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

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: IRF4 and MYC protein levels in MM cell‐lines following treatment with JQ1, OTX015, SGC‐CBP30, ISOX‐DUAL and JQ1+ SGC‐CBP30. Changes in MYC and IRF4 protein levels were analyzed by Western Blot following IC 50 drug treatments for 48 and 72 h in KMS‐12‐BM, SKMM‐1, NCI‐H929 and OCI‐AML3. The control (CTRL) is 2 mM DMSO treatment. β ‐actin was used as loading control. Quantification was performed by using LI‐COR machine and protein levels were expressed relative to the control treatment

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Western Blot

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: IRF4 and MYC mRNA expression in MM cell‐lines following treatment with JQ1, OTX015, SGC‐CBP30, ISOX‐DUAL and JQ1+ SGC‐CBP30. IRF4 and MYC mRNA expression was analyzed by qPCR following IC 50 drug treatments for 48 and 72 h in KMS‐12‐BM (green bars), SKMM‐1 (orange bars), NCI‐H929 (black bars) and OCI‐AML3 (blue bars) cells. The control (CTRL) is 2 mM DMSO treatment. Transcript levels were normalized against β ‐actin expression and expressed relative to the control treatment. Data are shown as mean ± SEM. A t ‐test was performed with reference to the control. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Expressing

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: IRF4 downstream gene mRNA expression in MM cell‐lines following treatment with JQ1, OTX015, SGC‐CBP30, ISOX‐DUAL and JQ1+ SGC‐CBP30. KLF2 and PRDM1mRNA expression was analyzed by qPCR following IC 50 drug treatments for 48 and 72 h in KMS‐12‐BM (green bars), SKMM‐1 (orange bars), and NCI‐H929 (black bars) cells. The control (CTRL) is 2 mM DMSO treatment. Transcript levels were normalized against β ‐actin expression and expressed relative to the control treatment. Data are shown as mean ± SEM. A t ‐test was performed with reference to the control. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Expressing

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: Computational model of the molecular regulatory network in MM cells. A, Systems Biology Graphical Notation (SBGBN) diagram of the model of IRF4, MYC and PRDM1 regulation. Positive regulation is indicated by lines capped with circles. Negative regulation is indicated by lines capped with bars. B, Experimentally measured expression of the indicated molecular species in H929, SKMM‐1, KMS cell‐lines exposed to SGC‐CBP30, JQ1, OTX015, ISOX‐DUAL, and JQ1+SGC‐CBP30 combination. Each shaded region represents the standard deviation of 3 experimental replicates. The modeled response is shown with a solid line. The model assumes a half‐life for IRF4 of 7 h. The squared distance between the mean experimental result and modeled response for each timepoint is shown in the bottom right with colors consistent with other panels

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Expressing, Standard Deviation

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: Analysis of IRF4 stability in MM cell‐lines and updated computational model of the molecular regulatory network in MM cell. A, KMS‐12‐BM, SKMM‐1, NCI‐H929 were incubated with 10 μg/ml cycloheximide for the indicated time points and cell lysates analyzed by Western blotting for protein levels of IRF4 and MYC. β ‐actin was used as a loading control. B, Experimentally measured expression of the indicated molecular species in H929, SKMM‐1, KMS cell‐lines exposed to SGC‐CBP30, JQ1, OTX015, ISOX‐DUAL, and JQ1+SGC‐CBP30 combination. Each shaded region represents the standard deviation of 3 experimental replicates. The modeled response is shown with a solid line. The model uses the experimentally determined IRF4 half‐life. The squared distance between the mean experimental result and modeled response for each timepoint is shown in the bottom right with colors consistent with other panels

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Incubation, Western Blot, Expressing, Standard Deviation

Journal: Hematological Oncology

Article Title: Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4‐MYC oncogenic axis in multiple myeloma

doi: 10.1002/hon.3016

Figure Lengend Snippet: Computational model simulating the effect of a drug acting on MYC transcription, IRF4 transcription or both. Systems Biology Graphical Notation (SBGBN) diagram of the model of IRF4, MYC and PRDM1 regulation. Positive regulation is indicated by lines capped with circles. Negative regulation is indicated by lines capped with bars. Drugs are shown impacting IRF4 transcription (A) and MYC transcription (B). Experimentally measured expression of the indicated molecular species in H929, SKMM‐1, KMS cell‐lines exposed to SGC‐CBP30, JQ1, OTX015, ISOX‐DUAL, and JQ1+SGC‐CBP30 combination. The impact of single targeting IRF4 (A, left) and MYC (B, middle) is shown, along with the combination (A + B, right). Each shaded region represents the standard deviation of 3 experimental replicates. The modeled response is shown with a solid line. The model uses the experimentally determined IRF4 half‐life. The squared distance between the mean experimental result and modeled response for each timepoint is shown in the bottom right with colors consistent with other panels

Article Snippet: Primary antibodies: IRF4 (ab133590, Abcam), MYC (sc‐40, Santa‐Cruz Biotechnology) and β ‐actin (A2066, Sigma‐Aldrich).

Techniques: Expressing, Standard Deviation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: Renal myeloid IRF4 was efficiently deleted in LysM-Cre IRF4f/f mice. (A) Renal myeloid cell IRF4 mRNA levels were significantly reduced in LysM-Cre IRF4f/f (MΦ IRF4−/−) mice compared with IRF4f/f (WT) mice. ***P<0.001, n=6 in WT group and n=7 in MΦ IRF4−/− group. (B and C) Immunofluorescence staining demonstrated that most renal F4/80-positive cells (macrophages/dendritic cells) were also IRF4 positive in WT mouse kidneys at day 0 (B) and 5 days (C) after AKI, whereas only a small portion of F4/80-positive cells were IRF4 positive in MΦ IRF4−/− mouse kidneys, indicating effective IRF4 deletion in renal myeloid cells.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: Immunofluorescence, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: Myeloid IRF4 deletion led to attenuated renal macrophage response to early acute ischemic injury. Male IRF4f/f (WT) and LysM-Cre IRF4f/f (MΦ IRF4−/−) mice underwent AKI and were sacrificed at indicated times. (A) WT and MΦ IRF4−/− mice had similar BUN at all early time points after AKI (n=9). (B) Then 1 day after ischemic injury, the mRNA levels of anti-inflammatory Th2/M2 cytokines (YM-1, IL-10, and FIZZ1) were markedly lower in renal myeloid cells from MΦ IRF4−/− mice than WT mice. ***P<0.001, n=9 in WT group and n=7 in MΦ IRF4−/− group. (C) The mRNA levels of proinflammatory Th1/M1 cytokines/chemokines, including iNOS, TNF-α, IL-1α, IL-1β, IL-6, and IL-23, were significantly lower in renal myeloid cells from MΦ IRF4−/− mice than WT mice 1 day after ischemic injury. ***P<0.001, n=9 in WT group and n=7 in MΦ IRF4−/− group. (D) Flow cytometric analysis showed the number of renal macrophages was similar at baseline between MΦ IRF4−/− mice and WT mice, but less increased at days 1 and 2 after ischemic injury in MΦ IRF4−/− mice. Similarly, increased renal neutrophils seen in WT mice day 1 after ischemic injury were attenuated in MΦ IRF4−/− mice. **P<0.01, n=4. (E) CD68 immunofluorescence staining indicated the increased renal macrophages seen in WT mice were markedly attenuated in MΦ IRF4−/− mice from day 1 until day 28. **P<0.01, ***P<0.001, n=4.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: Immunofluorescence, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: Myeloid IRF4 deletion led to relatively better kidney function and decreased renal fibrosis 4 weeks after AKI. Male IRF4f/f (WT) and LysM-Cre IRF4f/f (MΦ IRF4−/−) mice underwent AKI and were sacrificed 4 weeks later. (A and B) MΦ IRF4−/− mice had lower serum creatinine and albuminuria (A) and less tubular injury (B). *P<0.05, **P<0.01, ***P<0.001, n=9 in (A) and n=4 in (B). (C) MΦ IRF4−/− mice had less renal fibrosis as indicated by Sirius red staining. Original magnification ×160. ***P<0.001, n=6. (D) Quantitative PCR analysis showed decreases in renal mRNA levels of profibrotic and fibrotic components, including TGF-β1, TGF-β2, α-SMA, CTGF, collagen I (col I), col III, col IV, and fibronectin (FN) in MΦ IRF4−/− mice. **P<0.01, ***P<0.001; n=7 in WT group and n=8 in MΦ IRF4−/− group. (E) Immunoblotting showed decreased renal protein levels of α-SMA and CTGF in MΦ IRF4−/− mice. **P<0.01, n=4 in WT group and n=5 in MΦ IRF4−/− group. (F) Immunostaining showed that MΦ IRF4−/− mice had decreased renal expression of profibrotic and fibrotic components, including col I, col IV, α-SMA (marker of myofibroblasts), and CTGF. Original magnification ×160 for all. *P<0.05, **P<0.01, ***P<0.001, n=4 for all.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: Staining, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Expressing, Marker

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: Myeloid IRF4 deficiency protected against renal fibrosis in UUO. Male IRF4f/f (WT) and LysM-Cre IRF4f/f (MΦ IRF4−/−) mice underwent UUO for 7 days. (A) Quantitative PCR analysis showed lower renal mRNA levels of profibrotic and fibrotic components, including α-SMA, CTGF, TGF-β1, TGF-β2, collagen I (col I), col III, col IV, and fibronectin (FN) in MΦ IRF4−/− mice. **P<0.01, ***P<0.001; n=8. (B) Sirius red staining showed less renal fibrosis in MΦ IRF4−/− mice. ***P<0.001, n=4.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: IRF4 deletion led to impaired BMDM migration in vitro. BMDMs were isolated from IRF4f/f (WT) and LysM-Cre IRF4f/f (MΦ IRF4−/−) mice, and were immediately used for in vitro Transwell migration assays. (A) Chemotactic responses to 10% FBS, fMLP, and CX3CL1 were markedly lower for IRF4−/− BMDMs than WT BMDMs. ***P<0.001, n=4–6. (B) Chemotactic responses to CCL19 and CCL21 were also significantly lower for IRF4−/− BMDMs than WT BMDMs. ***P<0.001, n=5.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: Migration, In Vitro, Isolation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

doi: 10.1681/ASN.2020071010

Figure Lengend Snippet: Myeloid IRF4 deletion led to attenuated macrophage infiltration in vivo. BMDMs were isolated from WT and MΦ IRF4−/− mice, labeled with the monocyte tracking dye PKH26 and injected into WT mice that would undergo AKI 16 hours later, and labeled cells in the kidney were evaluated 3 days after ischemic injury. (A) The number of renal-infiltrated, PKH26, and F4/80 double-positive cells were significantly lower in mice receiving IRF4−/− monocytes than in mice receiving WT monocytes. ***P<0.001, n=4. (B) Flow cytometric analysis determined that F4/80 and PKH26 double-positive cell numbers were significantly lower in mice receiving IRF4−/− monocytes than in mice receiving WT monocytes. ***P<0.001, n=8.

Article Snippet: Antibodies Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) was purchased from AbD Serotec (now Bio-Rad), mouse anti–α-smooth muscle actin (α-SMA, a marker of myofibroblasts, A5228) was from Sigma; rabbit anti-murine collagen type I (600–401–103–0.1) and collage type IV (600–401–106–0.1) were from Rockland Immunochemicals; mouse anti–4-hydroxynonenal (4-HNE, a marker of oxidative stress, MAB3249), goat anti-CCL19 (AF880), goat anti-CCL21 (AF457) were from R&D; mouse anti-C/EBP homologous protein (2895), mouse anti–α-Tubulin (3873), rabbit anti–phospho-AKT (4060), rabbit anti-AKT (4691), rabbit anti–phospho-ERK (4370), rabbit anti-ERK (9102), rabbit anti–phospho-p38 (4511), rabbit anti-p38 (9212), rabbit anti–phospho-JNK (4668), rabbit anti-JNK (9252), and rabbit anti-PTEN (9552) were from Cell Signaling Technology; goat anti-human connective tissue growth factor (CTGF, SC-14939) was from Santa Cruz Biotechnology; rabbit anti-IRF4 was from St John’s Laboratory ( STJ93764 ); and rabbit anti-CX3CL1 (ab25088) and rabbit anti-CD68 (ab125212) were from Abcam.

Techniques: In Vivo, Isolation, Labeling, Injection