculated using Student's t-test. (D and E) Autophagy defects in dSesn -null animals were corrected by heterozygotic mutations of GATOR2 components. Thoraces from 5 day-old flies of indicated strains were analyzed through immunoblotting of dAtg8 and Actin proteins (D). Relative levels of unprocessed (dAtg8-I) and processed (dAtg8-II) dAtg8 proteins were quantified by densitometry and presented as a bar graph (E, n = 3, means ± s.e.m.). P values were calculated using Student's t-test. (F and G) Proposed model of the Sestrin2-GATOR-RagB pathway that mediates stress-induced mTORC1 silencing. In unstressed conditions, GATOR1 is constitutively inhibited by GATOR2, and RagB recruits mTORC1 to lysosomal surface and activates it as well as its downstream targets (F). Upon stress-induced expression of Sestrin2, GATOR1 is released from GATOR2-mediated inhibition and inactivates RagB (G). In the absence of active RagB, mTORC1 is released from lysosomal surface and mTORC1 signaling is subsequently inactivated. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Stable Transfection, Transduction, shRNA, Infection, Expressing, Transgenic Assay, Dissection, Microscopy, Light Microscopy, Western Blot, Inhibition