Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: HBEGF serves as the primary downstream target of the CagA‐FTO axis. (A) Workflow showed the screening strategy for candidate targets of the CagA‐FTO axis. (B) Western blotting to detect the protein level of FTO and HBEGF in GC cells after exposed to cagA + or ΔcagA H. pylori . (C) Western blotting to detect the protein level of FTO and HBEGF in GC cells with or without FTO knockdown. (D) Western blotting is used to detect the protein levels of FTO and HBEGF in GC cells overexpressing wild‐type FTO‐WT , FTO ‐ mut1 , or FTO ‐ mut2 . (E) Western blotting showed protein levels of FTO and HBEGF in NC and FTO knockdown GC cells exposed to cagA + or ΔcagA H. pylori . (F‐H) ELISA assay showed secretion level of HBEGF in the culture supernatants of H. pylori ‐infected (F), FTO knockdown (G) and FTO overexpressing (H) GC cells. (I) Western blotting to detect the expression levels of EMT markers in GC cells with HBEGF knockdown and cagA + H. pylori exposure. (J‐K) MeRIP‐qPCR showed enrichment of m 6 A modification on HBEGF mRNA after FTO knockdown (J) and FTO overexpression (wild‐type FTO or mutated FTO , K). HECBPA was used as a positive control. ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 were determined through one‐way ANOVA (F and K) and unpaired t ‐tests (G, H and J). Abbreviations: E‐Ca, E‐cadherin; EMT, epithelial‐mesenchymal transition; FTO mut , mutant FTO ; FTO, fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; MeRIP‐seq, Methylated RNA immunoprecipitation sequencing; HBEGF, heparin‐binding EGF like growth factor; HECBPA, 2’‐O‐methyladenosine‐3’‐phosphate‐5’‐phosphate; IgG, Immunoglobulin G; m 6 A‐IP, Methylated RNA immunoprecipitation; N‐Ca, N‐cadherin; Vim, Vimentin.

Article Snippet: The concentration of HBEGF was quantified using an HBEGF ELISA Kit (#EK14428, Signalway Antibody, College Park, MD, USA) according to the manufacturer's instructions.

Techniques: Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Modification, Over Expression, Positive Control, Mutagenesis, Methylation, RNA Immunoprecipitation, Sequencing, Binding Assay

Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: FTO increases the stability of HBEGF mRNA via YTHDF2. (A) Venn diagram showing 135 common genes among three times of repetition from MS results. (B) RIP‐qPCR verified the enrichment of HBEGF mRNA on YTHDF2 protein in MKN45 cells. (C) RNA pull‐down showing the binding of HBEGF mRNA to YTHDF2. (D) Western blotting showed an upregulated protein level of HBEGF with YTHDF2 knockdown in MKN45 cells. (E‐G) qRT‐PCR analysis of HBEGF mRNA in the indicated group when MKN45 cells were exposed to actinomycin D, with or without YTHDF2 knockdown (E), with or without FTO knockdown (F) and with or without FTO overexpression (G). (H) Expression correlation between HBEGF and YTHDF2 levels in GC tissues from the TCGA‐STAD cohort ( n = 408). (I) Log‐rank test indicating the OS of individuals with high ( n = 257) or low ( n = 618) YTHDF2 expression in the GSE62254 . (J) Western blotting showed protein level of HBEGF in MKN45 cells with FTO knockdown or YTHDF2 knockdown. (K) Schematic showing the construction of Flag‐tagged wild‐type or m 6 A modification site‐mutant HBEGF probes. (L‐N) Western blotting showed protein level of Flag‐tagged HBEGF in MKN45 cells among indicated groups, with or without FTO knockdown (L), with or without FTO overexpression (M) and with or without YTHDF2 knockdown (N). ∗ P < 0.05 and ∗∗∗ P < 0.001 according to the unpaired t ‐test (B), two‐way ANOVA (E to G), Pearson correlation tests (H) and log‐rank test (I). Abbreviations: 3’UTR, 3’untranslation region; 5’UTR, 5’untranslation region; CDS, Coding DNA Sequence; Flag‐mut, Flag‐labeled mutant HBEGF ; Flag‐WT, Flag‐labeled wild‐type HBEGF ; FTO mut , mutant FTO ; FTO , fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; HBEGF , heparin‐binding EGF like growth factor; MS, mass spectrometry; NC , negative control; YTHDF2, YTH N(6)‐methyladenosine RNA binding protein 2.

Article Snippet: The concentration of HBEGF was quantified using an HBEGF ELISA Kit (#EK14428, Signalway Antibody, College Park, MD, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, Western Blot, Knockdown, Quantitative RT-PCR, Over Expression, Expressing, Modification, Mutagenesis, Sequencing, Labeling, Mass Spectrometry, Negative Control, RNA Binding Assay

Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: MA treatment impeded the progression of GC following H. pylori eradication. (A) Western blotting showed protein levels of FTO and HBEGF after cagA + H. pylori infection and kanamycin therapy in MKN45 cells. The numbers below the bands represented the relative gray values of quantification. (B) Schematic showing construction of the CagA‐tet‐on system for the cycling CagA induction process. (C‐D) Western blotting showed protein levels of FTO and HBEGF in MKN45 (C) and AGS (D) cells after 20 cycles of CagA induction and withdrawal of DOX for 48 hours. (E) Dot‐blotting assay showed m 6 A levels in MKN45 (left) and AGS (right) cells treated with MA or DMSO. (F‐G) qRT‐PCR (F) and western blotting (G) showed the expression level of HBEGF when GC cells were treated with MA or DMSO. (H) Western blotting showed protein levels of EMT markers in response to MA treatment. The numbers below the bands represented the relative gray values of quantification. (I) Migration and invasion assay showed the suppressive effect of MA on MKN45 cells. Quantification results are shown below. n = 3. Scale bar, 200 µm. (J‐M) Representative image of immunofluorescence staining in GC organoids exposed to indicated groups, N‐ca (J), E‐ca (K), Snail (L) and Vim (M). Scale bar, 200 µm. (N) Quantification results of fluorescence intensity among EMT markers using ImageJ software. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 according to unpaired t ‐tests (F and I) and one‐way ANOVA (N). Abbreviations: CagA − , CagA uninduced; CagA + , CagA induced; DAPI, 4',6‐diamidine‐2‐phenylindole; DMSO, Dimethyl sulfoxide; DOX, doxycycline; E‐Ca, E‐cadherin; EMT, epithelial‐mesenchymal transition; FTO, fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; HBEGF, heparin‐binding EGF like growth factor; Kna, kanamycin; MA, meclofenamic acid; MB, Methylene blue stain; N‐Ca, N‐cadherin; Vim, Vimentin.

Article Snippet: The concentration of HBEGF was quantified using an HBEGF ELISA Kit (#EK14428, Signalway Antibody, College Park, MD, USA) according to the manufacturer's instructions.

Techniques: Western Blot, Infection, Quantitative RT-PCR, Expressing, Migration, Invasion Assay, Immunofluorescence, Staining, Fluorescence, Software, Binding Assay

Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: Graph illustration. CagA‐positive H. pylori increases FTO expression, which results in “hit‐and‐run” GC progression. However, the synergism of the FTO inhibitor MA with H. pylori eradication prevents GC development. Abbreviations: CagA, cytotoxin‐associated gene A; CEA, carcinoembryonic antigen; CK20, cytokeratin 20; CK7, cytokeratin 7; EpCAM, epithelial cell adhesion molecule; FTO, fat mass and obesity‐associated protein; FTO, fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; HBEGF, heparin‐binding EGF like growth factor; JUN, Jun Proto‐Oncogene; m 6 A, N6‐methyladenosine; MA, meclofenamic acid; T4SS, the type IV secretion system; YTHDF2, YTH N(6)‐methyladenosine RNA binding protein 2.

Article Snippet: The concentration of HBEGF was quantified using an HBEGF ELISA Kit (#EK14428, Signalway Antibody, College Park, MD, USA) according to the manufacturer's instructions.

Techniques: Expressing, Binding Assay, RNA Binding Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling.

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Figure 5. Endothelial Notch signaling affects RPE barrier function by regulating the HBEGF paracrine of EC. (A) Analysis for previously published transcriptome sequencing data of normal HUVECs and DAPT treated HUVECs: the GSEA results for ECM (left) and heatmap of the expression of ECM- related genes (right). (B) mRNA and protein levels of HBEGF in normal HUVECs and DAPT-treated HUVECs. (C) mRNA and protein levels of HBEGF in normal HUVECs and NICD adenovirus-infected HUVECs. (D) The secretion levels of HBEGF protein in normal HUVECs and DAPT-treated HUVECs or NICD adenovirus-infected HUVECs were tested using an ELISA kit. (E) Evaluating the RPE barrier function in RPE cells cultured in normal medium, RPE cultured in the EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF)

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Sequencing, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant

Journal: Antioxidants (Basel, Switzerland)

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling.

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Figure 6. r-HBEGF improved the RPE barrier dysfunction of Notch signaling deletion mice. (A) In the SI-induced RPE barrier disfunction model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJf/f intravitreally injected with IgG or r-HBEGF, and control mice (RBPJf/+) at p30, were analyzed using ZO-1 staining. The number of RPE cells with intact tight junctions were quantitatively compared. Red circles indicate the location of the optic disk. Scale bars: 50 µm. (B) In the laser-induced CNV model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJf/f intravitreally injected with IgG or r-HBEGF and control mice (RBPJf/+) were analyzed using IB-4 staining. IB4 positive areas were quantitatively compared. Three biological replicates were performed and values are presented as the mean ± SEM (n = 3). Scale bars: 150 µm. * p < 0.05, ** p < 0.01.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Staining, Injection, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling.

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Figure 7. EC regulates MMP-9 expression in RPE cells via Endothelial Notch signaling-driven HBEGF secretion. (A) Protein interaction prediction analysis results of HBEGF and MMP-9, using the String database. (B) mRNA level, protein level, and enzyme activity level of MMP-9 in the RPE, RPE with HUVEC, and RPE with Notch signaling-inhibited HUVECs were tested via qRT-PCR, Western blot, and gelatin assay kit. (C) mRNA levels of MMP-9 in normal HUVECs and DAPT-treated HUVECs were tested via qRT-PCR. (D,E) mRNA and protein levels of MMP-9 in the RPE, RPE with HUVECs, RPE with siNC-treated HUVECs, and RPE with siHBEGF-treated HUVECs were tested via qRT-PCR and Western blot. (F) Protein levels of MMP-9 in RPE cells cultured in normal medium, RPE cultured in EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF) were tested using Western blot. Three biological replicates were performed and values are presented as the mean ± SEM (n = 3). NS, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Cell Culture, Recombinant