Journal: Gut Microbes

Article Title: Helicobacter pylori infection aggravates hepatic steatosis by lactylation-driven WTAP-mediated m 6 A modification

doi: 10.1080/19490976.2025.2599543

Figure Lengend Snippet: The role of WTAP in hepatic steatosis in vivo by AAV8 tail vein injection . A) Schematic diagram of study design using the AAV8-WTAP injection to mimic the overexpression of WTAP in C57BL/6 mice, n = 5 per group; B) Western blotting of WTAP expression in C57/BL6 mice treated with AAV8-WTAP or AAV8-Blank. n = 5 per group; C) Representative images of the gross appearance in the liver histology (1 cm), quantification of the liver index (%), H&E (100 μm), F4/80 antibody (100 μm), Masson staining (100 μm), and ORO staining (100 μm); D-I) Serum concentration of ALT (U/L) (D), AST (U/L) (E), triglyceride (mg/dL) (F), cholesterol (mg/dL) (G), LDL (mg/dL) (H), and HDL (mg/dl) (I) in C57/BL6 mice treated with AAV8-WTAP or AAV8-Blank. n = 5 per group; J) Representative quantitative PCR analysis with key genes involved in lipogenesis, FAO and OXPHOS metabolism in C57/BL6 mice treated with AAV8-WTAP or AAV8-Blank. n = 5 per group; K) Schematic diagram of study design using AAV8-shWTAP to mimic the inhibition of WTAP in C57BL/6 mice; L) Western blotting of WTAP expression in C57/BL6 mice treated with AAV8-shWTAP, AAV8-shWTAP + H. pylori SS1 infection, AAV8-shBlank, and AAV8-shWTAP + H. pylori infection. n = 5 per group; M) Representative images of the gross appearance in the liver histology (1 cm), quantification of the liver index (%), H&E (100 μm), F4/80 antibody (100 μm), Masson staining (100 μm), and oil red staining (100 μm). N -Q) Serum concentration of ALT (U/L) ( N ), AST (U/L) (O), triglyceride (mmol/L) ( P ), and cholesterol (mmol/L) (Q) in C57/BL6 mice treated with AAV8-shWTAP, AAV8-shWTAP + H. pylori SS1 infection, AAV8-shBlank, and AAV8-shWTAP + H. pylori infection. n = 5 per group. Statistical analysis was performed using Two-tailed Student's t -test (two groups) and one-way analysis of variance (ANOVA) (multiple groups) followed by Bonferroni's test. *p < 0.05; **p < 0.01; and ***p < 0.001 .

Article Snippet: Samples were then incubated with primary antibodies, including anti-F4/80 antibodies (Servicebio, GB113373 ; 1:200), anti-WTAP antibodies (Proteintech, 10200-1-AP; 1:200), anti-H3K18la-antibodies (PTM-bio, PTM-1427RM; 1:200), anti-Pan-Kla-antibodies (PTM-bio, PTM-1401RM; 1:200) overnight at 4 °C.

Techniques: In Vivo, Injection, Over Expression, Western Blot, Expressing, Staining, Concentration Assay, Real-time Polymerase Chain Reaction, Inhibition, Infection, Two Tailed Test

Journal: Gut Microbes

Article Title: Helicobacter pylori infection aggravates hepatic steatosis by lactylation-driven WTAP-mediated m 6 A modification

doi: 10.1080/19490976.2025.2599543

Figure Lengend Snippet: H. pylori infection promotes glycolysis and regulates WTAP expression via histone lactylation . A) Volcano plot of significantly-changed metabolites and KEGG analysis of remarkably changed pathways from untargeted metabolomics analysis; B) Lactic acid level of liver tissues in C57BL/6 mice with or without H. pylori SS1 infection, n = 5 per group; C) The glycolytic capability of HepG2 cells co-cultured with or without H. pylori OMVs using ECAR curve, n = 3 per group; D) Mitochondrial oxidative capacity was measured after HepG2 cells co-cultured with or without H. pylori OMVs using OCR curve, n = 3 per group; E) Intracellular ROS levels of HepG2 cells co-cultured with or without H. pylori 26695 OMVs in a dose or time-dependent manner, n = 3 per group; F-G) mRNA and protein expression of LDHA and LDHB in liver tissues or HepG2 cells following H. pylori 26695 infection using qRT-PCR (F) and Western blot (G) analysis, n = 3 per group. H) mRNA and protein expression of WTAP in HepG2 cells after lactate treatment, n = 3 per group; I) Representative images of Silver staining-MS of lactylated proteins; J) Pan-lysine lactylation levels in cells were measured at indicated times with 10 ug/ml H. pylori 26695 OMVs or after the indicated dose treatment for 24 h and lysine lactylation levels of H3 were detected under the same conditions. K) ChIP-qPCR using anti-H3K18la antibodies validates H3K18la enrichment in peak 1 and peak 2 within the promoter region of WTAP in HepG2. L) WTAP mRNA and protein level after 2-DG and oxamate treatment, n = 3 per group. M) ChIP-qPCR using anti-H3K18la antibodies validates H3K18la enrichment in HepG2 cells after 2-DG or oxamate treatment or si-LDHA/B transfection. N ) WTAP mRNA and protein level in HepG2 cells after si-LDHA/B transfection with or without Nala addition, n = 3 per group. Statistical analysis was performed using Two-tailed Student's t -test (two groups) and one-way analysis of variance (ANOVA) (multiple groups) followed by Bonferroni's test. *p < 0.05; **p < 0.01; and ***p < 0.001 .

Article Snippet: Samples were then incubated with primary antibodies, including anti-F4/80 antibodies (Servicebio, GB113373 ; 1:200), anti-WTAP antibodies (Proteintech, 10200-1-AP; 1:200), anti-H3K18la-antibodies (PTM-bio, PTM-1427RM; 1:200), anti-Pan-Kla-antibodies (PTM-bio, PTM-1401RM; 1:200) overnight at 4 °C.

Techniques: Infection, Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Silver Staining, ChIP-qPCR, Transfection, Two Tailed Test

Journal: Gut Microbes

Article Title: Helicobacter pylori infection aggravates hepatic steatosis by lactylation-driven WTAP-mediated m 6 A modification

doi: 10.1080/19490976.2025.2599543

Figure Lengend Snippet: Direct lactylation of WTAP enhanced the capture of m6A-modified RNA. A) MOE is used to predict the binding affinity between the WTAP protein and L-lactate; B) Illustration of WTAP Kla sites derived from HepG2 cells identified by LC-MS; C) Lactylation of WTAP in 293 T cells was confirmed by IP method. 293 T cells transfected with FLAG-WTAP, treated with ddH2O or 25 mM L-lactic acid for 24 h. The cells were lysed for SDS pre-treated immunoprecipitation with anti-FLAG antibody followed by western blotting; D) Lactylation of WTAP in HepG2 was confirmed by IP method. HepG2 co-cultured with OMVs or treated with 25 mM LA for 24 h. The cells were lysed for SDS pre-treated immunoprecipitation with anti-METTL3 antibody followed by western blotting; E) FLAG-WTAP, with Myc-METTL3 or Myc-METTL14 were transfected into 293 T cells for 24 h. Then cells were treated with 25 mM L-lactic acid for 24 h. Lysates were used for IP with anti-FLAG antibody, followed by western blotting; F) FLAG-WTAP, with Myc-METTL3 or Myc-METTL14 were transfected into 293 T cells for 24 h. Then cells were treated with 25 mM L-lactic acid for 24 h. Cells were harvested for immunofluorescence staining and further photographed by laser scanning confocal microscopy. Scale bars, 20 mm; G) HepG2 were treated with 25 mM L-lactic acid for 24 h. Cells were harvested for immunofluorescence staining (20 mm) and further photographed by laser scanning confocal microscopy; H) Western blotting analysis of the distribution in nuclear and cytoplasmic fractions of WTAP in HepG2 treated with or without 25 mM L-lactic acid for 24 h. I) Western blotting analysis of WTAP in HepG2 with or without lactic acid treated with 0.1 mg/mL CHX for indicated time; J) FLAG-WTAP was transfected into 293 T cells for 24 h. Then cells were treated with L-lactic acid or Nala for 24 h. Cells were 254 nm UV-crosslinked before harvesting. Lysates were used for SDS pre-treated immunoprecipitation with anti-FLAG antibody, followed by western blotting to detect indicated targets. K) FLAG-WTAP (WT), FLAG-WTAP (K99R), FLAG-WTAP (K134R), and FLAG-WTAP (K99R and K134R) were transfected into 293 T cells for 24 h. Then cells were treated with L-lactic acid for 24 h. Cells were 254 nm UV-crosslinked before harvesting. Lysates were used for SDS pre-treated immunoprecipitation with anti-FLAG antibody, followed by western blotting to detect indicated targets; L) Western blotting analysis of WTAP in 293 T cells transfected with FLAG-WTAP (WT) and FLAG-WTAP (K99R and K134R) with or without lactic acid treated with 0.1 mg/mL CHX for indicated time.

Article Snippet: Samples were then incubated with primary antibodies, including anti-F4/80 antibodies (Servicebio, GB113373 ; 1:200), anti-WTAP antibodies (Proteintech, 10200-1-AP; 1:200), anti-H3K18la-antibodies (PTM-bio, PTM-1427RM; 1:200), anti-Pan-Kla-antibodies (PTM-bio, PTM-1401RM; 1:200) overnight at 4 °C.

Techniques: Modification, Binding Assay, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Transfection, Immunoprecipitation, Western Blot, Cell Culture, Immunofluorescence, Staining, Confocal Microscopy

Journal: Gut Microbes

Article Title: Helicobacter pylori infection aggravates hepatic steatosis by lactylation-driven WTAP-mediated m 6 A modification

doi: 10.1080/19490976.2025.2599543

Figure Lengend Snippet: H. pylori- induced hepatic steatosis in HFDHP mice is alleviated after H. pylori eradication. A) Schematic diagram of the animal experiment; C57BL/6 mice with HFD diets were divided into HFDHP ( H. pylori SS1 infection) and HFDHPe ( H. pylori eradication) groups; n = 5 per group; B−F) Serum concentration of glucose (mg/dL) (B), insulin (pg/mL) (C), triglyceride (mg/dL) (D), cholesterol (mg/dL) (E), and LDL (mg/dL) (F) in H. pylori -infected C57/BL6 mice eradicated with triple therapy or placebo. n = 5 per group; G) Representative images of the gross appearance in the liver histology (1 cm), quantification of the liver index (%), H&E (100μm), F4/80 antibody (100μm), Masson staining (100μm), and ORO staining (100μm). Histogram plots of weight (g), liver index (%), average liver balloon diameters (μm), number of F4/80 positive cells (%), CVF (%), and oil red lipid contents (%) of H. pylori -infected C57/BL6 mice eradicated with triple therapy or placebo. n = 5 per group; H) Representative quantitative PCR analysis with key genes involved in lipogenesis, FAO and OXPHOS metabolism in H. pylori -infected C57/BL6 mice eradicated with triple therapy or placebo. n = 5 per group; I−J) Immunofluorescence staining of Pan-Kla (I) and H3k18la (J) expression in H. pylori -infected C57/BL6 mice eradicated with triple therapy or placebo. n = 5 per group; K) Western blotting of WTAP and GLUT3 expression in H. pylori -infected C57/BL6 mice eradicated with triple therapy or placebo. n = 5 per group. Statistical analysis was performed using Two-tailed Student's t -test. *p < 0.05; **p < 0.01; and ***p < 0.001 .

Article Snippet: Samples were then incubated with primary antibodies, including anti-F4/80 antibodies (Servicebio, GB113373 ; 1:200), anti-WTAP antibodies (Proteintech, 10200-1-AP; 1:200), anti-H3K18la-antibodies (PTM-bio, PTM-1427RM; 1:200), anti-Pan-Kla-antibodies (PTM-bio, PTM-1401RM; 1:200) overnight at 4 °C.

Techniques: Infection, Concentration Assay, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Western Blot, Two Tailed Test