Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: Upregulation of IRF4 was related to kidney damage in SLE patients. A Schematic diagram of MeRIP-seq detecting the m6A level of IRF4; B qRT-PCR for IRF4 mRNA expression in PBMCs of healthy controls (HC), SLE-AP, and SLE-SP; C Western blotting for IRF4 protein expression in PBMCs of HC, SLE-AP and SLE-SP; Correlation between IRF4 expression and the proportions of peripheral T lymphocytes ( D ), T helper cells ( E ), suppressor T cells ( F ), NK cells ( G ) and B lymphocytes ( H ) in SLE patients ( n =12); I Correlation between IRF4 expression and the proportions of peripheral B lymphocytes in SLE-AP ( n =5). * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: Comparison of clinical manifestations and laboratory indexes between low-level and high-level IRF4 group

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Comparison, Expressing

Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: IRF4 expression was elevated in plasma cells of MRL/lpr mice. Flow cytometry for the proportion of spleen B-cell subsets ( A , B ), IRF4 expression of spleen B cell subsets ( C ), and the proportion of IRF4+ cells of spleen B cell subsets ( D ) in MRL/lpr mice; E qRT-PCR for IRF4 mRNA expression in kidneys of C57BL/6 ( n =6) and MRL/lpr mice ( n =7); F Western blotting for IRF4 protein expression in the kidneys of C57BL/6 ( n =6) and MRL/lpr mice ( n =7). Flow cytometry for the proportion ( G ), and mean fluorescence intensity of IRF4 ( H ) in kidney plasma cells of C57BL/6 ( n =7) and MRL/lpr mice ( n =7). I Immunofluorescence staining for IRF4 expression and its co-localization with kidney plasma cells of C57BL/6 and MRL/lpr mice, the Scale bar was 50 μm; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Expressing, Clinical Proteomics, Flow Cytometry, Quantitative RT-PCR, Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: IRF4 depletion alleviated kidney damage of MRL/lpr mice. A Western blotting for IRF4 protein expression in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus; ELISA or biochemical kit for levels of plasma creatinine ( B ), urine ALB ( C ), plasma ANA ( D ) and plasma urea nitrogen ( E ) in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. F Immunofluorescence staining for kidney IgG and C3 deposition in kidneys MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; G HE, PAS and MASSON staining for pathological condition in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; H Flow cytometry for proportion of kidney plasma cells in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Western Blot, Expressing, Injection, Control, Virus, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunofluorescence, Staining, Flow Cytometry

Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: IRF4 depletion partially reversed METTL3-aggravated kidney damage of MRL/lpr mice. A Western blotting for METTL3 and IRF4 protein expression in kidneys of MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus; ELISA or biochemical kit for levels of plasma creatinine ( B ), urine ALB ( C ), plasma ANA ( D ), and plasma urea nitrogen ( E ) in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus. F Immunofluorescence staining for kidney IgG and C3 deposition in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus, Scale bar, 50 μm; G HE, PAS and MASSON staining for pathological condition in kidneys of MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus, Scale bar, 50 μm; H Flow cytometry for proportion of kidney plasma cells in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus;* P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Western Blot, Expressing, Injection, Virus, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunofluorescence, Staining, Flow Cytometry

Journal: BMC Medicine

Article Title: METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

doi: 10.1186/s12916-024-03735-y

Figure Lengend Snippet: METTL3 promoted m6A RNA modifications on IRF4 mRNA, inducing IRF4 expression. Romas cells were transduced by lentiviruses of METTL3 overexpression and its control virus; A Western blotting for METTL3 and IRF4 protein expression in ( n =3); B qRT-PCR for IRF4 mRNA expression ( n =3); C MeRIP-qPCR assays, along with ( D ) agarose gel images for detection of m6A enrichment in IRF4 mRNA. RNA-sequencing on the kidneys of mice injected with AAV9-IRF4-sh and the control virus were conducted; E Volcano plot and ( F ) Heatmap of differentially expressed genes in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus; G KEGG analysis of differentially expressed genes; H qRT-PCR for verification of Cxcl1 , Bcl3 , Fos , Socs3 , and Jun mRNA expression. I Mechanism diagram of METTL3-IRF4 axis promoting kidney injury during SLE. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China).

Techniques: Expressing, Over Expression, Control, Virus, Western Blot, Quantitative RT-PCR, Agarose Gel Electrophoresis, RNA Sequencing, Injection

Journal: bioRxiv

Article Title: Selective alteration to CD4 T cell differentiation by heterozygous IRF4 L116R protects against neuroinflammation

doi: 10.1101/2025.06.25.661400

Figure Lengend Snippet: (A) Western blot of analysis showing IRF4 expression in splenocytes from Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R and Irf4 -/- mice. (B) Representative flow cytometric profiles of CD3 vs CD19 expression in splenocytes for indicated genotypes. (C-D) Quantification of T (CD3+) and B (CD19+) cells in the spleen. (E) Representative flow cytometric plots of CD4+ and CD8+ T cells in the spleens for Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R and Irf4 -/- mice. (F-G) Quantification of CD4+ and CD8+ cells as a proportion of CD3+ T cells in the spleen for indicated genotypes. (H) Representative flow cytometric profiles of spleen CD4+ naïve (CD62L + CD44 - ), central memory (CD62L + CD44 + ), effector memory (CD62L - CD44 + ) and terminally differentiated effector memory (CD62L - CD44 - ) for Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R and Irf4 -/- mice. (I-L) Quantification of naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) cells as a proportion of conventional (FOXP3-) CD4+ T cells in the spleen. (M) Representative histograms of FOXP3 expression in splenic CD4+ T cells for indicated genotypes. (N) Quantification of FOXP3+ Tregs as a proportion of CD4+ T cells in the spleen for indicated genotypes. Each dot is representative of a single mouse, n= 4. One-way ANOVA multiple comparisons adjusted with Tukey’s HSD are applied between groups.

Article Snippet: Following semi-dry electrophoretic transfer of proteins onto polyvinyldifluoride (PVDF) membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk in tris-buffered saline with Tween-20 (TBST) and incubated overnight with primary antibodies against IRF4 (1:1000 dilution, 62834, Cell Signaling Technologies) and GAPDH (1:4000 dilution, ab8245, Abcam).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Selective alteration to CD4 T cell differentiation by heterozygous IRF4 L116R protects against neuroinflammation

doi: 10.1101/2025.06.25.661400

Figure Lengend Snippet: (A) Representative histograms of FOXP3 expression in MACS sorted CD4+ Tnaive cells from Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R and Irf4 -/- mice under Treg differentiation stimuli. (B) Quantification of FOXP3+ cells following Treg differentiation for the indicated genotypes. (C , E) Representative flow cytometric plots of IL-17A and IFNγ expression in CD4+ Tnaive cells from Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R and Irf4 -/- mice under Th1 or Th17 differentiation stimuli, respectively. (D , F) Quantification of IFNγ and IL-17A expression. Each dot is representative of a single mouse, n=3-4. One-way ANOVA multiple comparisons are applied between groups using linear mixed models in R.

Article Snippet: Following semi-dry electrophoretic transfer of proteins onto polyvinyldifluoride (PVDF) membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk in tris-buffered saline with Tween-20 (TBST) and incubated overnight with primary antibodies against IRF4 (1:1000 dilution, 62834, Cell Signaling Technologies) and GAPDH (1:4000 dilution, ab8245, Abcam).

Techniques: Expressing

Journal: bioRxiv

Article Title: Selective alteration to CD4 T cell differentiation by heterozygous IRF4 L116R protects against neuroinflammation

doi: 10.1101/2025.06.25.661400

Figure Lengend Snippet: (A) EAE score and (B) percentage of disease-free mice 28 days post-induction with MOG peptide for Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R mice. Mice which become symptomatic are indicated in brackets. Representative flow cytometry plots for CD4 vs CD44 (C) , IL-17A vs CD44 (E) , IFNγ vs CD44 (G) and GM-CSF vs CD44 (I) expression in CD4+ T cells following PMA/ionomycin stimulation of blood samples collected on day 21 (EAE disease peak). Quantification of CD44+CD4+ (D) , CD44+IL-17A+ (F) , CD44+IFNγ+ (H) and CD44+GM-CSF+ (J) cells as a frequency of CD4+ T cells in the blood. Each dot is representative of a single mouse with symptomatic mice represented by triangles, n= 5-6 per genotype. Logrank (Mantel-Cox) test is applied for disease free survival curve comparison. One-way ANOVA multiple comparisons adjusted with Tukey’s HSD are applied between groups.

Article Snippet: Following semi-dry electrophoretic transfer of proteins onto polyvinyldifluoride (PVDF) membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk in tris-buffered saline with Tween-20 (TBST) and incubated overnight with primary antibodies against IRF4 (1:1000 dilution, 62834, Cell Signaling Technologies) and GAPDH (1:4000 dilution, ab8245, Abcam).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: bioRxiv

Article Title: Selective alteration to CD4 T cell differentiation by heterozygous IRF4 L116R protects against neuroinflammation

doi: 10.1101/2025.06.25.661400

Figure Lengend Snippet: (A) Absolute CD4+ T cell numbers, (B) CD44+CD4+ cell frequency of CD4+ T cells, and (C) absolute CD44+CD4+ T cell numbers in the CNS of Irf4 +/+ , Irf4 L116R/+ , Irf4 L116R/L116R mice 28 days post-induction with MOG peptide. Representative flow cytometry plots for IL-17A vs CD44 (D) , IFNγ vs CD44 (F) and GM-CSF vs CD44 (H) expression in CD4+ from PMA/Ionomycin stimulated CNS cells. Quantification of CD44+IL-17A+ (E) , CD44+IFNγ+ (G) and CD44+GM-CSF+ (I) cells as a frequency of CD4+ T cells in the CNS. Flow cytometric profiles (J) and quantification (K) of FOXP3 expression in CD4+ T cells in the CNS. Each dot is representative of a single mouse with symptomatic mice represented by triangles, n= 4-6 per genotype. One-way ANOVA multiple comparisons adjusted with Tukey’s HSD are applied between groups.

Article Snippet: Following semi-dry electrophoretic transfer of proteins onto polyvinyldifluoride (PVDF) membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk in tris-buffered saline with Tween-20 (TBST) and incubated overnight with primary antibodies against IRF4 (1:1000 dilution, 62834, Cell Signaling Technologies) and GAPDH (1:4000 dilution, ab8245, Abcam).

Techniques: Flow Cytometry, Expressing