Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A-D) BEAS-2B:doxMR1-GFP cells were treated with doxycycline, IFNγ, and/or 6-FP overnight. (A) MR1 expression was calculated relative to HPRT1 expression and NT control, paired by experimental replicate. gMFI of (B) MR1-GFP, (C) surface MR1 α-26.5 stain, and (D) surface MHC-Ia α-W6/32 stain. Data are experimental replicates. (E) ELISPOT of BEAS-2b cells treated with filtered M. smegmatis supernatant and MAIT cells. Data points are experimental replicates of no-antigen background-subtracted IFNγ SFU. (F) Putative transcription factor binding sites were acquired through the Eukaryotic Promoter Database browser using the Search Motif Tool to perform on-the-fly scanning for transcription factor motifs using the FindM tool from the Signal Search Analysis (SSA) Server toolkit – . Highlighted proteins are involved in IRF1-(green) or NLRC5 enhanceosome-(blue) mediated HLA transcription. RT-qPCR of (G) primary human AECs or (H) BEAS-2b cells treated with recombinant IFNγ for 12 hours. IRF1 (left) and NLRC5 (right) expression were calculated relative to HPRT1 expression and NT control, paired by individual donor or experimental replicate. (I) IRF1 mRNA expression from BEAS-2b cells treated with IFNγ over the indicated time periods, calculated relative to t=0 control. Pairwise T tests were performed by experiment (A-E, H) or donor (G) .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Expressing, Control, Staining, Enzyme-linked Immunospot, Binding Assay, Quantitative RT-PCR, Recombinant

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A) RT-qPCR of BEAS-2b cells treated with IRF1, NLRC5, and/or missense siRNA for 36 hours, then incubated with IFNγ for 12 hours. MR1 expression was calculated relative to HPRT1 expression and missense NT control, paired by experimental replicate. (B-C) RT-qPCR of Cas9 + or NLRC5 -/- clone G1 BEAS-2b cells treated with IRF1 or missense siRNA for 36 hours, then incubated with IFNγ for 12 hours. (B) MR1 and (C) HLA-A expression were calculated relative to HPRT1 expression and Cas9 + or NLRC5 -/- clone G1 missense NT controls, paired by experimental replicate. (D) Cells in B-C were used as antigen-presenting cells in ELISPOT assay, with filtered M. smegmatis supernatant as the antigen source. Data points are experimental replicates of Cas9 + or NLRC5 -/- clone G1 missense control no-antigen background-subtracted IFNγ SFU. (E-F) RT-qPCR of Cas9 + or IRF1 -/- clone G7 BEAS-2b cells treated with IFNγ for 12 hours. (E) MR1 and (F) HLA-A expression were calculated relative to HPRT1 expression and Cas9 + or IRF1 -/- clone G7 NT controls, paired by experimental replicate. (G-H) Flow cytometry of Cas9 + or IRF1 -/- clone A10 BEAS-2b cells treated with IFNγ for 12 hours. gMFI of (G) surface MR1 (α-26.5 Ab) and (H) MHC-Ia (α-W6/32 Ab) are paired by experimental replicate. Statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Quantitative RT-PCR, Incubation, Expressing, Control, Enzyme-linked Immunospot, Flow Cytometry

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A-B) RT-qPCR of BEAS-2b cells treated with IRF1, NLRC5, and/or missense siRNA for 36 hours, then incubated with IFNγ for 12 hours. (A) IRF1 and (B) NLRC5 expression were calculated relative to HPRT1 expression and missense NT control, paired by experimental replicate. (C-D) RT-qPCR of Cas9 + or NLRC5 -/- BEAS-2b cells treated with IRF1 or missense siRNA for 36 hours, then incubated with IFNγ for 12 hours. Gene expression of (C) NLRC5 -/- clone G1 or (D) NLRC5 -/- clone H2 were calculated relative to HPRT1 expression and Cas9 + or NLRC5 -/- clone missense NT controls, paired by experimental replicate. (E) Flow cytometry of cells from C-D. gMFI of surface MR1 (α-26.5 Ab) are from IFNγ-treated Cas9 + (left), NLRC5 -/- clone G1 (middle), and NLRC5 -/- clone H2 (right). (F) RT-qPCR of Cas9 + or IRF1 -/- clone A10 BEAS-2b cells treated with IFNγ for 12 hours. MR1 expression was calculated relative to HPRT1 expression and Cas9 + or IRF1 -/- clone A10 NT controls, paired by experimental replicate. (G) Flow cytometry of Cas9 + or IRF1 -/- clone G7 BEAS-2b cells treated with IFNγ for 12 hours. gMFI of surface MR1 (left, α-26.5 Ab) and MHC-Ia (left, α-W6/32 Ab) are paired by experimental replicate. Statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Quantitative RT-PCR, Incubation, Expressing, Control, Gene Expression, Flow Cytometry

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A-B) Flow cytometry of (A) primary human AECs infected with S. pneumoniae ( Sp ) for one hour and incubated overnight with MAIT cell clone (n=3), or (B) AECs treated with IFNγ for 12 hours (n=3). gMFI of stained pSTAT1 expression is paired by individual donor. (C-F) RT-qPCR of primary human AECs infected with S. pneumoniae ( Sp ) for one hour and incubated overnight with MAIT cell clone. Expression of (C) β2m, (D) HLA-A (E) IRF1 , and (F) NLRC5 were calculated relative to HPRT1 expression and NT control, paired by individual donor (n=4 (C) or n=5 (D-F) donors). Pairwise statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Flow Cytometry, Infection, Incubation, Staining, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A) Flow cytometry of primary human AECs infected with S. pneumoniae ( Sp ) for one hour and incubated overnight with MAIT cell clone. gMFI of stained pSTAT1 expression is paired by individual donor (n=3). (B-D, G) RT-qPCR of primary human AECs infected with S. pneumoniae ( Sp ) for one hour and incubated overnight with MAIT cell clone. Expression of (B) β2m, (C) NLRC5 (D) HLA-A , and (G) IRF1 were calculated relative to HPRT1 expression and NT control, paired by individual donor (n=4 (B) or n=5 (C, D, G) donors). (E-F, H-I) RT-qPCR of wildtype BEAS-2b cells treated as indicated below. Gene expression was calculated relative to HPRT1 expression and NT controls, paired by experiment. (F) HLA-A and (H) IRF1 expression of BEAS-2b cells infected with M. smegmatis ( Ms ) for one hour and incubated overnight with MAIT cell clone. (G) HLA-A and (I) IRF1 expression of BEAS-2b cells treated with 5-OP-RU (left) or 6-FP (right) for one hour and incubated overnight with MAIT cell clone. Pairwise statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Flow Cytometry, Infection, Incubation, Staining, Expressing, Quantitative RT-PCR, Control, Gene Expression

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A-D) RT-qPCR of wildtype BEAS-2b cells treated as indicated below. Gene expression was calculated relative to HPRT1 expression and NT controls, paired by experiment. (A) HLA-A and (C) IRF1 expression of BEAS-2b cells infected with M. smegmatis ( Ms ) for one hour and incubated overnight with MAIT cell clone. (B) HLA-A and (D) IRF1 expression of BEAS-2b cells treated with 5-OP-RU (left) or 6-FP (right) for one hour and incubated overnight with MAIT cell clone. (E-F) RT-qPCR of Cas9 + or IRF1 -/- clone A10 BEAS-2b cells (E) infected with M. smegmatis or (F) treated with 5-OP-RU for one hour, then incubated overnight with MAIT cell clone. MR1 expression was calculated relative to HPRT1 expression and Cas9 + or IRF1 -/- NT controls, paired by experimental replicate. Pairwise statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Infection, Incubation

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: (A) RT-qPCR of wildtype BEAS-2b cells treated with recombinant cytokines for 12 hours. MR1 expression was calculated relative to HPRT1 expression and NT controls. (B-C, F-H) RT-qPCR of BEAS-2b cells treated with IFNγ or IFNβ for 12 hours. Expression of (B) MR1, (C) HLA-A (F) IRF1 , (G) NLRC5 , and (H) β2m were calculated relative to HPRT1 and NT control, paired by experiment. (D) Flow cytometry of BEAS-2b cells treated with IFNγ or IFNβ for 12 hours. gMFI of surface MR1 (left, α-26.5 Ab) and MHC-Ia (right, α-W6/32 Ab) are paired by experimental replicate. (E) ELISPOT of BEAS-2b cells treated with IFNγ or IFNβ for 12 hours, infected with a titration of M. smegmatis for one hour, then incubated with MAIT cells overnight. Data points are average SFU of no-antigen background-subtracted IFNγ SFU. Nonlinear regression agonist response curves were computed in GraphPad Prism 10.4.0 and analyzed with extra sum-of-squares F test. Statistical analyses are in .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Quantitative RT-PCR, Recombinant, Expressing, Control, Flow Cytometry, Enzyme-linked Immunospot, Infection, Titration, Incubation

Journal: bioRxiv

Article Title: IFNγ regulates MR1 transcription and antigen presentation

doi: 10.1101/2025.02.09.637183

Figure Lengend Snippet: IFNγ from MAIT cells or other cellular sources induces IRF1 expression and subsequent MR1 transcription. Increased MR1 expression and antigen presentation enhances MAIT cell responses to antigens from exogenous sources or pathogens like S. pneumoniae or M. smegmatis .

Article Snippet: RT-qPCR was performed using TaqMan (Applied Biosystems) gene expression assays: HPRT1 (Hs02800695_m1), MR1 (Hs01042278_m1), HLA-A,H (Hs01058806_g1), IRF1 (Hs00971965_m1), NLRC5 (Hs01072123_m1), and β2m (Hs00187842_m1).

Techniques: Expressing, Immunopeptidomics

Journal: Chemistry & biology

Article Title: Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase.

doi: 10.1016/j.chembiol.2010.11.014

Figure Lengend Snippet: Figure 6. SET7/9 Is Inhibited by Phosphorylation of the Target Peptide or Protein (A) Relative activity of SET7/9 on CelluSpot array derived from H3 peptides containing different combinations of PTMs (Table S2). Phosphorylation of T3, S10, and T11 abolished SET7/9 activity. (B) Influence of phosphorylation of nonhistone substrates on SET7/9 methyla- tion activity. GST-tagged nonhistone substrates of SET7/9 were incubated with PKA. All domains except Cullin1 were phosphorylated (Figure S5). Then, phosphorylated and mock treated domains were methylated by SET7/9. A complete loss or strong reduction of SET7/9 activity was observed with ZDN8, IRF1, MINT, PPARBP, and MeCP2. See also Figure S6.

Article Snippet: Cloning, Expression, and Purification of Proteins The sequence encoding human A kinase anchor protein 6 (AKA6) (residues 485–776; NCBI accession number NP_004265.3), Bromodomain containing 1 (BRD1) (residues 681–891; NCBI accession number NP_055392.1), Centromeric protein C1 (CENPC1) (residues 279–474; NCBI accession number NP_001803.2), Dnmt1 (residues 622–780; NCBI accession number NP_001370.1), Methyl CpG binding protein 2 (MeCP2) (residues 201–486; NCBI accession number NP_004983.1), Msx2-interacting protein (MINT) (residues 1994–2281; NCBI accession number NP_055816.2), PPAR binding protein (PPARBP) (residues 847–1084; NCBI accession number NP_004765.2), Retinoblastoma binding protein 5 (RBBP5) (residues 323– 538; NCBI accession number NP_005048.2), Zinc finger DHHC domain containing 8 (ZDHHC8) (residues 227–400; NCBI accession number NP_037505.1), PRP4 pre-mRNA processing factor 4 homolog B (PRP4) (residues 93–396; NCBI accession number NP_789770.1) CDC2 related protein kinase 7 (CRKRS) (residues 2–339; NCBI accession number NP_057591.1), Cullin1 (residues 2–250; NCBI accession number NP_003583.2), TTK (residues 603–799; NCBI accession number NP_003309.2), and IRF1 (residues 71–240; 118 Chemistry & Biology 18, 111–120, January 28, 2011 a2011 Elsev NCBI accession number NP_002189.1) were amplified from HEK293 cDNA and cloned into pENTR/D-TOPO (Invitrogen) gateway entry vector according to the manufacturer’s instructions. pGEX-6P-2 vector (GE healthcare) was converted into Gateway destination vector (pGEX-6P-2-GW-AJ) using Gateway vector conversion system (Invitrogen).

Techniques: Phospho-proteomics, Activity Assay, Derivative Assay, Incubation, Methylation

Journal:

Article Title: Interferon Regulatory Factor 7 Mediates Activation of Tap-2 by Epstein-Barr Virus Latent Membrane Protein 1

doi: 10.1128/JVI.75.1.341-350.2001

Figure Lengend Snippet: Activation of Tap-2 promoter reporter construct by IRF-7. (A) Schematic diagrams of Tap-2 reporter constructs. Top line, Tap-2 genomic region; open rectangle, ISRE. A 573-bp fragment from the Tap-2 promoter region was cloned; the ISRE sequence and mutations are shown. (B) Schematic diagrams of various IRF-7 expression plasmids. IRF-7A, -7B, and -7C are splicing variants of IRF-7. IRF-7DN lacks the DNA-binding domain. (C) Activation of Tap-2 reporter construct by IRF-7 in B cells. Akata cells were transfected with the reporter construct Tap2-CAT together with vector pcDNA-3 (column 1) or expression plasmids for IRF-1 (column 2), IRF-2 (column 3), IRF-3 (column 4), ICSBP (column 5), IRF-7A (column 6), IRF-7B (column 7), IRF-7DN (column 8), or LMP-1 (column 9). Columns 10 to 16, pcLMP-1 was cotransfected with IRF-1, IRF-2, IRF-3, ICSBP, IRF-7A, IRF-7B, and IRF-7DN, respectively. (D) Mutations in ISRE abolish activation by IRF-7. Akata cells were transfected with the reporter construct mTap2-CAT and pcDNA-3 (column 1) or IRF-7A (column 2) or IRF-7A plus pcLMP-1 (column 3) or IRF-1 expression plasmid (column 4). CAT assay results were normalized to β-galactosidase activity. CAT activity is expressed relative to the vector control. Standard deviations are shown.

Article Snippet: The IRF-1 (C-20) and IRF-2 (C-19) antibodies were purchased from Santa Cruz Biotechnology, Inc. LMP-1 monoclonal antibody CS1-4 was purchased from Dako.

Techniques: Activation Assay, Construct, Clone Assay, Sequencing, Expressing, Binding Assay, Transfection, Plasmid Preparation, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF 168 facilitates oestrogen receptor ɑ transcription and drives breast cancer proliferation

doi: 10.1111/jcmm.13694

Figure Lengend Snippet: Reduction of RNF 168 level reduces recruitment of RNF 168 to ER ɑ promoter—a potential mechanism for ER ɑ signalling regulation. A, Genomic organization of ER α promoter structure of human ER α genes is shown, among which promoter A, promoter B and promoter E2 are used in MCF ‐7 cells. B, Ch IP assay shows that RNF 168 is recruited to ER α promoter B and E2. MCF 7 cells were fixed for 30 min. Rabbit Ig G was used as the negative control, while ER α antibody was used as the positive control. The primer sequences were shown in Table S1. Then enriched DNA fragments were subject to PCR reaction and detected by DNA gel electrophoresis. C, Ch IP assay shows that for RNF 168 depletion decreases RNF 168 recruitment to ER α promoter regions. MCF 7 cells were transfected with si RNF 168 or siControl for 48 h. After that, cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. The relative ER α promoter enrichment was measured by real‐time PCR . * P < .05; ** P < .01; *** P < .001 for binding comparison. D, Ch IP assay shows that RNF 168, ER α, ER α co‐activators ( SRC 1 and SRC 3) and H3K27ac co‐occupy at ER α promoter B and E2. MCF 7 cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. Then enriched DNA fragments were subject to PCR reaction and detected by DNA gel electrophoresis. E, Ch IP assay shows that for RNF 168 depletion decreases ER α, ER α co‐activators ( SRC 1 and SRC 3) and H3K27ac recruitment to ER α promoter regions. MCF 7 cells were transfected with si RNF 168 or siControl for 48 h. After that, cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. The relative ER α promoter enrichment was measure by real‐time PCR . * P < .05; ** P < .01; *** P < .001 for binding comparison

Article Snippet: Anti‐SRC1 (128E7), anti‐SRC3 (5E11) and anti‐H3K27ac (D5E4) antibodies were acquired from Cell Signaling Technology.

Techniques: Negative Control, Positive Control, DNA Gel Electrophoresis, Transfection, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Venn analysis identifying profoundly up-regulated Irf1 target genes in murine lung endothelial cells (MLECs). ( B ) Web-based term association analysis to identify which Irf1 target gene(s) have the strongest association with endothelial regeneration-associated search terms. ( C ) qPCR analysis of Irf1 and Lif gene expression in MLECs isolated from mice pre- and post-LPS. ( D ) Representative immunoblots and densitometric quantification of Irf1 and Lif protein levels in MLECs pre- and post-LPS challenge. ( E ) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) isolated from mice pre- and post-LPS. ( F ) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs pre- and post-LPS challenge. ( G ) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) transfected with control or IRF1 vector. ( H ) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs with control vector or IRF1 overexpression. ( I ) Schematic of the human LIF promoter region depicting the highly-conserved IRF1 ISRE binding site at −102 ∼ −127 bp. The WT and MUT ISRE sequences used in panel ( K ) are provided. ( J ) HLMVECs transduced with either control or IRF1 plasmids, subjected to vehicle or LPS conditions for 8 h, followed by ChIP-qPCR assays for detection of IRF1 binding to the −102 bp binding site within the LIF promoter region. ( K ) HLMVECs co-transfected with control or IRF1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. ** P <0.01 [one-way ANOVA (C) and two-way ANOVA (E–H) with Bonferroni post-hoc tests].

Article Snippet: For the IRF1 promoter experiments, the WT human IRF1 promoter clone (1562-bp fragment, -1379/+182; HPRM44152, GeneCopoeia) or its GAS1 mutant (MUT GAS1) were subcloned into the pGL3-Luc vector.

Techniques: Gene Expression, Isolation, Western Blot, Transfection, Control, Plasmid Preparation, Over Expression, Binding Assay, Transduction, ChIP-qPCR, Luciferase, Construct

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs isolated from Ifr1 fl/fl control mice pre- and post-LPS. ( B ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs in Ifr1 fl/fl control mice and endothelial cell-specific Irf1 knockout mice at baseline. ( C ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation and Irf1 protein levels in the indicated MLEC cell lines following LPS (10 μg/ml) treatment. ( D ) Representative immunoblots and densitometric quantification of STAT1 Ser727 phosphorylation and IRF1 protein levels in the indicated HLMVEC cell lines following LPS (10 μg/ml) treatment. ( E ) Schematic of the human IRF1 promoter region depicting the two highly-conserved STAT1 binding site at −44 ∼ −56 bp and −177 ∼ −188 bp. The WT and MUT GAS1 sequences used in panel ( G ) are provided. ( F ) HLMVECs subjected to vehicle or LPS conditions for 8 hours, followed by ChIP-qPCR assays for detection of STAT1 binding to the two binding sites within the IRF1 promoter region. (G) HLMVECs co-transfected with control or STAT1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or 6 independent biological replicates per group. Data represented as means ± SDs. * P <0.05, ** P <0.01 [(A, C, D) one-way ANOVA with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests; (B) two-tailed Student’s t -tests; (E, H, I) two-way ANOVA with Bonferroni post-hoc tests].

Article Snippet: For the IRF1 promoter experiments, the WT human IRF1 promoter clone (1562-bp fragment, -1379/+182; HPRM44152, GeneCopoeia) or its GAS1 mutant (MUT GAS1) were subcloned into the pGL3-Luc vector.

Techniques: Western Blot, Phospho-proteomics, Isolation, Control, Knock-Out, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation, Luciferase, Construct, Two Tailed Test

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: R-based bioinformatics analysis of published microarray data (GEO acc no. GSE5883) in which cultured human lung microvascular endothelial cells (HLMVECs) were left untreated (Ctrl) or exposed to LPS (10 ng) for 4 h (n = 4 biological replicates per group). ( A ) Gene set enrichment analysis (GSEA)-based identification of two discreet gene co-expression modules associated with LPS exposure: M1 and M2. Red coloring denotes a positive NES score, while blue coloring denotes a negative NES score. ( B ) Network plots for the two gene co-expression modules M1 and M2. ( C ) Reactome enrichment analysis for the two gene co-expression modules M1 and M2. The color saturation denotes the prediction confidence. ( D ) Volcano plots of differentially expressed genes (DEGs) with staining for M1 module membership (blue, top panel) or M2 module membership (pink, bottom panel). ( E ) Heatmap of the top 50 M2 module up-regulated DEGs ordered by descending log2 fold-change. Up-regulation is denoted by green coloring, while down-regulation is denoted by red coloring. IRF1 is marked by a red rectangle.

Article Snippet: For the IRF1 promoter experiments, the WT human IRF1 promoter clone (1562-bp fragment, -1379/+182; HPRM44152, GeneCopoeia) or its GAS1 mutant (MUT GAS1) were subcloned into the pGL3-Luc vector.

Techniques: Microarray, Cell Culture, Expressing, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Schematic illustration depicting Irf1 fl/fl × Cdh5 -CreERT2 mice crosses to construct endothelial cell-specific Irf1 knockout mice ( Irf1 EC−/− ). Tamoxifen was administered for five consecutive days, left to rest for 4 weeks prior to a sub-lethal (8 mg/kg) LPS challenge. ( B ) Irf1 protein levels in endothelial cells isolated from flushed lungs from control and Irf1 EC−/− mice. ( C ) Quantification of protein levels. ( D ) Time course illustrating lung vessel permeability post-LPS in control and Irf1 EC−/− mice. ( E ) Flow cytometric analysis of CD31+CD45− endothelial cells in mice post-LPS injury. ( F ) Representative images of BrdU-APC, CD31-AF488, and DAPI co-staining in lung cryosections obtained from control Irf1 fl/fl mice and Irf1 EC−/− mice 3 days post-LPS. BrdU+ MLEC quantification in lung cryosections obtained from mice post-LPS ( n = 10 cryosections per mouse). ( G ) Re-introduction of Irf1 through liposome vector plasmids (50 μg) in Irf1 EC−/− mice post-LPS. ( H ) Lung vessel permeability at day 3 following LPS challenge in Irf1 EC−/− mice with re-introduced Irf1 . ( I ) Flow cytometric analysis of CD31+CD45− endothelial cells at day 3 following LPS challenge in Irf1 EC−/− mice with re-introduced Irf1 . ( J ) BrdU+ MLEC quantification in lung cryosections obtained at day 3 following LPS challenge in Irf1 EC−/− mice with re-introduced Irf1 ( n = 10 cryosections per mouse). All experiments: n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. * P <0.05, ** P <0.01 [two-tailed Student’s t -tests ( C, J ) and two-way ANOVA ( D, E–H ) with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests].

Article Snippet: For the IRF1 promoter experiments, the WT human IRF1 promoter clone (1562-bp fragment, -1379/+182; HPRM44152, GeneCopoeia) or its GAS1 mutant (MUT GAS1) were subcloned into the pGL3-Luc vector.

Techniques: Construct, Knock-Out, Isolation, Control, Permeability, Staining, Plasmid Preparation, Two Tailed Test

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Confocal microscopy illustrating FLAG staining along with CD31+ and DAPI staining in lung cryosections obtained from mice receiving either a scrambled control (Ctrl), FLAG- Irf1 , or FLAG- Irf1 /shLif construct. Scale bar = 50 and 20 μm (enlarged panel). Co-localization coefficient for FLAG-Irf1 staining in CD31+ MLECs. ( B ) FLAG-Irf1 and Lif protein levels in MLECs derived from mice receiving either a Ctrl, FLAG- Irf1 , or FLAG- Irf1 /shLif construct. ( C ) Lung vessel permeability pre- and post-LPS in mice receiving either a Ctrl, FLAG- Irf1 , or FLAG- Irf1 /shLif construct. ( D ) Flow cytometric analysis of CD31+CD45− endothelial cells pre- and post-LPS in mice receiving either a Ctrl, FLAG- Irf1 , or FLAG- Irf1 /shLif construct. ( E ) BrdU+ MLEC quantification in lung cryosections obtained from mice receiving either a Ctrl, FLAG- Irf1 , or FLAG- Irf1 /shLif construct ( n = 6 cryosections per mouse). ( F ) Survival curves following LPS challenge in mice receiving either a Ctrl, FLAG- Irf1 , or FLAG- Irf1 /shLif construct ( n = 30 mice per group). All experiments except (F): n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. * P <0.05, ** P <0.01 [(A–E) two-way ANOVA with Bonferroni post-hoc test; (F) Log-rank test].

Article Snippet: For the IRF1 promoter experiments, the WT human IRF1 promoter clone (1562-bp fragment, -1379/+182; HPRM44152, GeneCopoeia) or its GAS1 mutant (MUT GAS1) were subcloned into the pGL3-Luc vector.

Techniques: Confocal Microscopy, Staining, Control, Construct, Derivative Assay, Permeability

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: STAT1/IRF1 axis mediates the upregulation of CXCL10 and CXCL11 induced by autophagy targeting. (A‐B) Western blotting showing the expression levels of STAT1, pSTAT1, and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors or the knockout of RB1CC1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (C‐D) Western blotting showing the expression levels of STAT1 and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors and the silencing of STAT1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (E‐F) Expression of CXCL10 and CXCL11 mRNA by RT‐qPCR and ELISA quantification of CXCL10 and CXCL11 protein levels in the supernatants of shControl, shSTAT1 and shIRF1 Nalm6 and Raji cells ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA tests. (G‐H) ChIP‐qPCR showing the binding of STAT1 and IRF1 to the promoter region of CXCL10 and CXCL11 ( n = 3). Values are shown as the mean ± SD. Statistical differences for each cell line are calculated with unpaired Student's t tests. (I) Graphic abstract: in the proposed model, inhibition of cancer cell‐autonomous autophagy leads to accumulation of cytosolic DNA, which thereby not only suppresses cancer cell survival by inducing TNFR1‐TNF‐α mediated apoptosis but also promotes the CAR‐T cell recruitment in tumor microenvironment via STAT1/IRF1‐dependent activation of chemokine signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T, chimeric antigen receptor T; ChIP, chromatin immunoprecipitation; CXCL CXC, chemokine ligand; ELISA, enzyme‐linked immunosorbent assay; FC, fold change; IRF, interferon regulatory factor; ns: not significant; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; sh short hairpin; STAT, signal transducers and activators of transcription.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Western Blot, Expressing, Knock-Out, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay, Inhibition, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: Naïve CD4+ T cells isolated from wildtype mice were primed with IL-27 in the presence of anti-CD3 and anti-CD28 antibodies (a) Volcano plot analysis for samples collected at 2 hours post cell stimulation. Depicted is time point of log2 fold-change (x-axis) versus -log10 p-value (y-axis, representing the probability that the gene is differentially expressed). Black dotted line marks p-value 0.05 and red dots marks fold change higher or lower than two. Irf1 and Batf marked in blue. (b) Irf1 and Batf mRNA expression measured by qPCR over 72 hours following cell stimulation. Analysis of Tr1 differentiation in Irf1−/− cells 72 hours after cell priming with IL-27 measured by (c) flow cytometry (d) qPCR (left, n=3 samples) and ELISA (right, n=5 samples). Analysis of Tr1 differentiation in Batf−/− cells 72 hours after cell priming with IL-27 measured by (e) flow cytometry (f) qPCR (left, n=3 samples) and ELISA (right, n=5 samples). Dots represent biological replicates. Data are representative of three independent experiments (b), representative of four independent experiments (c, e), or are pooled from three independent experiments (d, f). *P < 0.001, **P < 0.0001 (unpaired t-test, error bars represent mean ±s.e.m.).

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Isolation, Cell Stimulation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: (a) Irf1 mRNA expression in Stat1−/− (left) and Stat3−/− (right) cells primed in the presence of IL-27. (b) Batf mRNA expression in Stat1−/− (left) and Stat3−/− (right) cells primed in the presence of IL-27. (c) Effects of IRF1 and BATF retroviral overexpression on Il10, Il21, Maf and AhR expression in cells treated in TH0 or Tr1 conditions; mRNA levels were quantified using qPCR. Data are pooled from 3 independent experiments (a (Stat1−/−), b, c; n=3 samples, dots represent biological replicates) or representative of 4 independent experiments (a (Stat3−/−); n=3 samples, dots represent technical replicates). NS, not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (unpaired t-test, error bars represent mean ±s.e.m.).

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Expressing, Retroviral, Over Expression

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: (a) EAE scores in WT and Irf1−/− mice were immunized with MOG35-55, n=24 mice (WT) or n=27 mice (Irf1−/−) (left); linear regression curve including the 95% confidence band of the regression line (middle); maximum scores (right). (b) Cytokine expression in CD4+ T cells isolated from the CNS of WT and Irf1−/− mice at the peak of disease. (c) Frequency of IL-17A+ and IFN-γ+ cells within the CD4+ T cells isolated from the CNS (n=5 mice). (d) EAE course in Rag2−/− mice with 2D2 or Irf1−/−2D2 CD4+ T cells transferred, followed by MOG35-55 immunization; n=8 mice/group. (e) Spontaneous EAE scores in 2D2 (n=5 mice) and Irf1−/− 2D2 mice (n=7 mice). (f) Spleens and lymph nodes from WT and Irf1−/− immunized mice were cultured with different concentration of MOG35-55. 3H thymidine was added to assess cell proliferation; n=5 samples. (g) Cytokine expression in CD4+ T cells isolated from WT and Irf1−/− mice immunized with MOG35-55 and cultured with MOG35-55±IL23. (h) Quantification of (g); n=5 mice. Data are pooled from four independent experiments (a), representative of two independent experiments (b, f, g), or pooled from two independent experiments (c, d, h). Dots represent individual mice. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data represent mean ±s.e.m (linear regression (a (EAE plot), d; unpaired t-test (a (max score), c, f, h).

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Expressing, Isolation, Cell Culture, Concentration Assay

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: (a) Il10 expression in CD4+ T cells isolated from the CNS and dLNs (draining lymph nodes) of WT and Irf1−/− mice immunized with MOG35-55. Floating bars indicate min to max with a line at mean. (b) Gene expression fold change between CD4+ T cells isolated from wildtype mice immunized with MOG35-55 and re-stimulated in the presence of MOG35-55 ± IL-27. Genes with at least 2-fold change (MOG+IL-27/MOG) are shown. (c) Differential gene expression in WT, Irf1−/− and Batf−/− CD4+ T cells isolated from mice immunized with MOG35-55 and restimulated with MOG35-55 and IL-27. Gene-set created in (b) was used to generate the heatmap. (d) Tr1-polarized CD4+ T cells isolated from WT, Irf1−/− or Batf−/− mice were transferred into WT mice 10 days after EAE induction; n=7 mice (No Tr1, Batf−/−), n=8 (Irf1−/−), n=10 (WT Tr1). (e) IL-10 expression in mesenteric lymph nodes (MLN) from WT, Irf1−/− and Batf−/− mice injected with anti-CD3 antibody or an isotype control (IC). (f) Splenocytes from mice in (e) were cultured for 72 hours, ELISA was used to measure IL-10 in the cultures. Dots represent individual mice. Data are pooled from three independent experiments (a), pooled from two independent experiments (b, c, d), representative of two independent experiments with n=3–4 mice pooled/group per experiment (e) or representative of two independent experiments (f). *P < 0.05, **P < 0.01 and ***P < 0.0001. Data represent mean ±s.e.m (unpaired t-test (a, f); linear regression analysis (d)).

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Expressing, Isolation, Gene Expression, Injection, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: (a) Schematic drawing of the Il10 locus; known CNS and HSS sites are marked. ChIP analysis of (b) IRF1 (n=2 samples) and (c) BATF (n=3 samples) interactions within the Il10 promoter in WT Tr1 cells polarized for 72 hrs. (d) Sequential ChIP analysis of BATF and IRF1 interactions in the CNS-9 and HSS+2.98 regions of the Il10 promoter in WT and Batf−/− cells differentiated in the presence of IL-27. (e) ChIP analysis of IRF1 binding in the CNS-9, HSS-0.12, HSS+2.98 sites of the Il10 promoter in WT and Batf−/− cells differentiated in Tr1 conditions; n=2 samples. (f) ChIP analysis of BATF binding in the CNS-9, and HSS+2.98 sites of the Il10 promoter in WT and Irf1−/− cells differentiated in Tr1 conditions; n=3 samples. (g) ChIP analysis of epigenetic marks recruitment to the Il10 promoter in WT, Irf1−/− and Batf−/− cells differentiated in Tr1 conditions. Data are representative of three independent experiments with similar results (b, c, e, f) or two independent experiments (d, g). *P < 0.05 (unpaired t-test, error bars represent mean ±s.e.m.).

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Binding Assay

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: Luciferase activity in 293T cells transfected with different IL-10 luciferase reporters in the presence of constructs encoding IRF1, IRF1 DNA-binding mutant W11R, c-Maf and BATF. RLU (Relative Light Units) are shown. (a) Proximal promoter indicates the proximal 1.5 kb region of the promoter. (b) CNS-9 and HSS+2.98 reporters contain the Il10 promoter fragments cloned upstream of the Il10 minimal promoter. (c) Il10 expression (mRNA) in WT and Irf1−/− CD4+ T cells retrovirally transduced with c-Maf and cultured in TH0 or Tr1 conditions (d) Il10 expression (mRNA) in WT and Batf−/− CD4+ T cells retrovirally transduced with c-Maf and cultured in TH0 or Tr1 conditions (e) ChIP analysis of c-Maf interactions with c-Maf-binding sites (MARE-1--4) in the Il10 promoter in WT and Irf1−/− Tr1 polarized cells. (f) ChIP analysis of AhR interactions with AhR-binding site XRE-1 in the Il10 promoter in WT, Irf1−/− and Batf−/− Tr1 polarized cells. Data are representative of three independent experiments (a, b; n=3 samples), representative of two independent experiments (c, d; n=3 samples) or pooled from two independent experiments (e, f). Dots represent technical replicates (a, b, c, d) or biological replicates (e, f). NS, not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Luciferase, Activity Assay, Transfection, Construct, Binding Assay, Mutagenesis, Clone Assay, Expressing, Transduction, Cell Culture

Journal: Nature immunology

Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

doi: 10.1038/ni.3683

Figure Lengend Snippet: (a) Heatmap of normalized ATAC-seq peak intensities (log2-fold-changes relative to the mean for each peak). Limited to (6017) peaks that are condition-dependent with log2|FC|>3 and FDR=1% for at least one pairwise comparison of interest. (b) PCA of the 72h conditions, using all ATAC-seq peaks in the dataset (180,478 DESeq2-normalized peak intensities). (c) Number of differentially accessible peaks detected using DESeq2, comparing Tr1 KO (knockouts) to control cells at 72h, log2|FC|>1 and FDR=10% (Subsampled, each comparison had n=2 (KO), n=6 (control)). Estimates of log2-fold-changes in gene expression from RNA-seq data, comparing either Batf−/− or Irf1−/− and control Tr1 cells (log2|FC|>1, FDR=10%) for DNA-binding proteins (d) and cytokines and cytokine receptors (e). RNA-seq and ATAC-seq datasets were integrated to generate putative transcriptional regulatory networks for Irf1−/− (f–g) and Batf−/− (h–i) Tr1 cells. Nodes represent differentially expressed transcription factors and target genes, colored according to relative gene expression (log2(KO/Control)) at 72h; red and blue indicate high and low relative expression, respectively. Edges are drawn between TFs and putative gene targets if that differentially expressed TF’s motif was enriched in ATAC-seq peaks cis to genes that were increased or decreased in response to KO (Praw<10−3, hypergeometric CDF). Edge colors (blue - inhibitory, red - activating) are based on correlation between TF and putative target gene expression. “ON” indicates genes de-repressed in the knockout cells, while “OFF” indicates genes repressed in the knockout cells.

Article Snippet: The following primers-probes mixtures were purchased from Applied Biosystems: Il10 (Mm01288386_m1), Irf1 (Mm01288580_m1), Batf (Mm00479410_m1), Il21 (Mm00517640_m1), Maf (Mm02581355_s1), AhR (Mm00478937_m1), Gapdh (Mm99999915_g1).

Techniques: Comparison, Control, Gene Expression, RNA Sequencing, DNA Binding Assay, Expressing, Targeted Gene Expression, Knock-Out

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: Sequences of the Primers Used for qRT-PCR

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Sequencing

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P <0.01 vs scramble). ( C ) IRF1 expression was significantly upregulated in M1 subset and downregulated in M2 subset in THP-1 and Raw264.7 cells (* P <0.05, ** P <0.01 vs M0). ( D ) IRF1 expression was significantly upregulated in M1 macrophages and downregulated in M2 macrophages in murine peritoneal macrophages (* P <0.05 vs M0). ( E ) Western blotting of IRF1 in M1, M2 and M0 macrophages (* P <0.05). ( F ) IRF1 expression was detected after transfection with miR-106b-5p mimics or inhibitor in THP1 and Raw264.7 cells. RF1 expression decreased after miR-106b-5p mimics treatment, and increased after miR-106b inhibitor treatment (* P <0.05, ** P <0.01 vs NC). ( G ) Protein expression of IRF1 after transfection with miR-106b-5p mimics or inhibitor; * P <0.05.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition, Expressing, Western Blot, Transfection

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P <0.001). ( F ) IRF1 expression in the syngeneic intracranial glioma models (Western blotting; n=3).

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p enhanced the proliferation of glioma-infiltrating macrophages to increase tumor growth. ( A ) The time points of tumor cell inoculation and measurements; ( B, C ) volumes of subcutaneous GL261 tumor in the miR-106b-5p mimics/inhibitor group and NC group (* P <0.05 vs control, n=10). ( D ) Subcutaneous GL261 tumor in C57BL/6J mice. ( E ) IRF1 protein expression (IHC; 100×, n=3). ( F ) Ki67 protein expression (IHC; 100×, n=3). ( G ) CD163 protein expression (IHC; 100×, n=3).

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Control, Expressing

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P <0.05, ** P <0.01 vs control). ( C ) The mRNA expressions of IFN-β and IRF5 significantly decreased in THP-1 cells treated with conditioned medium from U251 cell as glioma-infiltrating macrophage model (* P <0.05, ** P <0.01 vs control). ( D ) The mRNA expressions of IFN-β and IRF5 significantly increased in M1 macrophages, and decreased in M2 macrophage (* P <0.05, ** P <0.01 vs M0). ( E ) The mRNA expressions of IFN-β and IRF5 increased after overexpression of IRF1 or inhibition of miR-106b-5p (* P <0.05, ** P <0.01 vs control). ( F ) The mRNA expression of IRF5, IL-10 and CD163 in THP1 and Raw264.7 cells transfected with IRF5 or siIRF5 and miR-106b-5p mimics or inhibitor. ( G ) The mRNA expression of IFN-β, IL-10 and CD163 in THP1 and Raw264.7 cells transfected with IFN-β or siIFN-β and miR-106b-5p mimics or inhibitor.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Over Expression, Expressing, Control, Inhibition, Transfection

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Expressing, Blocking Assay