Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: The abnormal m6A modification and protein levels of GLIS1 in human aged kidney tissues. A Total m6A RNA levels in the H/aged ( n = 10) and H/control group ( n = 10) detected by m6A methylation quantification kit; B differential methylated mRNA expressions between H/aged (A) and H/control (Y) group by using m6A-mRNA epitranscriptomic microarray analysis ( n = 3); C analysis of significant changes in methylated mRNA expression levels between the H/aged and H/control group; D the Gene Ontology (GO) biological process enrichment analysis pathways of predicted mRNA targets; E enriched m6A modification of GLIS1 in the H/aged and H/control group by MeRIP assay ( n = 4); F , G protein level of GLIS1 in the H/aged and H/control group by western blot, and its semi-quantitative analyses ( n = 6). The data are expressed as the mean ± SD of three independent experiments. *** P < .001 indicates a significant difference versus the H/control group by Student’s t -test

Article Snippet: Human m6A epitranscriptomic microarray and mRNA microarray analyses were performed based on Arraystar’s standard protocols.

Techniques: Modification, Control, Methylation, Microarray, Expressing, Western Blot

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: METTL3 directly interacted with GLIS1 in kidney aging. A The mRNA level of METTL3 in the vector and siMETTL3 group in HK-2 cells by RT-qPCR ( n = 3); B , C protein level of GLIS1 by western blot in the vector and siMETTL3 group in HK-2 cells, and its semi-quantitative analysis ( n = 3); D protein levels of METTL3, METTL14, and WTAP by western blot in the 24-month-old and 6-month-old group ( n = 6); E , F the expression of METTL3 in the 24-month-old and 6-month-old group by IHC assay, and its semi-quantitative analysis ( n = 6), scale bar = 50 μm; G immunostaining for METTL3 (green), with DAPI (blue) counterstaining by IF staining in the 24-month-old and 6-month-old group ( n = 6), scale bar = 50 μm; H immunostaining for METTL3 (green), GLIS1 (red), with DAPI (blue) counterstaining by IF staining in HK-2 cells ( n = 3), scale bar = 50 μm; I , J METTL3 RIP and RT-PCR confirmed the interaction between METTL3 and GLIS1 mRNA, and its semi-quantitative analysis ( n = 3); K the lower m6A level of GLIS1 in siMETTL3 group compared with the vector group in HK-2 cells by using MeRIP-qPCR ( n = 3); L the mRNA level of GLIS1 in the vector and siMETTL3 group in HK-2 cells by RT-qPCR ( n = 3); M mutations at the two putative m6A sites in GLIS1 (A to G); N m6A level of GLIS1 in HK-2 cells with co-expression of siMETTL3 and GLIS1-WT/Muts by MeRIP-qPCR ( n = 3); O the mRNA level of GLIS1 in the vector and GLIS1-Mut3 group by RT-qPCR ( n = 3). The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the vector group ( A , C , K ) or 6-month-old group ( E ) or IgG group ( J ) by Student’s t -test; ## P < .01, ### P < .001 versus the vector-WT group, ++ P < .01, +++ P < .001 versus the siMETTL3-WT group, ** P < .01 versus vector group by two way-ANOVA ( N )

Article Snippet: Human m6A epitranscriptomic microarray and mRNA microarray analyses were performed based on Arraystar’s standard protocols.

Techniques: Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: METTL3 ameliorated age-related renal fibrosis in accelerated aging mouse model. A , B Downregulated protein levels of METTL3, METTL14, and GLIS1 in an accelerated aging mouse model were reversed in the presence of AAV-METTL3, and the upregulated protein levels of P16 INK4A and FN by western blot in the accelerated aging mouse model were reduced by introducing AAV-METTL3 ( n = 6); C the enriched m6A modification of GLIS1 in the control, AAV-Vector and AAV-METTL3 group by MeRIP-qPCR assay ( n = 6); D GLIS1 mRNA levels in the control, AAV-Vector, and AAV-METTL3 group detected by RT-qPCR ( n = 5); E protein levels of GLIS1, P16 INK4A , and FN in the control, AAV-Vector and AAV-METTL3 group by Masson staining and IHC assay and their semi-quantitative analyses ( n = 6), scale bar = 50 μm; F immunostaining for PPARα (red) and CPT1A (green), HK2 (pink), and PDK1 (yellow), with DAPI (blue) counterstaining by IF staining in the control, AAV-Vector, and AAV-METTL3 group ( n = 6), scale bar = 50 μm. The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the AAV-vector group by one-way ANOVA

Article Snippet: Human m6A epitranscriptomic microarray and mRNA microarray analyses were performed based on Arraystar’s standard protocols.

Techniques: Western Blot, Modification, Control, Plasmid Preparation, Quantitative RT-PCR, Staining, Immunostaining

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: YTHDF1 identified with m6A-meditated GLIS1 mRNA and participated the translation process of GLIS1 protein. A The expression of YTHDF1 in the 24-month-old and 6-month-old group by IHC assay, and its semi-quantitative analysis ( n = 6), scale bar = 50 μm; B immunostaining for YTHDF1 (green), with DAPI (blue) counterstaining by IF staining in the 24-month-old and 6-month-old group ( n = 6), scale bar = 50 μm; C , D protein level of YTHDF1 in the 24-month-old and 6-month-old group by western blot, and its semi-quantitative analysis ( n = 6); E mRNA level of YTHDF1 by RT-qPCR in HK-2 cells within vector or siYTHDF1 ( n = 3); F protein level of GLIS1 in HK-2 cells within vector or siYTHDF1, and its semi-quantitative analysis ( n = 3); G double immunostaining for GLIS1 (red) and YTHDF1 (green) by IF staining in HK-2 cells ( n = 3), scale bar = 50 μm; H , I RIP and RT-PCR assays confirmed the interaction between YTHDF1 and GLIS1 mRNA, and its semi-quantitative analysis ( n = 3); J the expression of GLIS1 with RPL22-FLAG label by ribosomal immunoprecipitation in HK-2 cells ( n = 3); K the mRNA level of GLIS1 by RT-qPCR in HK-2 cells within vector or siYTHDF1 ( n = 3); L , M relative luciferase activity of the GLIS1-WT or GLIS1-Mut 3′UTR luciferase reporter in the vector and siMETTL3 group, n = 3. The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the 6-month-old group, or vector group, or IgG group by Student’s t -test

Article Snippet: Human m6A epitranscriptomic microarray and mRNA microarray analyses were performed based on Arraystar’s standard protocols.

Techniques: Expressing, Immunostaining, Staining, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Double Immunostaining, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Luciferase, Activity Assay

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: Differentially expressed miRNA in exosomes from tuberculous pleural effusion and transudative pleural effusion (as control) by miRNAs sequencing. A Heatmap of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients; n = 9. RPKM values are represented by gradient colors and shown for each sample. Red represents a higher RPKM; blue represents a lower RPKM. Results are based on nine RNA sequencing samples. B Venn diagram showing the overlap between differentially expressed miRNAs in TPE exosomes and transudate exosomes. C Volcanic map of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients. Adjusted P value < 0.05 and fold change > 1 was set as restrictive conditions to identify the differentially expressed genes. D Pathway enrichment analysis showed the significant target genes of differentially expressed miRNAs associated with various KEGG pathways. E The expression of three differentially expressed miRNAs in TPE exosomes and transudate exosomes was verified by RT-qPCR and normalized by the U6. Data are expressed as mean ± SEM. n = 6, **** P < 0.0001 (Paired student’s t-test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Control, Sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: miR-503-5p regulated Smurf1/Smad7 signaling pathway. A , B Venn diagram showing overlap between differentially up-regulated miRNAs in TPE exosomes and miRNAs targeting Smurf1 or Smad7 predicted by the miRTarBase. The online analysis database “miRTarBase” ( https://miRTarBase.cuhk.edu.cn/ ) was used. C Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6. D - E Human PMCs were transfected with miR-25-3p mimics or miR-503-5p mimics or miR-92a-3p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which intracellular mRNA levels of Smurf1 were measured by RT-qPCR and normalized to GAPDH ( D ). The protein expression of Smurf1 and TGF-β receptor (TGFBR) were detected by western blotting. Bar graphs revealed changes in relative ratio of Smurf1 and TGFBR to GAPDH. F Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which mRNA expression of Smurf1, TGFBR and COL1A1 were detected by qRT-PCR. G Human PMCs were incubated with miR-503-5p mimics. After 24 h, Smurf1 protein was detected by immunofluorescence staining and nuclei with DAPI staining. Bar scale: 50 μm. H - K Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h. miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6 ( H ). The protein expression of S Smurf1, TGFBR and COL1A1 were detected by western blotting ( I ). Bar graphs revealed changes in the relative ratio to GAPDH ( J ). mRNA levels of S Smurf1, TGFBR and COL1A1 were measured by RT-qPCR and normalized to GAPDH ( K ). Data are mean ± SEM. n = 3. * P < 0.05 (student’s t-test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Immunofluorescence, Staining

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: Triple miRNAs inhibitor attenuated TPE-Exo induced pleural fibrosis. C57BL/6 mice were intra-pleural injected by using PBS (100 µl/mouse), TPE-Exo (100 µl/mouse), TPE-Exo plus control inhibitor, or TPE-Exo plus triple miRNAs inhibitor with carbon particles (0.1 mg/mouse) as descriptions in the Methods. TPE-Exo from 50 ml TPE was administered at days 1, 5, 9. In TPE-Exo plus triple miRNAs inhibitor group, TPE-Exo was co-incubated with triple miRNAs inhibitor which restrained expressions of miR-150-3p, miR-424-3p and miR-503-5p. All mice were euthanized at day 21, and tissues were taken for analysis. A Representative Masson’s trichrome staining images of visceral pleura from lung sections, parietal pleura from chest wall and diaphragm sections. Original magnification, ×400. B Changes in pleural thickness. C Changes in collagen percentages of visceral and parietal pleura. Data are expressed as mean ± SEM. n = 6 mice. *** P < 0.001 (One-way ANOVA followed by the Bonferroni’s test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Injection, Control, Incubation, Staining