Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: Design and characterization of mouse T cell-targeted TGF- β agonists. a. Schematic of the plasmid construct encoding the MSA–TGM1-targeting-arm fusion protein. b. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. c. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. d. Representative flow cytometry plots showing mouse CD4 and CD8α expression in mouse CD4- or CD8α-transduced HEK293 cells. e. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. f. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. g. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. h. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. i. Dose-dependent curves showing Foxp3 cell percentages in in vitro -cultured CD4 T cells, and Granzyme B–producing cell percentages in in vitro -cultured CD8 T cells treated with the indicated proteins on day 3. Data are presented as mean ± s.e.m.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Plasmid Preparation, Construct, SDS Page, Molecular Weight, Recombinant, Flow Cytometry, Expressing, In Vitro, Cell Culture

Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: a. Frequencies of immune cell subsets. b. SCENIC prediction of IRF4 and Blimp1 transcription factor activity. c. Immunoglobulin gene expression. d. UMAP plots showing cell cycle gene signature scores. e. Quantification of cell cycle gene signature scores. f. Scaled signature scores for TGF-β–inducible genes, senescence genes, and apoptosis-related genes, aggregated by sample.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Activity Assay, Gene Expression

Journal: Advanced Science

Article Title: Targeting Immunoproteasome in Polarized Macrophages Ameliorates Experimental Emphysema Via Activating NRF1/2‐P62 Axis and Suppressing IRF4 Transcription

doi: 10.1002/advs.202405318

Figure Lengend Snippet: ONX‐0914 Suppresses M1 Polarization via NRF1 and NRF2‐P62 Signaling Pathway. A) Differential binding of transcription factors between M1+ONX‐0914 and M1 predicted by TOBIAS. This plot indicates a significant increase in NRF1 (MAFG::Nfe2l1) and NRF2 (MAF::Nfe2l2) binding score following ONX‐0914 treatment. B) Mouse genome NRF1 and NRF2 motifs obtained from JASPAR 2022 database. C) Gene Ontology enrichment analysis of genes upregulated by ONX‐0914 treatment in M1 phenotype of PMs. The y‐axis represents the GO terms, and the x‐axis indicates the gene count. D) Heatmaps displaying proteasome‐related genes, the NRF1 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group (n = 4). E) Volcano plot showing DEGs between ONX‐0914‐treated and untreated M1 AMs. Genes meeting dual thresholds of FDR <0.05 and log 2 (fold change) >1 are highlighted in red, indicating significant upregulation or downregulation after ONX‐0914 treatment. F) Protein expression level of NRF1 analyzed by Western blotting in ONX‐0914‐treated M1 AMs. Data was obtained from 3 independent samples in each group. G) Heatmaps displaying anti‐oxidative stress‐related genes, the NRF2 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group of PMs (n = 4). H) Representative IF images of NRF2 after applying 0.2 µM ONX‐0914 to AMs for 24 h. I) After AMs were pretreated with 0.2 µM ONX‐0914 for 6 h, and then stimulated with 250 µg mL −1 dosage of CSE for 24 h, the gene Nfe2l2 was examined by RT‐qPCR (n = 3). J and K) Transcriptional changes in genes associated with NRF1‐increased binding sites (J) and NRF2‐increased binding sites (K) after ONX‐0914 treatment in M1 of AMs. The plot demonstrates that the regulatory activity of upregulated genes significantly surpasses that of downregulated genes (n = 3). L) After silencing the expression of NRF1 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l1, Sqstm1 , and M1 marker genes ( Nos2, Il12b ) were detected by RT‐qPCR (n = 3). M) After silencing the expression of NRF2 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l2, Sqstm1 , and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). N) Integrative Genomics Viewer (IGV) plot illustrates the binding landscape of the NRF2 transcription factor to Sqstm1 in M0, M0+ONX‐0914, M1 and M1+ONX‐0914. The upper 4 tracks display the signal intensity of NRF2 binding to Sqstm1 , and the lower 4 tracks display the specific locations where NRF2 binding peaks were identified, which suggest the potential regulatory roles in Sqstm1 gene expression after ONX‐0914 treatment in M1 macrophages. O) CUT&Tag‐qPCR shows that the binding affinity of NRF2 to Sqstm1 enhancer and promoter is significantly increased under ONX‐0914 treatment in M1 macrophages. P) RT‐qPCR analysis detected Nfe2l2 , Sqstm1 and M1 marker genes Nos2 , Il12b , Il1b , and Il6 in each group (n = 3). After silencing the expression of NRF2 with siRNA and overexpression of P62 with p3xFLAG‐P62 var2 plasmid, 0.2 µM ONX‐0914 was used to treat M1‐polarized AMs, and the expression of Nfe2l2 , Sqstm1 , and M1 marker genes ( Nos2 , Il12b , Il1b and Il6 ) were detected by RT‐qPCR (n = 3). Q) After silencing the expression of NRF2 with siRNA and using 0.2 µM ONX‐0914 pretreated CSE (250 µg mL −1 ) stimulation of AMs, Sqstm1 and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The plasmids used in this study included p3xFLAG‐P62 var2 (#204576, Addgene) and pMIG‐IRF4 (#58987, Addgene).

Techniques: Binding Assay, Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Marker, Gene Expression, Over Expression, Plasmid Preparation

Journal: Advanced Science

Article Title: Mechanisms of Baicalin Alleviates Intestinal Inflammation: Role of M1 Macrophage Polarization and Lactobacillus amylovorus

doi: 10.1002/advs.202415948

Figure Lengend Snippet: Baicalin inhibits LPS‐induced M1 macrophage polarization in vitro. A) Macrophages in CON, LPS, and BL + LPS group. B) mRNA expression of STAT1 , STAT2 , STAT3 , and TLR2 in CON, LPS, and BL + LPS group ( n = 4). C) mRNA expression of TLR4 , IRF4 , IRF7 , IRF8 , IRF9 , CD86 , IL‐1β , NLRP3 , IL‐12 , and MMP9 in CON, LPS, and BL + LPS group ( n = 4). D) Immunofluorescence analyses of CD86, IL‐1β, NLRP3, and TLR4 protein expression in RAW264.7 cells. Nuclei are counterstained with DAPI (blue). (E) Mean gray value of CD86, IL‐1β, NLRP3, and TLR4 in immunofluorescence analyses ( n = 3).

Article Snippet: The primary antibodies used were obtained from the following suppliers: anti‐ACTB (Sangon Biotech, #D110001), NLRP3 (Beyotime, #AF2155), IL‐1β (Bioss, #bs‐25615R), Occludin (Sangon Biotech, #D110001), CD163 (Sangon Biotech, #D260965), STAT2 (Proteintech, #16674‐1‐AP), TLR4 (Beyotime, #AF8187), Foxp3 (Beyotime, #AF6927), RORγt (Bioss, #bs‐10647R), IL‐17F (Bioss, #bs‐7333R), CTLA4 (ABclonal, #A13966), IRF4 (Proteintech, #66451‐1‐Ig), IRF7 (Proteintech, #22392‐1‐AP), and TLR9 (Beyotime, #AF8193).

Techniques: In Vitro, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: Mechanisms of Baicalin Alleviates Intestinal Inflammation: Role of M1 Macrophage Polarization and Lactobacillus amylovorus

doi: 10.1002/advs.202415948

Figure Lengend Snippet: Lactobacillus amylovorus SKLAN202301ZF regulates macrophage polarization and T cell differentiation. A) mRNA expression of STAT1 , STAT2 , STAT3 , TLR2 , TLR3 , TLR4 , IRF7 , and IRF8 ( n = 12). B) mRNA expression of IRF9 , PIAS1 , PIAS3 , CD226 , Unc93b1 , RORγt , CD86 , and CD163 ( n = 12). C) mRNA expression of NLRP3 , IL‐1β , IL‐17A , IL‐17F , IL‐18 , CCL17 , MMP9 ( n = 12). D) Western blotting was conducted to measure the expression of CTLA4, IRF4, IRF7, TLR9, FOXP3, RORγt, IL‐1β, NLRP3, and ACTB in the colon, E) and the ratio of cleaved to full‐length forms for these proteins was calculated ( n = 4). ACTB was used as a loading control.

Article Snippet: The primary antibodies used were obtained from the following suppliers: anti‐ACTB (Sangon Biotech, #D110001), NLRP3 (Beyotime, #AF2155), IL‐1β (Bioss, #bs‐25615R), Occludin (Sangon Biotech, #D110001), CD163 (Sangon Biotech, #D260965), STAT2 (Proteintech, #16674‐1‐AP), TLR4 (Beyotime, #AF8187), Foxp3 (Beyotime, #AF6927), RORγt (Bioss, #bs‐10647R), IL‐17F (Bioss, #bs‐7333R), CTLA4 (ABclonal, #A13966), IRF4 (Proteintech, #66451‐1‐Ig), IRF7 (Proteintech, #22392‐1‐AP), and TLR9 (Beyotime, #AF8193).

Techniques: Cell Differentiation, Expressing, Western Blot, Control

Journal: PLOS ONE

Article Title: Investigation of the anti-tumor mechanism of tirabrutinib, a highly selective Bruton’s tyrosine kinase inhibitor, by phosphoproteomics and transcriptomics

doi: 10.1371/journal.pone.0282166

Figure Lengend Snippet: Microarray analysis was conducted in the TMD8 xenograft model after 21 days of tirabrutinib administration. (A) Volcano plot for differential gene expression between the vehicle and tirabrutinib 10 mg/kg group with Welch’s t test. (B) Heat map of top altered gene group including 59 upregulated probes and 87 downregulated probes (fold change, >4 or <0.25; P < 0.00001) in the vehicle, tirabrutinib 3 mg/kg, and 10 mg/kg groups. (C) Gene set enrichment analysis (GSEA) plots involved in IRF4, MYC, and mTORC1 signaling in the tirabrutinib 10 mg/kg group versus vehicle group.

Article Snippet: Rabbit monoclonal anti- phosphorylated BTK (Tyr-223) antibody (p-BTK; Novus Biologicals, #NB100-79907), rabbit monoclonal BTK antibody (Cell Signaling Technology, Inc., #3533), rabbit polyclonal phosphorylated AKT (Ser-473) antibody (p-AKT; Cell Signaling Technology, Inc., #9271), rabbit monoclonal AKT (pan) (C67E7) antibody (Cell Signaling Technology, Inc., #4691), rabbit polyclonal phosphorylated p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204) antibody (p-ERK1/2; Cell Signaling Technology, Inc., #9101), rabbit monoclonal p44/42 MAPK (ERK1/2) (137F5) antibody (Cell Signaling Technology, Inc., #4695), rabbit monoclonal phosphorylated PLCγ2 (Tyr-759) (E9E9Y) antibody (p-PLCγ2; Cell Signaling Technology, Inc., #50535), rabbit monoclonal PLCγ2 (E5U4T) antibody (Cell Signaling Technology, Inc., #55512), mouse monoclonal phosphorylated IκBα (Ser-32/36) (5A5) antibody (p-IκBα; Cell Signaling Technology, Inc., #9246), rabbit monoclonal IκBα (44D4) antibody (Cell Signaling Technology, Inc., #4812), rabbit polyclonal phosphorylated PKCβ (phospho-Thr-641) antibody (p-PKCβ; Signalway Antibody LLC, #11172), rabbit monoclonal IRF4 (D9P5H) antibody (Cell Signaling Technology, Inc., #15106), rabbit monoclonal BCL6 (D65C10) antibody (Cell Signaling Technology, Inc., #5650), rabbit monoclonal c-MYC (D84C12) antibody (Cell Signaling Technology, Inc., #5605), and rabbit monoclonal GAPDH (14C10) antibody (Cell Signaling Technology, Inc., #2118) were used.

Techniques: Microarray, Gene Expression

Journal: Cancer Immunology Research

Article Title: The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages

doi: 10.1158/2326-6066.cir-20-0555

Figure Lengend Snippet: Figure 4. Lenalidomide changes the polarization state by interfering with the IRF4/IRF5 homeostasis. A, ChIP sequencing (ChIP-seq) analysis demonstrates the IKZF1 binding profile at the IRF4, IRF5, and IRF3 promoters. ChIP-seq data were from the ENCODE project (GM12878). Protein levels of IKZF1, IRF4, IRF5, IRF3, and b-actin were determined by Western blotting in M2-like macrophages after treatment with increasing concentrations of lenalidomide (as indicated; B) and after different incubation periods with 1 mmol/L lenalidomide (C). D, Protein levels of IKZF1, IRF4, IRF5, IRF3, and b-actin were determined in M2-like macrophages after treatment with either lenalidomide (1 mmol/L) or specific siRNA. Lane M shows the protein marker. E, M2-like macrophages were treated for 72 hours with lenalidomide (blue, 1 mmol/L) or untreated (orange). ChIP using antibodies against tri-methyl-histone H3 (K4), tri-methyl-histone H3 (K27), or normal IgG as control was performed. Enrichment in the proximal region of the IRF4 or IRF5 promoter was quantified by qPCR (n ¼ 3). TSS, transcription start site. F, Macrophages (defined as CD163þCD15) from patients with multiple myeloma before (orange, n ¼ 12) and during IMiD therapy (blue, n ¼ 10) were analyzed by flow cytometry for expression of IRF4 or IRF5. MFI, median fluorescence intensity. Error bars show SEM (, P < 0.05; , P < 0.01).

Article Snippet: Western blotting and chromatin immunoprecipitation Antibodies specific for IKZF1 (14859S, Cell Signaling Technology), IRF3 (11904S, Cell Signaling Technology), IRF4 (4299S, Cell Signaling Technology), IRF5 (13496S, Cell Signaling Technology), c-Jun (60A8, Cell Signaling Technology), NF-kB p65 (D14E12, Cell Signaling Technology), Tri-Methyl-Histone H3 (K4) (9751P, Cell Signaling Technology), Tri-Methyl-Histone H3 (K27) (9733S, Cell Signaling Technology), IgG (2729P, Cell Signaling Technology), Tubulin (Abcam, ab7291), b-actin (AC-15, Abcam), goat anti-mouse IgGHRP (Dako, P0447), and goat anti-rabbit IgG-HRP (Cell Signaling Technology) were used for Western blotting and chromatin immunoprecipitation (ChIP).

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Incubation, Marker, Control, Cytometry, Expressing

Journal: Cancer Immunology Research

Article Title: The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages

doi: 10.1158/2326-6066.cir-20-0555

Figure Lengend Snippet: Figure 5. Lenalidomide treatment of CrbnI391V mice promotes the tumoricidal activity of macrophages. A, B6.CrbnI391V mice received daily intraperitoneal (i.p.) injections with either DMSO or lenalidomide (len; 10 mg/kg body weight, in PBS containing 10% DMSO) for 7 days. Subsequently, mice were sacrificed, and BM was extracted from femur, tibia, and hip bone and analyzed in vitro. B, BM macrophages (Ly6G, CD115þ, and F4/80þ) from solvent-treated mice (n ¼ 5, orange) or lenalidomide-treated mice (n ¼ 7, blue) were analyzed by flow cytometry for the expression of IKZF1, IRF4, and IRF5. C, Expression of effector molecules by BM macrophages (solvent treated: orange, n ¼ 5 and lenalidomide treated: blue, n ¼ 7) was measured by qPCR [iNOS and arginase (Arg1); arbitrary units (A.U.)] or by flow cytometry (PD-L1). Horizontal lines show mean values. Isolated BM macrophages from solvent-treated mice or lenalidomide-treated mice were coincubated with labeled myeloma cells (5TGM1, red; D) or with labeled lymphoma cells (291PC, red; E) in an E:T of 1:1 in the presence or absence of a CD38- or CD20-specific antibody, respectively. After 3 hours, ADCP was analyzed by confocal microscope. Macrophages were stained with CD11b (green). Magnifications (630) present representative areas from one of five independent experiments performed with different mice. Scale bar, 20 mm. Error bars show SEM (, P < 0.05; , P < 0.01).

Article Snippet: Western blotting and chromatin immunoprecipitation Antibodies specific for IKZF1 (14859S, Cell Signaling Technology), IRF3 (11904S, Cell Signaling Technology), IRF4 (4299S, Cell Signaling Technology), IRF5 (13496S, Cell Signaling Technology), c-Jun (60A8, Cell Signaling Technology), NF-kB p65 (D14E12, Cell Signaling Technology), Tri-Methyl-Histone H3 (K4) (9751P, Cell Signaling Technology), Tri-Methyl-Histone H3 (K27) (9733S, Cell Signaling Technology), IgG (2729P, Cell Signaling Technology), Tubulin (Abcam, ab7291), b-actin (AC-15, Abcam), goat anti-mouse IgGHRP (Dako, P0447), and goat anti-rabbit IgG-HRP (Cell Signaling Technology) were used for Western blotting and chromatin immunoprecipitation (ChIP).

Techniques: Activity Assay, In Vitro, Solvent, Cytometry, Expressing, Isolation, Labeling, Microscopy, Staining