anti irf1 primary antibody  (Cell Signaling Technology Inc)

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a GSEA using GO pathways was performed between shAPC or scramble control transfected CT26 subcutaneous tumors, using gene sets associated with type II interferon response and antigen processing and presentation. b Pathway responsive genes for activity inference from gene expression (progeny) analysis performed between tumors formed in Apc-silenced and control groups. c Total cell lysates from a series of IFNγ concentrations were subjected to immunoblot analysis with antibodies to the indicated proteins. Data represent three independent experiments. d Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression (RT-qPCR) in CT26-shApc or CT26-scramble cells. n = 6 per group, one-way ANOVA. e Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. f MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. g Numbers of OVA-tetramer positive CD8 + T cells in TILs of MC38-OVA-shAPC subcutaneous tumors as detected by flow cytometry. n = 3 for each group, one-way ANOVA. h Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in the indicated cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. i APC-silenced CT26 cells were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then subcutaneously xenotransplanted to Balb/c mice; tumor growth was monitored at the indicated times. n = 8 for each group, two-way ANOVA. j Scatterplot showing numbers of CD8 + cells in the indicated groups. n = 8, 7, 8 for each group, one-way ANOVA. k AKP organoids were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then orthotopically inoculated into C57BL/6 mice; tumor growth was monitored and scored by colonoscopy. n = 6 for each group, one-way ANOVA. l Representative immunofluorescence staining of CK (red) and CD8 (yellow) in tumor tissues, with scatterplot showing numbers of CD8 + cells in three groups. n = 6 for each group, one-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Control, Transfection, Activity Assay, Gene Expression, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Over Expression, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Apc-silenced CT26 cells transfected with negative control (N.C) or two single guide RNAs (sgRNAs) targeting Ptpn13 were stimulated with different concentrations of IFNγ. Total cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Data are representative of three independent experiments. b Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in Ptpn13-knockout or negative control sgRNA-transfected CT26-shAPC cells with 12 h exposure to IFNγ (50 ng/mL). n = 6 per group, one-way ANOVA. c Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. d Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. e Numbers of OVA-tetramer positive CD8 + T cells in TILs of Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC subcutaneous tumors, as detected by flow cytometry. n = 3 for each group, one-way ANOVA. f Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in intestinal tumors of TAM- or oil-treated APV mice. n = 6 per group, one-way ANOVA. g MFI of H-2Kb/2Db positive cells in intestinal tumors of TAM- or oil-treated APV mice as detected by flow cytometry. n = 6 per group, unpaired t -test. h Scatterplot showing correlation between MFI of HLA-ABC and Ptpn13 IF staining in CRC primary tissues. n = 80, Pearson’s r . i, j CRC-patient-derived organoids (PDO) were cultivated and transfected with Ptpn13-knockout or negative control sgRNA. i Representative immunofluorescence staining of Epcam (green), HLA-ABC (red) and CD8 (cyan). j MFI of HLA-ABC IF staining in Ptpn13-knockout or negative control sgRNA-transfected PDOs. One-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Knock-Out, Flow Cytometry, Staining, Derivative Assay, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Flag-tagged Stat1 expression vector (0.5 μg) was transfected into CT26 cells. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. b Schematic diagram showing the GST-fused PTPN13 motifs and His-tagged STAT1 used in the GST pull-down assays. c GST pull-down assays examining the interactions between GST-fused PTPN13 fragments and His-tagged STAT1 protein. Data are representative of three independent experiments. d Phosphorylated STAT1 was immunoprecipitated with anti-Flag from IFNγ-stimulated 293T transfectants and incubated with 0.2 mg/mL recombinant GST-PTPase domain of PTPN13. Immunoprecipitates were immunoblotted with anti-phospho-STAT1. Equal loading was verified by reprobing with anti-STAT1. e Total cell lysates of CT26 cells were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. f CT26 cells were treated with the indicated concentrations of IFNγ, and cell lysates were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. g A Flag-tagged Stat1 vector (0.5 μg) was transfected into CT26-shApc cells or their control cells. Total cell lysates from the indicated cells were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. h Alphafold3-predicted binding pattern of human APC (brown) and the PDZ2a domain of PTPN13 (cyan). The APC V2843 residue is labeled, and APC Q2829–V2843 residues are shown as sticks and colored in yellow. Hydrogen bonds are shown as yellow dotted lines. i CT26 cells were transfected with HA-tagged Apc-WT or Apc V2860A mutant plasmids, and total cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-PTPN13 and anti-CTNNB1. Data are representative of three independent experiments. j IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. k CRISPR/Cas9-based establishment of APC V2860A point mutation. l CT26 cells transfected with APC-WT or APC V2860A mutant plasmids (CT26-APC V2860A -1/2) were incubated with or without IFNγ at the indicated concentrations for 2 h, and total cell lysates were subjected to immunoblot analysis. m IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids (CT26-APC V2860A -1/2) for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. n CT26 cells harboring gRNA-induced mutant APC V2860A (CT26-APC V2860A -1/2) and the WT control were subcutaneously injected (2 × 10 6 cells) into Balb/c mice, and tumor growth was monitored. n = 8 for each group, two-way ANOVA. o Immunofluorescence of CD8 + cell infiltration in subcutaneous tumors of CT26-APC V2860A and the control group. n = 8 for each group, one-way ANOVA. Data are representative of three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Incubation, Recombinant, Control, Binding Assay, Residue, Labeling, Mutagenesis, Quantitative RT-PCR, CRISPR, Western Blot, Injection, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Schematic diagram showing the indicated residues at the APC C terminus. b Kinetics of the interaction between PDZ-2a and the indicated residues of APC were explored by surface plasmon resonance-based binding assays. c Binding affinities of PDZ2a to APC C-terminal peptides of different lengths, as measured by an FP assay. d The 2.1-Å complex structure of the PDZ2a domain (1364–1446 aa) and the APC11 peptide. PDZ2a is shown in cyan and presented as a surface diagram, whereas the peptide is shown in yellow and presented as a stick diagram. e Detailed interactions between the APC11 peptide and PDZ2a within the complex. The PDZ2a residues involved are labeled and shown as magenta sticks, and the peptide-interacting water molecules are shown as green balls. Hydrogen bonds are shown as yellow dotted lines. f Binding affinity of PDZ2a to the WT APC11 peptide and the APC11M mutant (V2843A) as measured by an FP assay. g GST-fused STAT1 was incubated with HA-tagged PDZ2a and with TAT-APC11 or TAT-APC11M peptides, immunoprecipitated with GST beads, and immunoblotted with anti-GST and anti-HA antibodies. Data are representative of three independent experiments. h CT26 cells transfected with a Flag-tagged Stat1 vector were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 4 h. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. i Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. j Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in Apc-silenced CT26 cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. k Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of the MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kd/2Dd antibody or isotype control antibodies. Data were calculated from three independent experiments. One-way ANOVA. l Apc-silenced MC38-OVA 257-264 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of OVA 257-264 -specific MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kb/SIINFEKL antibody or isotype control antibodies. Data represent three independent experiments. One-way ANOVA. m DLD1 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. n IRF1, LMP2, TAP1, TAP2, HLA-A, HLA-B, HLA-C , and B2M mRNA expression (RT-qPCR) in the indicated cells after exposure to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: SPR Assay, Binding Assay, FP Assay, Labeling, Mutagenesis, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Staining, Control

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Specific regulon activity increases in T cell subclusters with aging. (A) The “signature genes” in the X-axis are the set of transcription factors and their downstream target genes. Dot plots show the expression levels of these “signature genes” across the 24 cellular clusters. The size of the dots represents the proportion of regulon in specific cell types, and the spectrum of color indicates the Z-score. (B) Feature plots showing specific regulon activity ( Ets1 , Junb , Irf1 , Mta3 , Fli1 , Elf1 , Elf2 or Bclaf1 ) in T cell subclusters. (C) Expression values are represented as normalized log 2 -transformed counts. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the young group (two-sided Wilcoxon rank-sum tests). (D) Ifnγ , Il-17a , Bclaf1 , Elf1 , Elf2 , Fli1 , Irf1 , Junb , Mta3 , and Ets1 mRNA levels in the spleen and mediastinal lymph nodes by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to the control. The mRNA relative expression was normalized to the 2-month-old group. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with Student’s t-test.

Article Snippet: Primary antibodies against IRF1 (#11335, Proteintech, USA) and CD4 (#100507, BioLegend, USA), fluorophore-conjugated antibodies for CD4-PE (#12-0042-82, eBioscience, USA) and CD44-PE-Cy5 (#15-0441-81, eBioscience, USA), and IFNγ-FITC (#505806, BioLegend, USA) were used.

Techniques: Activity Assay, Expressing, Transformation Assay, Quantitative RT-PCR, Control

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: IFNγ transcribed by IRF1 increases in pulmonary T cells with aging. (A) Ifnγ , Irf1 , Fli1, Elf1 , Junb , Bclaf1 , Mta3 , and Ets1 mRNA levels in CD4 + T EM sorted from 2-month-old WT and 2-year-old WT mouse spleens by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. Three biological replicates were used per experiment. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with one-way ANOVA. (B) IRF1-like sequence binding site (highlighted in yellow) in the IFNγ promoter region and the IRF1 sequence highlighted in red. (C) IRF1 and IFNγ mRNA levels in 293T cells transfected with basic and IRF1-overexpression plasmid by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. (D) Western blots of 293T cell extracts transfected with basic and IRF1 -overexpression plasmid showing IRF1 and IFNγ. β-actin was the loading control. (E) Protein levels relative to β-actin were assessed by densitometric analysis and normalized to the basic-transfected group. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. (F) A model of IFN-γ promoter truncated primers. (G) ChIP assays were performed with chromatin prepared from 293T cells transfected with basic and IRF1-overexpression plasmid. The chromatin was immunoprecipitated with normal rabbit IgG or antibodies against IRF1, and precipitated genomic DNA was analyzed as relative enrichment through real-time PCR using different primers for the different regions of the IFNγ promoter. The β-actin promoter (-393 to -175 bp) was used as a negative control. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. **p < 0.01, ***p < 0.001 compared with the basic-transfected group. Statistical analysis was performed with Student’s t-test. (H) Schematic diagram of the structure of the pGL4.10-IFNγ promoter reporter plasmid and deletion construct of the pGL4.10-IFNγ promoter reporter plasmid. (I) IFNγ promoter activity was measured by a luciferase reporter gene assay. Six biological replicates were used per experiment (N = 6). Values are the mean ± SEM of three determinations. ***p < 0.001 compared with the corresponding group. Statistical analysis was performed with one-way ANOVA.

Article Snippet: Primary antibodies against IRF1 (#11335, Proteintech, USA) and CD4 (#100507, BioLegend, USA), fluorophore-conjugated antibodies for CD4-PE (#12-0042-82, eBioscience, USA) and CD44-PE-Cy5 (#15-0441-81, eBioscience, USA), and IFNγ-FITC (#505806, BioLegend, USA) were used.

Techniques: Quantitative RT-PCR, Control, Expressing, Transfection, Sequencing, Binding Assay, Over Expression, Plasmid Preparation, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Construct, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Differentiation dynamics of T cells with aging. Pseudotemporal trajectories identified transcriptional dynamics of T cells. (A) Monocle pseudotime analysis revealing the progression of the seven stages of T cell differentiation, which included the four T cell subtypes (naïve CD4 + , CD4 + T EM , naïve CD8 + , and CD8 + T EM ). (B) Expression levels of IRF1 and IFNγ in differentiation trajectories, the red point indicating the cells of the older mice and the blue point indicating the cells of the young mice. Dot size indicates the expression levels. Values are log10 (value + 0.1). (C) Heatmap showing the scaled expression of differentially expressed transcription factor genes along with the pseudotime, cataloged into three major gene clusters. According to pseudotemporal trajectories illustrated by (A) and (B), clusters 1 to 3 were used to label the specific branch. The box was used to choose a branch point to inspect in illustrations. (D) Pathway enrichment analysis associated with transcription factor genes as in (C). (E) Heatmap showing the scaled expression of differently expressed genes in the four branches. From the center to the left of the heatmap, the kinetic curve from the root along the trajectory to CD4 + T EM . From the center to the right, the curve from the root to CD8 + T EM . According to pseudotemporal trajectories illustrated by (A) and (B), pre-branch, cell fate 1, and cell fate 2 were used to label the specific branch. The genes were clustered hierarchically, and clusters 1 to 4 cells were automatically classified according to similar lineage-dependent gene expression patterns. The box was used to choose a branch point to inspect in illustrations. (F) KEGG analysis of differently expressed genes associated with three gene clusters as in (E).

Article Snippet: Primary antibodies against IRF1 (#11335, Proteintech, USA) and CD4 (#100507, BioLegend, USA), fluorophore-conjugated antibodies for CD4-PE (#12-0042-82, eBioscience, USA) and CD44-PE-Cy5 (#15-0441-81, eBioscience, USA), and IFNγ-FITC (#505806, BioLegend, USA) were used.

Techniques: Cell Differentiation, Expressing, Gene Expression

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Specific regulon activity increases in T cell subclusters with aging. (A) The “signature genes” in the X-axis are the set of transcription factors and their downstream target genes. Dot plots show the expression levels of these “signature genes” across the 24 cellular clusters. The size of the dots represents the proportion of regulon in specific cell types, and the spectrum of color indicates the Z-score. (B) Feature plots showing specific regulon activity ( Ets1 , Junb , Irf1 , Mta3 , Fli1 , Elf1 , Elf2 or Bclaf1 ) in T cell subclusters. (C) Expression values are represented as normalized log 2 -transformed counts. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the young group (two-sided Wilcoxon rank-sum tests). (D) Ifnγ , Il-17a , Bclaf1 , Elf1 , Elf2 , Fli1 , Irf1 , Junb , Mta3 , and Ets1 mRNA levels in the spleen and mediastinal lymph nodes by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to the control. The mRNA relative expression was normalized to the 2-month-old group. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with Student’s t-test.

Article Snippet: Pulmonary CD4 + effector memory T cells were treated for 48 h with anti-IRF1 primary antibody (#11335, Proteintech, USA).

Techniques: Activity Assay, Expressing, Transformation Assay, Quantitative RT-PCR, Control

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: IFNγ transcribed by IRF1 increases in pulmonary T cells with aging. (A) Ifnγ , Irf1 , Fli1, Elf1 , Junb , Bclaf1 , Mta3 , and Ets1 mRNA levels in CD4 + T EM sorted from 2-month-old WT and 2-year-old WT mouse spleens by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. Three biological replicates were used per experiment. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with one-way ANOVA. (B) IRF1-like sequence binding site (highlighted in yellow) in the IFNγ promoter region and the IRF1 sequence highlighted in red. (C) IRF1 and IFNγ mRNA levels in 293T cells transfected with basic and IRF1-overexpression plasmid by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. (D) Western blots of 293T cell extracts transfected with basic and IRF1 -overexpression plasmid showing IRF1 and IFNγ. β-actin was the loading control. (E) Protein levels relative to β-actin were assessed by densitometric analysis and normalized to the basic-transfected group. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. (F) A model of IFN-γ promoter truncated primers. (G) ChIP assays were performed with chromatin prepared from 293T cells transfected with basic and IRF1-overexpression plasmid. The chromatin was immunoprecipitated with normal rabbit IgG or antibodies against IRF1, and precipitated genomic DNA was analyzed as relative enrichment through real-time PCR using different primers for the different regions of the IFNγ promoter. The β-actin promoter (-393 to -175 bp) was used as a negative control. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. **p < 0.01, ***p < 0.001 compared with the basic-transfected group. Statistical analysis was performed with Student’s t-test. (H) Schematic diagram of the structure of the pGL4.10-IFNγ promoter reporter plasmid and deletion construct of the pGL4.10-IFNγ promoter reporter plasmid. (I) IFNγ promoter activity was measured by a luciferase reporter gene assay. Six biological replicates were used per experiment (N = 6). Values are the mean ± SEM of three determinations. ***p < 0.001 compared with the corresponding group. Statistical analysis was performed with one-way ANOVA.

Article Snippet: Pulmonary CD4 + effector memory T cells were treated for 48 h with anti-IRF1 primary antibody (#11335, Proteintech, USA).

Techniques: Quantitative RT-PCR, Control, Expressing, Transfection, Sequencing, Binding Assay, Over Expression, Plasmid Preparation, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Construct, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Differentiation dynamics of T cells with aging. Pseudotemporal trajectories identified transcriptional dynamics of T cells. (A) Monocle pseudotime analysis revealing the progression of the seven stages of T cell differentiation, which included the four T cell subtypes (naïve CD4 + , CD4 + T EM , naïve CD8 + , and CD8 + T EM ). (B) Expression levels of IRF1 and IFNγ in differentiation trajectories, the red point indicating the cells of the older mice and the blue point indicating the cells of the young mice. Dot size indicates the expression levels. Values are log10 (value + 0.1). (C) Heatmap showing the scaled expression of differentially expressed transcription factor genes along with the pseudotime, cataloged into three major gene clusters. According to pseudotemporal trajectories illustrated by (A) and (B), clusters 1 to 3 were used to label the specific branch. The box was used to choose a branch point to inspect in illustrations. (D) Pathway enrichment analysis associated with transcription factor genes as in (C). (E) Heatmap showing the scaled expression of differently expressed genes in the four branches. From the center to the left of the heatmap, the kinetic curve from the root along the trajectory to CD4 + T EM . From the center to the right, the curve from the root to CD8 + T EM . According to pseudotemporal trajectories illustrated by (A) and (B), pre-branch, cell fate 1, and cell fate 2 were used to label the specific branch. The genes were clustered hierarchically, and clusters 1 to 4 cells were automatically classified according to similar lineage-dependent gene expression patterns. The box was used to choose a branch point to inspect in illustrations. (F) KEGG analysis of differently expressed genes associated with three gene clusters as in (E).

Article Snippet: Pulmonary CD4 + effector memory T cells were treated for 48 h with anti-IRF1 primary antibody (#11335, Proteintech, USA).

Techniques: Cell Differentiation, Expressing, Gene Expression

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Specific regulon activity increases in T cell subclusters with aging. (A) The “signature genes” in the X-axis are the set of transcription factors and their downstream target genes. Dot plots show the expression levels of these “signature genes” across the 24 cellular clusters. The size of the dots represents the proportion of regulon in specific cell types, and the spectrum of color indicates the Z-score. (B) Feature plots showing specific regulon activity ( Ets1 , Junb , Irf1 , Mta3 , Fli1 , Elf1 , Elf2 or Bclaf1 ) in T cell subclusters. (C) Expression values are represented as normalized log 2 -transformed counts. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the young group (two-sided Wilcoxon rank-sum tests). (D) Ifnγ , Il-17a , Bclaf1 , Elf1 , Elf2 , Fli1 , Irf1 , Junb , Mta3 , and Ets1 mRNA levels in the spleen and mediastinal lymph nodes by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to the control. The mRNA relative expression was normalized to the 2-month-old group. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with Student’s t-test.

Article Snippet: Recombinant mouse IFNγ, anti-IFNγ primary antibody, and anti-IRF1 primary antibody : AT2 cells were treated for 48 h with different concentrations (0, 20, 40, 60, 80, and 100 ng/mL) of mouse IFNγ (Chamot Biotechnology Inc., Shanghai, China), sorted cells, and anti-IFNγ primary antibody (#15365, Proteintech, USA).

Techniques: Activity Assay, Expressing, Transformation Assay, Quantitative RT-PCR, Control

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: IFNγ transcribed by IRF1 increases in pulmonary T cells with aging. (A) Ifnγ , Irf1 , Fli1, Elf1 , Junb , Bclaf1 , Mta3 , and Ets1 mRNA levels in CD4 + T EM sorted from 2-month-old WT and 2-year-old WT mouse spleens by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. Three biological replicates were used per experiment. Values are the mean ± SEM of three determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 2-month-old group. Statistical analysis was performed with one-way ANOVA. (B) IRF1-like sequence binding site (highlighted in yellow) in the IFNγ promoter region and the IRF1 sequence highlighted in red. (C) IRF1 and IFNγ mRNA levels in 293T cells transfected with basic and IRF1-overexpression plasmid by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was normalized to the basic-transfected group. (D) Western blots of 293T cell extracts transfected with basic and IRF1 -overexpression plasmid showing IRF1 and IFNγ. β-actin was the loading control. (E) Protein levels relative to β-actin were assessed by densitometric analysis and normalized to the basic-transfected group. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. (F) A model of IFN-γ promoter truncated primers. (G) ChIP assays were performed with chromatin prepared from 293T cells transfected with basic and IRF1-overexpression plasmid. The chromatin was immunoprecipitated with normal rabbit IgG or antibodies against IRF1, and precipitated genomic DNA was analyzed as relative enrichment through real-time PCR using different primers for the different regions of the IFNγ promoter. The β-actin promoter (-393 to -175 bp) was used as a negative control. Three biological replicates were used per experiment (N = 3). Values are the mean ± SEM of six determinations. **p < 0.01, ***p < 0.001 compared with the basic-transfected group. Statistical analysis was performed with Student’s t-test. (H) Schematic diagram of the structure of the pGL4.10-IFNγ promoter reporter plasmid and deletion construct of the pGL4.10-IFNγ promoter reporter plasmid. (I) IFNγ promoter activity was measured by a luciferase reporter gene assay. Six biological replicates were used per experiment (N = 6). Values are the mean ± SEM of three determinations. ***p < 0.001 compared with the corresponding group. Statistical analysis was performed with one-way ANOVA.

Article Snippet: Recombinant mouse IFNγ, anti-IFNγ primary antibody, and anti-IRF1 primary antibody : AT2 cells were treated for 48 h with different concentrations (0, 20, 40, 60, 80, and 100 ng/mL) of mouse IFNγ (Chamot Biotechnology Inc., Shanghai, China), sorted cells, and anti-IFNγ primary antibody (#15365, Proteintech, USA).

Techniques: Quantitative RT-PCR, Control, Expressing, Transfection, Sequencing, Binding Assay, Over Expression, Plasmid Preparation, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Construct, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Aging and Disease

Article Title: IFNγ Transcribed by IRF1 in CD4 + Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/AD.2023.0320

Figure Lengend Snippet: Differentiation dynamics of T cells with aging. Pseudotemporal trajectories identified transcriptional dynamics of T cells. (A) Monocle pseudotime analysis revealing the progression of the seven stages of T cell differentiation, which included the four T cell subtypes (naïve CD4 + , CD4 + T EM , naïve CD8 + , and CD8 + T EM ). (B) Expression levels of IRF1 and IFNγ in differentiation trajectories, the red point indicating the cells of the older mice and the blue point indicating the cells of the young mice. Dot size indicates the expression levels. Values are log10 (value + 0.1). (C) Heatmap showing the scaled expression of differentially expressed transcription factor genes along with the pseudotime, cataloged into three major gene clusters. According to pseudotemporal trajectories illustrated by (A) and (B), clusters 1 to 3 were used to label the specific branch. The box was used to choose a branch point to inspect in illustrations. (D) Pathway enrichment analysis associated with transcription factor genes as in (C). (E) Heatmap showing the scaled expression of differently expressed genes in the four branches. From the center to the left of the heatmap, the kinetic curve from the root along the trajectory to CD4 + T EM . From the center to the right, the curve from the root to CD8 + T EM . According to pseudotemporal trajectories illustrated by (A) and (B), pre-branch, cell fate 1, and cell fate 2 were used to label the specific branch. The genes were clustered hierarchically, and clusters 1 to 4 cells were automatically classified according to similar lineage-dependent gene expression patterns. The box was used to choose a branch point to inspect in illustrations. (F) KEGG analysis of differently expressed genes associated with three gene clusters as in (E).

Article Snippet: Recombinant mouse IFNγ, anti-IFNγ primary antibody, and anti-IRF1 primary antibody : AT2 cells were treated for 48 h with different concentrations (0, 20, 40, 60, 80, and 100 ng/mL) of mouse IFNγ (Chamot Biotechnology Inc., Shanghai, China), sorted cells, and anti-IFNγ primary antibody (#15365, Proteintech, USA).

Techniques: Cell Differentiation, Expressing, Gene Expression

Journal: Aging and disease

Article Title: IFNγ Transcribed by IRF1 in CD4⁺ Effector Memory T Cells Promotes Senescence-Associated Pulmonary Fibrosis

doi: 10.14336/ad.2023.0320

Figure Lengend Snippet: Figure 6. IFNγ transcribed by IRF1 increases in pulmonary T cells with aging. (A) Ifnγ, Irf1, Fli1, Elf1, Junb, Bclaf1, Mta3, and Ets1 mRNA levels in CD4+ TEM sorted from 2-month-old WT and 2-year-old WT mouse spleens by real-time RT-PCR, calculated as a ratio to β-actin mRNA and expressed relative to control. The mRNA relative expression was

Article Snippet: Immunofluorescent staining of pulmonary sections Primary antibodies against IRF1 (#11335, Proteintech, USA) and CD4 (#100507, BioLegend, USA), fluorophore-conjugated antibodies for CD4-PE (#12- 0042-82, eBioscience, USA) and CD44-PE-Cy5 (#15- 0441-81, eBioscience, USA), and IFNγ-FITC (#505806, BioLegend, USA) were used.

Techniques: Quantitative RT-PCR, Control, Expressing