Journal: iScience

Article Title: FTO inhibition attenuates renal fibrosis by downregulating ferroptosis activator ACSL4 and profibrotic factor TGFBI

doi: 10.1016/j.isci.2025.113515

Figure Lengend Snippet: FTO inhibition suppresses ferroptosis in kidney epithelial cells by ROS reduction Primary kidney epithelial cells were generated using mouse kidneys (A). Images of primary mouse kidney epithelial cells (B) and cell viability assay (C) showed that FB-23 and Lipro-1 protected primary mouse kidney epithelial cells from erastin-induced ferroptosis. FB-23 and Lipro-1 protected human kidney epithelial HK2 cells from erastin-induced decrease in cell viability (D) and increased ROS production (E) and membrane damage (F). Synergy map analysis revealed a high level of antagonism between erastin and FB23-2 (G). FTO protein expression was abolished by western blot in three independent FTO KO clones (H). Images of HK2 cells (I) and cell viability assay (J) showed FTO knockdown suppressed erastin-induced ferroptosis; cell membrane damage (K), lipid peroxidation (I), and ROS production (M) were decreased in FTO knockdown cells compared with WT cells. Both pharmacological (N) and genetic inhibition of FTO (O) significantly decreased the transcript level of kidney damage marker LCN2 in HK2, and LCN2 protein expression in FTO KO cells was diminished (P). ∗ p < 0.05; ∗∗ p < 0.01. Scale bars: 100 μm in (B and I). Data are represented as mean ± SD.

Article Snippet: Primary renal epithelial cells were generated from kidney tissues excised from 8- to 12-wk-old C57/BL6 female mice and cultured in Renal Epithelial Cell Growth Medium 2 (PromoCell C-26030) as previously described.

Techniques: Inhibition, Generated, Viability Assay, Membrane, Expressing, Western Blot, Clone Assay, Knockdown, Marker

Journal: iScience

Article Title: FTO inhibition attenuates renal fibrosis by downregulating ferroptosis activator ACSL4 and profibrotic factor TGFBI

doi: 10.1016/j.isci.2025.113515

Figure Lengend Snippet: FTO inhibition protects against ferroptosis in hESC-derived kidney organoids by downregulating ACSL4-mediated ferroptosis (A) Generation of kidney organoids from hESC cells. (B) Immunofluorescence staining for cell type-specific markers in kidney organoids. (C) Expression changes of stem cell markers and kidney epithelial markers during the differentiation period of 20 days determined by qRT-PCR. (D) FTO, ACSL4, and TGFBI expression levels were dramatically decreased in kidney organoids after lentiviral transfection of shFTO determined by western blot. FTO inhibition attenuated erastin-induced ferroptosis determined by LDH assay (E), lipid peroxidation determined by MDA assay (F), and ROS production (G) in kidney organoids. FTO inhibition significantly reduced TGFBI (H) and LCN2 (I) expression at transcript level determined by qPCR compared with vehicle control. Schematic diagram of the mechanisms of FTO upregulation (J) and downregulation (K) in RF. Scale bars: 50 μm in (A and B). ∗ p < 0.05; ∗∗ p < 0.01. Data are represented as mean ± SD.

Article Snippet: Primary renal epithelial cells were generated from kidney tissues excised from 8- to 12-wk-old C57/BL6 female mice and cultured in Renal Epithelial Cell Growth Medium 2 (PromoCell C-26030) as previously described.

Techniques: Inhibition, Derivative Assay, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Lactate Dehydrogenase Assay, Multiple Displacement Amplification, Control

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate attenuates ethanol‐induced gastric mucosal hemorrhage and oedema in vivo. (A) Establishment of ethanol‐induced gastric ulcer in rats by intragastric administration of anhydrous ethanol. (B) Flowchart of the animal experimental procedures. (C) Macroscopic appearance of the gastric mucosa in four groups. (D) Quantification of gastric damage expressed as the ulcer index (UI). The UI was calculated as follows: UI = 10 × (ulcerated area/total mucosal area). (E) Representative HE‐stained images of stomach tissues. Scale bar = 50 µm. (F) Histological evaluation of gastric lesions using a microscopic HE score (0–14). The score sums the severity of four features: inflammatory cells (0–3), mucosal edema (0–4), hemorrhage (0–4), and epithelial loss (0–3). (G) Representative CD31 IF staining images in gastric mucosa. Scale bar = 20 µm. (H and I) RT‐qPCR analysis of Ang1 and Et1 mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. (J and K) The nitrate levels of the serum and gastric mucosa in the four groups. Quantitative data are expressed as the mean ± SD. * p < 0.05, *** p < 0.001, and ns denotes no significance. HE, hematoxylin–eosin; Nit, nitrate; IF, immunofluorescence; CD31, platelet endothelial cell adhesion molecule‐1; Ang1, angiotensin‐1; Et1, endothelin‐1; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; SD, standard deviation.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: In Vivo, Staining, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Tff2 knockdown abolishes nitrate protection against ethanol‐induced gastric ulcers in vivo. (A) Establishment of Tff2‐KD rat by tail vein injection of recombinant AAV. (B) The timeline of dietary nitrate administration (3 weeks after injection and 7 days before ethanol gavage). (C and D) The macroscopic appearance and ulcer index of the gastric mucosa in Tff2‐KD and scramble groups with ethanol gavage. The UI was calculated as follows: UI = 10 × (ulcerated area/total mucosal area). (E and F) Representative histology images of stomach tissue and a histopathologic score of HE staining in Tff2‐KD and scramble groups. The HE score sums the severity of four features: inflammatory cells (0–3), mucosal edema (0–4), hemorrhage (0–4), and epithelial loss (0–3). Scale bar = 50 µm. (G and H) Representative gastric tissue images of AB–PAS staining and mucin histochemical analysis of Tff2‐KD and scramble groups. The mucin area was expressed as fold change relative to the scramble + EtOH group. Scale bar = 50 µm. Quantitative data are expressed as the mean ± SD. *** p < 0.001, and ns denotes no significance. AAV, adeno‐associated virus; HE, hematoxylin–eosin; AB–PAS, Alcian blue and periodic acid‐Schiff; EtOH, ethanol; KD, knockdown; Nit, nitrate; Tff2; trefoil factor 2.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Knockdown, In Vivo, Injection, Recombinant, Staining, Virus

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate promotes migration by TFF2 upregulation in vitro. (A) Images of the scratch healing process of GES‐1 cells in Ibidi culture inserts. Scale bar = 500 µm. (B and C) Quantitative analysis of the migration rate at 24 h and 48 h in (A). (D and E) Representative immunoblotting band of the TFF2 protein and analysis of band gray values. (F) IF staining of pMLC (pink) and DAPI (blue). Scale bar = 40 µm. (G) IF analysis of pMLC with MFI. (H) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts. Scale bar = 500 µm. (I and J) Quantitative analysis of the migration rate at 24 h and 48 h. (K) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts containing ethanol. Scale bar = 500 µm. (L and M) Quantitative analysis of the migration rate at 24 h and 48 h. Migration rate is quantified by the percentage of closed area to the initial scratch area. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and ns denotes no significance. TFF2, trefoil factor 2; GES‐1, human gastric epithelial; EtOH, ethanol; Nit, nitrate; Ctrl, control; MLC, myosin light chain 2; pMLC, phosphorylated myosin light chain 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; NC, negative control; SD, standard deviation.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Migration, In Vitro, Western Blot, Staining, Transfection, Negative Control, Control, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: The RNA binding protein Insulin Growth factor 2 binding Protein 3 (IGF2BP3) modulates IL-13/IL-4 signalling in human bronchial epithelial cells and is dysregulated in type 2 disease

doi: 10.1101/2025.07.19.665669

Figure Lengend Snippet: IGF2BP3 is increased in type 2 disease. A. Left: normalized expression levels of IGF2BP3 mRNA in severe asthma (SA) vs healthy control (HC) primary BECs at total and polyribosome-bound levels; centre: IGF2BP3 normalized microarray expression data comparing airway epithelial cells from healthy non-atopic (HNA) and atopic asthma (AA) children; right IGF2BP3 normalized microarray expression data comparing nasal lavage samples from children during Picornavirus-induced exacerbation (EX) vs 7-14 days post-exacerbation (post-EX). B. Normalized count levels comparing type 2 high vs type 2 low nasal brushings for genes that showed IGF2BP3-dependent IL-13-modulation in our Frac-seq data . C: Heatmap showing the normalized expression values of the common mRNA signature between differentially expressed genes in the GALA II cohort and our Frac-seq IGF2BP3 and IL-13-dependent DEGs. D. Model for IGF2BP3 effects on type 2 responses. Decreasing IGF2BP3 levels leads to increased stability of IL13RA1 and IL4R mRNA and increased surface expression of IL4R and IL13Rα1, leading to increased STAT6 phosphorylation and increased IL-13 (and possibly IL-4) transcriptional responses, contributing to an overall increased type 2 response. In disease, IGF2BP3 is upregulated potentially to supress excessive T2 responses.

Article Snippet: Primary bronchial epithelial cells (BECs) were commercially sourced (PromoCell), grown and expanded in growth factor-supplemented medium (PromoCell) on collagen-coated plates.

Techniques: Expressing, Control, Microarray, Phospho-proteomics

Journal: bioRxiv

Article Title: The RNA binding protein Insulin Growth factor 2 binding Protein 3 (IGF2BP3) modulates IL-13/IL-4 signalling in human bronchial epithelial cells and is dysregulated in type 2 disease

doi: 10.1101/2025.07.19.665669

Figure Lengend Snippet: Decreasing IGF2BP3 levels increases IL-13/IL-4-driven transcriptional responses. RT-qPCR results of primary BECs transfected with siRNAs against IGF2BP3 (siIGF2BP3) or scrambled control (siControl) and stimulated with IL-13 or IL-4. A: RT-qPCR results for IGF2BP3 knock down and the major signalling mediators of IL-13 and IL-4. B: RT-qPCR results for chemokines. C: RT-qPCR results for pro-inflammatory IL6 and IL8 mRNAs. Data (n=3) depicted as mean ±SEM. One-tailed ratio t-tests. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Primary bronchial epithelial cells (BECs) were commercially sourced (PromoCell), grown and expanded in growth factor-supplemented medium (PromoCell) on collagen-coated plates.

Techniques: Quantitative RT-PCR, Transfection, Control, Knockdown, One-tailed Test