Journal: Inflammopharmacology

Article Title: Herbal remedies for oral and dental health: a comprehensive review of their multifaceted mechanisms including antimicrobial, anti-inflammatory, and antioxidant pathways

doi: 10.1007/s10787-024-01631-8

Figure Lengend Snippet: Efficacy of herbal remedies against periodontal pathogens

Article Snippet: Lippia alba (Mill.) Cymbopogon citratus L. , Whole plant/essential oil extraction through steam distillation , An assay called MBEC-high-throughput (MBEC-HTP) to measure the amount of S. mutans ATCC 35668 strain biofilms that were eradicated CHO cells were subjected to cytotoxicity assessment using the MTT cell proliferation assay , Geraniol and citral were the main constituents of both oils; they were 18.9% and 15.9% in L. alba and 31.3% and 26.7% in C. citratus , respectively. No concentration of either essential oil was toxic to CHO cells over a 24-h period. The essential oils of L. alba and C. citratus demonstrated eradication activity against Streptococcus mutans biofilms of 95.8% and 95.4%, respectively, at 0.1 and 0.01 mg/dL concentrations and 93.1%, at 0.001 mg/dL concentration , (Tofiño-Rivera et al. ) .

Techniques: Diffusion-based Assay, Produced, Inhibition, Positive Control, Bacteria, Microbial Assay, In Vivo, Cytotoxicity Assay, Activity Assay, In Vitro, Derivative Assay, Concentration Assay, Time-Kill Assay, Extraction, Microdilution Assay, Sequencing, Expressing, Electron Microscopy, Confocal Laser Scanning Microscopy, MTT Assay, Enzyme-linked Immunosorbent Assay, Control, Functional Assay, Serial Dilution, Adsorption, Probiotics, Formulation, Comparison, Cannabis, Ex Vivo, Maltodextrin, Modification, Biofilm Production Assay, Permeability, Incubation, Membrane, Tube Formation Assay, Binding Assay, Migration, In Silico, Cell Counting, Recombinant, WST-1 Assay, Luminescence Assay, DNA Synthesis, Gene Expression, Irradiation, Serial Time-encoded Amplified Microscopy, Distillation, MTT Cell Proliferation, Sampling, Staining, Resazurin Assay, Solvent, Serial Dilution Assay, Dilution Assay, Antioxidant Activity Assay, Animal Model, Real-time Polymerase Chain Reaction, Isolation, Transmission Assay, Preserving, Colorimetric Assay, Saline, Activation Assay, Molecular Weight, Disruption, Labeling, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 2. Chemokine receptor expression on pulmonary neutrophils. Chemokine receptor expression on pulmonary neutrophils was assessed by flow cytometry (left column), immunohistochemistry (middle column), and confocal microscopy (right column). Flow cytometry: representative his- tograms are shown of neutrophils from BALF of a representative patient with COPD and a representative control subject (Control) and from pe- ripheral blood (Blood) of a control subject. Isotype expression overlapped with the specific expression in peripheral blood. Immunohistochemistry: representative sections of lung tissue from three COPD patients are shown. Diaminobenzidine was used as chromogen and hematoxylin for counter- staining. Black arrows mark chemokine receptor-expressing neutrophils. Confocal microscopy: Neutrophils isolated from BALF from COPD pa- tients were fixed in paraformaldehyde and permeabilized with Triton X-100. After washing, the neutrophils were incubated with mouse anti- CXCR3, anti-CXCR4, anti-CCR1, anti-CCR2, anti-CCR3, or anti-CCR5 or the respective isotype controls. After staining with anti-mouse Alexa Fluor 555 along with Con A conjugated to Alexa Fluor 488, confocal laser-scanning microscopy was performed with 63 objectives. Red color represents chemokine receptors and green color shows Con A (membrane and cytoplasm staining).

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Expressing, Cytometry, Immunohistochemistry, Confocal Microscopy, Flow Cytometry, Control, Staining, Isolation, Incubation, Confocal Laser Scanning Microscopy, Membrane

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 4. Chemokine receptors on infiltrated neutro- phils are functional. A, Chemotaxis. Neutrophils were iso- lated from peripheral blood and BALF of COPD patients and SF of RA patients. Chemokine receptors were blocked by pretreatment of neutrophils with anti-CCR1-, anti- CCR2-, anti-CCR3-, anti-CCR5-, anti-CXCR3-blocking Abs (each 10 g/ml) or AMD3100 (3 M) for 30 min. Neutrophils (0.5 106 cells) were labeled with calcein and were placed in the upper compartment of a Transwell filter system. CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each at 100 nM) were placed in the lower com- partment. The Transwells were incubated for 60 min at 37°C. The percent cell migration was calculated by flow cytometry, i.e., the amount of fluorescence in each of these compartments was measured and related to the fluores- cence of the total input (set at 100%). Bars represent the means SEM of independent experiments with cells from three different donors. , p 0.05 Ab-treated vs non- treated neutrophils. B, Chemokinesis. B shows dose-de- pendent migratory responses of BALF-isolated neutrophils from COPD patients (n 3). Chemotaxis was analyzed with the respective chemokines (CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 at the indicated concentra- tions) present in the lower compartment of the Transwell chamber (lower). Chemokinesis was analyzed with che- mokines present in both compartments (both) or just in the upper compartment (upper) of the chamber. , p 0.05 lower chamber vs upper chamber or both chambers.

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Functional Assay, Chemotaxis Assay, Blocking Assay, Labeling, Incubation, Migration, Cytometry, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 5. Neutrophil effector functions. A, Respi- ratory burst. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA pa- tients. Chemokine receptors were blocked by pretreat- ment of neutrophils with chemokine receptor-blocking Abs (each 10 g/ml) for 30 min. Neutrophils were loaded with DHR and were treated with CCL3, CCL2, CCL11, CCL5, CXCL11, or CXCL12 (each 100 nM) for 30 min at 37°C to activate the CCR1, CCR2, CCR3, CCR5, CXCR3, or CXCR4 function. After the chemo- kine stimulation, fMLP (1 M) was added for 5 min at 37°C. Afterward, the reactions were stopped by trans- ferring the sample tubes onto ice, and cells were im- mediately analyzed on the flow cytometer as described previously (35). Background MFI from a control tube without fMLP was subtracted from the MFI of the fMLP-stimulated cells. Results are expressed as the MFI of the total neutrophil population. , p 0.05 vs neutrophils from peripheral blood. B, Phagocytosis. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA patients. Che- mokine receptors were blocked by pretreatment of neu- trophils with chemokine receptor-blocking Abs (each 10 g/ml) for 30 min. P. aeruginosa bacteria were in- cubated with the fluorescent ligand LY for 60 min at room temperature. After preopsonization, the bacteria (2 107/ml) in HBSS-gel were incubated at 37°C for 2 h with neutrophils (2 106/ml) in the presence of the CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each 100 nM). After the incubation period, the neutro- phils were separated from the free bacteria by three cen- trifugations at 200 g for 5 min. The LY fluorescence of the isolated neutrophils was analyzed by flow cytom- etry. , p 0.05 vs neutrophils from peripheral blood. C, -Defensin release. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA patients. Chemokine receptors were blocked by pre- treatment of neutrophils with chemokine receptor- blocking Abs (each 10 g/ml) or AMD3100 (3 M) for 30 min. Afterward, neutrophils were incubated with CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each at 100 nM) for 1 h. -defensins were quantified in cell-free supernatants by sandwich ELISA with anti- human -defensin mAb, anti-human -defensin poly- clonal Ab, HRP-conjugated rabbit anti-goat IgG, and recombinant human -defensin-1. , p 0.05 vs neu- trophils from peripheral blood. Treating neutrophils with isotype control Abs instead of blocking Abs had no effect on respiratory burst, phagocytosis, or -defensin release (data not shown).

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Isolation, Blocking Assay, Cytometry, Control, Bacteria, Incubation, Sandwich ELISA, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 6. Bacterial killing. A, A clinical isolate of a mucoid P. aeruginosa from a CF patient’s sputum was subcultured overnight, grown to stationary phase, washed, and preopsonized by incubation for 60 min at 37°C in 20% pooled, fresh C5a-depleted human serum. After washing two times in PBS, the opsonized P. aeruginosa bacteria were resuspended in 1 ml of a mixture of HBSS-gel and tryptic soy broth. Afterward, neutrophils isolated from BALF of a patient with COPD were mixed with preopsonized bacteria (2 107 bacteria/ml) at a ratio of 1:5 (neutrophils/bacteria) in the absence or presence of CCL2, CCL4, CCL11, CCL15, CXCL11, or CXCL12 (each 100 nM). Aliquots of each mixture were removed after 0, 30, 60, 90, and 120 min of incubation at 37°C. P. aeruginosa colonies at each time interval were counted by serial dilution in distilled water and quantitative spread plating and were expressed as CFU per ml. B, After 60 min, anti-CXCR3-blocking Abs (10 g/ml) or AMD3100 (3 M) were added to the CXCL11- or CXCL12-treated aliquots. Treating neutrophils with isotype control Abs instead of blocking Abs had no effect on bacterial killing (data not shown). Bars represent the means SEM of three independent experiments with cells from different donors.

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Incubation, Bacteria, Isolation, Serial Dilution, Blocking Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 8. Effect of Toll-like/NOD2 receptor ligands on chemokine receptor expression of neutrophils. Neutrophils (106/ml) from the blood of healthy controls were preincubated with 50 ng/ml recombinant human GM-CSF for 90 min and were then incubated for 6 h at 37°C in HBSS with 100 ng/ml LPS, 10 g/ml Pam3CSK4, 10 g/ml LTA, 10 g/ml PGN, 10 g/ml MDP, 10 g/ml R-848, 50 g/ml poly(I:C), 100 g/ml CpG, 1 g/ml flagellin, or 50 g/ml zymosan A. After the incubation period, neutrophils were incubated with CCR1, CCR2, CCR3, CCR5, CXCR3, or CXCR4 Abs and analyzed by flow cytometry. The means SEM of five independent experiments with cells from different donors are shown. , p 0.05 vs HBSS-treated neutrophils.

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Expressing, Recombinant, Incubation, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 9. CXCR3 expression in neutrophils. A, Confocal microscopy. Neutrophils were isolated from peripheral blood of healthy controls (2 106/ml). Where indicated, neutrophils were permeabilized with an equal volume of cold 0.1% (w/v) Triton X-100. Neutrophils were stained with rabbit anti-CXCR3 Abs, anti-rabbit-Ig Alexa Fluor 555, and Con A conjugated to Alexa Fluor 488. Confocal laser-scanning microscopy was performed with the Leica TCS NT laser system using a Leica DM IRB microscope. Red color represents CXCR3 receptors and green color Con A. B, Mobilization of intracellular CXCR3 expression. Neutrophils (2 106/ml) were incubated in a shaking water bath at 37°C before addition of the (priming) agents CytB (5 g/ml, 5 min) or PAF (1 mol/L, 2 min) and further stimulation with fMLP (1 mol/L). After the incubation time, the CXCR3 surface expression was analyzed by flow cytometry. Green line histogram: isotype control; blue-filled histogram: CXCR3 surface expression. C, Coexpression of CXCR3 and CD63. Neutrophils were isolated and stimulated with CytB plus fMLP or PAF plus fMLP as described above and were costained with CXCR3, CD63, and CD66b. Where indicated, isotype controls were used instead of the specific Abs.

Article Snippet: The following mAbs were used: CD16-allophycocyanin mouse IgG1, CCR1-PE mouse IgG2b, CCR2-PE mouse IgG2b, CCR3-PE rat IgG2a, CCR4-PE IgG2b, CCR5-PE mouse IgG2b, CXCR3-FITC mouse IgG1 (all R&D Systems) and CD63-PE mouse IgG1, CD66b-PE mouse IgG2a, CD35-PE mouse IgG1, CD15-FITC mouse IgM, and CXCR4-PE mouse IgG2a (all from BD Pharmingen).

Techniques: Expressing, Confocal Microscopy, Isolation, Staining, Confocal Laser Scanning Microscopy, Microscopy, Incubation, Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

doi: 10.3390/ijms17121984

Figure Lengend Snippet: Regulation of the mechanistic/mammalian target of rapamycin (mTOR) inhibitor and stress sensor DNA damage-inducible transcript 4 (Ddit4) by liganded vitamin D receptors (VDRs) within primary keratinocytes (column 1.5). ( A ) Dose-dependent transcriptional induction of Cyp24a1 and Ddit4 by 1,25-dihydroxyvitamin D (1,25D 3 , 6 h) in wildtype (WT), but not VDR −/− , primary keratinocytes derived from neonatal pups; ( B ) Transient transfection of VDR −/− primary keratinocytes with a mVDR plasmid restores endogenous Ddit4 transcript levels. Combined treatment further enhanced Ddit4 mRNA levels (with mVDR transfection and 1,25D 3 of 10 nM for 6 h); ( C ) Immunofluorescence detection of Ddit4 within primary keratinocytes. WT, but not VDR −/− , primary keratinocytes exposed to 10 nM 1,25D 3 for 18 h resulted in increased intracellular accumulation of Ddit4 (white arrows). Ddit4 upregulation in response to fetal bovine serum deprivation for 24 h in both WT and VDR −/− keratinocytes. One-way ANOVA at an α = 0.05 (95% confidence interval) and Tukey’s multiple comparison post-tests were utilized. Significance is denoted with asterisks: * p < 0.05 ( n = 3–4 experiments). RT-qPCR: reverse transcription quantitative polymerase chain reaction.

Article Snippet: The untagged mouse vitamin D receptor expression vector (pCMV6) was purchased from Origene (BC006716).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Immunofluorescence, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

doi: 10.3390/ijms17121984

Figure Lengend Snippet: Ddit4 transcriptional activity is directly regulated by the VDR within primary epidermal keratinocytes (1.5 column). ( A ) Representative chromatin immunoprecipitation (ChIP) gel images of VDR and RNA polymerase 2 (RNApol2) interactions at the Ddit4 promoter region within WT primary keratinocytes. (AI) shows VDR immunoprecipitation, while (AII) shows RNApol2 immunoprecipitation results. Cells were treated with 1,25D 3 (10 nM) for 15 min, fixed and then the chromatin was purified. NS (non-specific), OC (osteocalcin), and VDR-S1 (VDR intron 3 and 4) genomic sites were used for control purposes. P (proximal), D (distal), VDRE (putative vitamin D response element); ( B ) ChIP-Quantitative PCR (qPCR) analysis of VDR immunoprecipitation; ( C ) ChiP-qPCR analysis of RNApol2 immunoprecipitation. One-way ANOVA at an α = 0.05 (95% confidence interval) and Tukey’s multiple comparison post-tests were utilized. Significance is denoted with asterisks: ** p < 0.01 ( n = 4 experiments).

Article Snippet: The untagged mouse vitamin D receptor expression vector (pCMV6) was purchased from Origene (BC006716).

Techniques: Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

doi: 10.3390/ijms17121984

Figure Lengend Snippet: Ddit4 −/− mouse embryonic fibroblasts are resistant to the pro-differentiation actions of vitamin D (1 column). ( A ) Representative images of WT and Ddit4 −/− mouse embryonic fibroblasts (MEFs). MEFs were untreated or treated with a serial dilution of 1,25D 3 (0.39–100 nM) for 24 h; ( B ) Cell counts performed after 1,25D 3 treatment to calculate the proliferation % relative to the untreated sample. One-way ANOVA at an α = 0.05 (95% confidence interval) and Tukey’s multiple comparison post-tests were utilized. Significance is denoted with asterisks: * p < 0.05, ** p < 0.01 ( n = 3 experiments per conditions and genotype); ( C ) Representative BrdU-labeled MEFs shown after 1,25D 3 treatment for 12 h. DAPI-positive cells were marked using the Imaris (Bitplane) software to perform quantitative analysis. Bar = 50 µm; ( D ) Quantification of BrdU-labeled MEFs after 1,25D 3 treatment. Significance is denoted with asterisks: ** p < 0.01, *** p < 0.001 ( n = 3 experiments per conditions and genotype), ns (not significant); ( E ) Quantitative PCR analysis of Vimentin in Wt and KO MEFs after 6 h with or without vitamin D, * p < 0.05 ( I ). Quantitative PCR analysis of MEFs treated or untreated with low or high concentrations of 1,25D 3 for 6 h ( II – IV ). Increased expression of Vdr ( II ) and induction of Cyp24a1 ( III , IV ) within Ddit4 −/− MEFs. ( n = 3 preparations of each cell line).

Article Snippet: The untagged mouse vitamin D receptor expression vector (pCMV6) was purchased from Origene (BC006716).

Techniques: Serial Dilution, Labeling, Software, Real-time Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

doi: 10.3390/ijms17121984

Figure Lengend Snippet: Early stress in VDR −/− hair follicles and epidermal regulation of Ddit4 by the VDR (column 1.5). Schema depicting the major findings. ( A ) VDR −/− hair follicles (HFs) exhibit DDIT4 + stress compartments, marked in red, during morphogenesis which prolongs the telogen phase; ( B ) The VDR regulates Ddit4 expression during epidermal homeostasis and wound healing responses. By day 48, VDR −/− hair follicles have degenerated resulting in loss of Ddit4expression; ( C ) Proposed functional role of Ddit4 and mTOR during epidermal maturation and hair growth. During epidermal maturation, VDR signaling induces Ddit4 expression to block mTOR cascades. During the initiation of anagen, Ddit4 levels are suppressed in order to stimulate the mTOR growth-promoting cascade; ( D ) Proposed involvement of DDIT4 in regulating the pro-differentiation effects of vitamin D in embryonic fibroblasts.

Article Snippet: The untagged mouse vitamin D receptor expression vector (pCMV6) was purchased from Origene (BC006716).

Techniques: Expressing, Functional Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 3. A, Neutrophil chemokine receptor expression in peripheral blood, BALF, and SF. Chemokine receptor expression levels on neutrophils from peripheral blood of healthy controls () or patients with lung diseases (white hatched bars), from BALF of healthy controls (u) or patients with lung diseases (gray hatched bars) and from peripheral blood and SF of patients with RA (f). Receptor expression on neutrophils was analyzed by flow cytometry. The upper panel shows the surface expression levels of chemokine receptors, the lower panel shows percentages of chemokine receptor-expressing neutrophils. Bars represent means SEM. , p 0.05: vs neutrophils in peripheral blood. B, Chemokines in bronchoalveolar lavage and synovial fluid. Levels of human CCL2, CCL3, CCL4, CCL5, CCL11, CXCL11, and CXCL12 were quantified in BALF of healthy controls, in BALF of patients with lung diseases, and in SF of patients with RA by sandwich ELISA. Bars represent means SEM. , p 0.05 vs BALF from healthy controls. C, Neutrophil chemokine receptor expression in BALF from patients with chronic inflammatory lung diseases. Chemokine receptor expression levels on neutrophils in BALF of healthy controls and patients with CF, COPD, or allergic asthma. Receptor expression on neutrophils was analyzed by flow cytometry. Bars represent means SEM. , p 0.05 vs neutrophils in peripheral blood.

Article Snippet: Recombinant human CCL2, CCL4, CCL11, CCL15, CXCL11, and CXCL12 were from R&D Systems.

Techniques: Expressing, Cytometry, Sandwich ELISA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 4. Chemokine receptors on infiltrated neutro- phils are functional. A, Chemotaxis. Neutrophils were iso- lated from peripheral blood and BALF of COPD patients and SF of RA patients. Chemokine receptors were blocked by pretreatment of neutrophils with anti-CCR1-, anti- CCR2-, anti-CCR3-, anti-CCR5-, anti-CXCR3-blocking Abs (each 10 g/ml) or AMD3100 (3 M) for 30 min. Neutrophils (0.5 106 cells) were labeled with calcein and were placed in the upper compartment of a Transwell filter system. CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each at 100 nM) were placed in the lower com- partment. The Transwells were incubated for 60 min at 37°C. The percent cell migration was calculated by flow cytometry, i.e., the amount of fluorescence in each of these compartments was measured and related to the fluores- cence of the total input (set at 100%). Bars represent the means SEM of independent experiments with cells from three different donors. , p 0.05 Ab-treated vs non- treated neutrophils. B, Chemokinesis. B shows dose-de- pendent migratory responses of BALF-isolated neutrophils from COPD patients (n 3). Chemotaxis was analyzed with the respective chemokines (CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 at the indicated concentra- tions) present in the lower compartment of the Transwell chamber (lower). Chemokinesis was analyzed with che- mokines present in both compartments (both) or just in the upper compartment (upper) of the chamber. , p 0.05 lower chamber vs upper chamber or both chambers.

Article Snippet: Recombinant human CCL2, CCL4, CCL11, CCL15, CXCL11, and CXCL12 were from R&D Systems.

Techniques: Functional Assay, Chemotaxis Assay, Blocking Assay, Labeling, Incubation, Migration, Cytometry, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 5. Neutrophil effector functions. A, Respi- ratory burst. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA pa- tients. Chemokine receptors were blocked by pretreat- ment of neutrophils with chemokine receptor-blocking Abs (each 10 g/ml) for 30 min. Neutrophils were loaded with DHR and were treated with CCL3, CCL2, CCL11, CCL5, CXCL11, or CXCL12 (each 100 nM) for 30 min at 37°C to activate the CCR1, CCR2, CCR3, CCR5, CXCR3, or CXCR4 function. After the chemo- kine stimulation, fMLP (1 M) was added for 5 min at 37°C. Afterward, the reactions were stopped by trans- ferring the sample tubes onto ice, and cells were im- mediately analyzed on the flow cytometer as described previously (35). Background MFI from a control tube without fMLP was subtracted from the MFI of the fMLP-stimulated cells. Results are expressed as the MFI of the total neutrophil population. , p 0.05 vs neutrophils from peripheral blood. B, Phagocytosis. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA patients. Che- mokine receptors were blocked by pretreatment of neu- trophils with chemokine receptor-blocking Abs (each 10 g/ml) for 30 min. P. aeruginosa bacteria were in- cubated with the fluorescent ligand LY for 60 min at room temperature. After preopsonization, the bacteria (2 107/ml) in HBSS-gel were incubated at 37°C for 2 h with neutrophils (2 106/ml) in the presence of the CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each 100 nM). After the incubation period, the neutro- phils were separated from the free bacteria by three cen- trifugations at 200 g for 5 min. The LY fluorescence of the isolated neutrophils was analyzed by flow cytom- etry. , p 0.05 vs neutrophils from peripheral blood. C, -Defensin release. Neutrophils were isolated from peripheral blood and BALF of COPD patients and SF of RA patients. Chemokine receptors were blocked by pre- treatment of neutrophils with chemokine receptor- blocking Abs (each 10 g/ml) or AMD3100 (3 M) for 30 min. Afterward, neutrophils were incubated with CCL2, CCL3, CCL4, CCL11, CXCL11, or CXCL12 (each at 100 nM) for 1 h. -defensins were quantified in cell-free supernatants by sandwich ELISA with anti- human -defensin mAb, anti-human -defensin poly- clonal Ab, HRP-conjugated rabbit anti-goat IgG, and recombinant human -defensin-1. , p 0.05 vs neu- trophils from peripheral blood. Treating neutrophils with isotype control Abs instead of blocking Abs had no effect on respiratory burst, phagocytosis, or -defensin release (data not shown).

Article Snippet: Recombinant human CCL2, CCL4, CCL11, CCL15, CXCL11, and CXCL12 were from R&D Systems.

Techniques: Isolation, Blocking Assay, Cytometry, Control, Bacteria, Incubation, Sandwich ELISA, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

doi: 10.4049/jimmunol.181.11.8053

Figure Lengend Snippet: FIGURE 6. Bacterial killing. A, A clinical isolate of a mucoid P. aeruginosa from a CF patient’s sputum was subcultured overnight, grown to stationary phase, washed, and preopsonized by incubation for 60 min at 37°C in 20% pooled, fresh C5a-depleted human serum. After washing two times in PBS, the opsonized P. aeruginosa bacteria were resuspended in 1 ml of a mixture of HBSS-gel and tryptic soy broth. Afterward, neutrophils isolated from BALF of a patient with COPD were mixed with preopsonized bacteria (2 107 bacteria/ml) at a ratio of 1:5 (neutrophils/bacteria) in the absence or presence of CCL2, CCL4, CCL11, CCL15, CXCL11, or CXCL12 (each 100 nM). Aliquots of each mixture were removed after 0, 30, 60, 90, and 120 min of incubation at 37°C. P. aeruginosa colonies at each time interval were counted by serial dilution in distilled water and quantitative spread plating and were expressed as CFU per ml. B, After 60 min, anti-CXCR3-blocking Abs (10 g/ml) or AMD3100 (3 M) were added to the CXCL11- or CXCL12-treated aliquots. Treating neutrophils with isotype control Abs instead of blocking Abs had no effect on bacterial killing (data not shown). Bars represent the means SEM of three independent experiments with cells from different donors.

Article Snippet: Recombinant human CCL2, CCL4, CCL11, CCL15, CXCL11, and CXCL12 were from R&D Systems.

Techniques: Incubation, Bacteria, Isolation, Serial Dilution, Blocking Assay, Control

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/JVI.01782-19

Figure Lengend Snippet: HIV + exosomes promote KSHV infection in oral epithelial cells. (A) Serial dilution of KSHV infection in OKF6/TERT2 cells. A freshly prepared KSHV stock solution was diluted to 1:100, 1:50, and 1:20 with the culture medium for infection in OKF6/TERT2 cells. At 20 h after infection, cells were stained using antibodies to LANA and GFP. Immunofluorescent images were taken using a fluorescence microscope. (B) OKF6/TERT2 cells were treated with exosomes from HIV + J1.1 (J1.1 exo) or Jurkat (Jurkat exo) cells at 4 × 10 9 exosomes/ml for 10 min, followed by KSHV infection for 20 h. Cells were fixed for immunofluorescent staining using antibodies to LANA (red) and GFP (green). Bars, 25 μm. (C) Flow cytometry of GFP + cells after KSHV infection in the presence of exosomes from J1.1 and Jurkat cells, respectively. The control (ctrl) consisted of KSHV alone. Data represent the results of one independent experiment ( n = 4) out of three biological repeats. *, P < 0.05, F test. (D) Flow cytometry histogram of the results presented in panel C. FITC-A, fluorescein isothiocyanate area.

Article Snippet: The antibodies used in immunoblotting and immunofluorescence microscopy were CD9 (clone TS9, catalog number 10626D), CD81 (clone M38, catalog number MA1-10290), and rabbit polyclonal claudin 1 (catalog number 51-9000) antibodies from Thermo Fisher Scientific; CD63 (clone NKI/C3, catalog number NBP2-32819) and KSHV K8 (clone 8C12G10G1, catalog number NB-100-1086) antibodies from Novus Biologicals; pY1175-EGFR (catalog number 05-483), EGFR (catalog number 4267), phopho-p38 (catalog number 9211), p38 (clone D13E1, catalog number 8690), and phospho-p38 (catalog number 9211) antibodies from Cell Signaling Technology; KSHV LANA antibody (clone LN53, catalog number MABE1109) from Millipore; and GFP antibody (catalog number 13970) from Abcam.

Techniques: Infection, Serial Dilution, Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/JVI.01782-19

Figure Lengend Snippet: HIV-associated saliva exosomes enhance KSHV infection in oral epithelial cells. (A) OKF6/TERT2 cells were treated with HIV + (pt 8 and pt 9) and HIV − (N1 and N2) saliva exosomes (100 μg/ml of exosome protein) for 10 min and then infected with KSHV for 20 h. Infection was quantified by GFP flow cytometry. *, P < 0.05. (B) Human oral epithelial cells (HOECs) were infected with KSHV virion solution without dilution to test the infectivity of KSHV in primary HOECs. LANA (red) and GFP (green) expression was detected using immunofluorescent staining. Nuclei stained blue (DAPI). Bars, 50 μm. Representative images are shown. (C) HOECs were treated with saliva exosomes (100 μg exosomal protein/ml) from healthy (N1 and N2) and HIV + (pt 8 and pt 9) donors, followed by KSHV infection. KSHV-infected GFP + HOECs were quantified using flow cytometry. Data represent the mean ± SD. *, P < 0.05. (D) HOECs were treated with saliva exosomes (100 μg/ml of exosome protein) purified from healthy ( n = 4) and HIV-infected ( n = 8) donors, followed by KSHV infection. KSHV-infected GFP + HOECs were determined by flow cytometry. (Right) Mean fluorescence intensity (MFI) plot of the data in the left panel. P values were determined by one-way ANOVA. (E) (Left) OKF6/TERT2 cells were infected with KSHV in the presence of Jurkat cell exosomes (Jurkat exo) or HIV + J1.1 cell exosomes (J1.1 exo) for 1 and 2 h, followed by immunofluorescent staining of ORF65 (red). Representative images are shown. (Right) MFI of ORF65 staining in OKF6/TERT2 cells. Data represent those from one independent experiment ( n = 4). *, P < 0.05, F test. post-inf, postinfection.

Article Snippet: The antibodies used in immunoblotting and immunofluorescence microscopy were CD9 (clone TS9, catalog number 10626D), CD81 (clone M38, catalog number MA1-10290), and rabbit polyclonal claudin 1 (catalog number 51-9000) antibodies from Thermo Fisher Scientific; CD63 (clone NKI/C3, catalog number NBP2-32819) and KSHV K8 (clone 8C12G10G1, catalog number NB-100-1086) antibodies from Novus Biologicals; pY1175-EGFR (catalog number 05-483), EGFR (catalog number 4267), phopho-p38 (catalog number 9211), p38 (clone D13E1, catalog number 8690), and phospho-p38 (catalog number 9211) antibodies from Cell Signaling Technology; KSHV LANA antibody (clone LN53, catalog number MABE1109) from Millipore; and GFP antibody (catalog number 13970) from Abcam.

Techniques: Infection, Flow Cytometry, Expressing, Staining, Purification, Fluorescence

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/JVI.01782-19

Figure Lengend Snippet: HIV + exosomes increase KSHV infection in oral mucosal tissue cultures. (A) Hematoxylin and eosin (H&E) staining of the organotypic culture of OKF6/TERT2 cells (top) and the oral buccal mucosal tissue consisting of primary human oral epithelial cells (MatTek Inc.) (bottom). Representative images are shown. (B) HIV + J1.1 T-cell exosomes (J1.1 exo) increased KSHV infection in OKF6/TERT cells grown in the organotypic culture model. GFP + cells represent KSHV-infected cells. Representative images are shown. (C) Cultured oral buccal mucosal tissues were treated with HIV + (J1.1 exo) and HIV − (Jurkat exo) exosomes, followed by KSHV infection. Tissue sections were stained with antibodies to LANA (red) and GFP (green). Nuclei stained blue (DAPI). Arrows, GFP; arrowheads, LANA. Bars, 25 μm. Representative images are shown. The lower left image represents the zoomed-in box of each merge photo to detail cellular expression of GFP and LANA. (D) Quantification of LANA-positive cells versus total cells in 3 to 5 sections of each paraffin block shown in panel C. Data are represented as the mean ± SD. *, P < 0.05. (E) The oral mucosal tissue (MatTek Inc.) treated with J1.1 cell exosomes and infected with KSHV was stained for LANA (red) and the tight junction protein claudin 1 (CLDN1; green). Arrows in the enlarged inset of the boxed area, LANA; arrowheads in enlarged inset, claudin 1. Bar, 25 μm.

Article Snippet: The antibodies used in immunoblotting and immunofluorescence microscopy were CD9 (clone TS9, catalog number 10626D), CD81 (clone M38, catalog number MA1-10290), and rabbit polyclonal claudin 1 (catalog number 51-9000) antibodies from Thermo Fisher Scientific; CD63 (clone NKI/C3, catalog number NBP2-32819) and KSHV K8 (clone 8C12G10G1, catalog number NB-100-1086) antibodies from Novus Biologicals; pY1175-EGFR (catalog number 05-483), EGFR (catalog number 4267), phopho-p38 (catalog number 9211), p38 (clone D13E1, catalog number 8690), and phospho-p38 (catalog number 9211) antibodies from Cell Signaling Technology; KSHV LANA antibody (clone LN53, catalog number MABE1109) from Millipore; and GFP antibody (catalog number 13970) from Abcam.

Techniques: Infection, Staining, Cell Culture, Expressing, Blocking Assay

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/JVI.01782-19

Figure Lengend Snippet: HIV TAR is critical for HIV + exosome-enhanced KSHV infection. (A) Immunoblots of HIV Tat and Nef proteins on total proteins (100 μg) extracted from exosomes isolated from the culture supernatants of Jurkat, J1.1, 2D10 , and C22G cells that lack Nef and a functional Tat gene. Total cell lysates of J1.1 cells activated with 5 ng/ml of TNF-α (J1.1 +TNF) were used as controls. (B) Immunoblots of HIV Tat and Nef proteins on total proteins (100 μg) extracted from saliva exosomes (sal exo) purified from HIV + donors (pt 6, pt 7, pt 8, and pt 9). Total cell lysates (100 μg of protein) of J1.1 cells treated with TNF-α (J1.1 +TNF) were used as controls. Tub, human tubulin, used as a loading control. (C) KSHV infection of OKF6/TERT2 cells in the presence of exosomes from Jurkat, C22G, 2D10, J1.1, and J1.1 T cells treated with TNF-α. GFP + cells were determined by flow cytometry ( n = 3). *, P < 0.05. (D) OKF6/TERT2 cells were transfected with the synthetic HIV TAR RNA (TAR), the mutant TAR (mutTAR), and TAR RNA together with the R06 aptamer (TAR+R06) or with the scrambled aptamer (TAR+Scrb), followed by KSHV infection for 20 h. KSHV infection was determined by GFP flow cytometry. Data represent those from one independent experiment ( n = 4) out of three repeats. *, P < 0.01; **, P < 0.02.

Article Snippet: The antibodies used in immunoblotting and immunofluorescence microscopy were CD9 (clone TS9, catalog number 10626D), CD81 (clone M38, catalog number MA1-10290), and rabbit polyclonal claudin 1 (catalog number 51-9000) antibodies from Thermo Fisher Scientific; CD63 (clone NKI/C3, catalog number NBP2-32819) and KSHV K8 (clone 8C12G10G1, catalog number NB-100-1086) antibodies from Novus Biologicals; pY1175-EGFR (catalog number 05-483), EGFR (catalog number 4267), phopho-p38 (catalog number 9211), p38 (clone D13E1, catalog number 8690), and phospho-p38 (catalog number 9211) antibodies from Cell Signaling Technology; KSHV LANA antibody (clone LN53, catalog number MABE1109) from Millipore; and GFP antibody (catalog number 13970) from Abcam.

Techniques: Infection, Western Blot, Isolation, Functional Assay, Purification, Control, Flow Cytometry, Transfection, Mutagenesis

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/JVI.01782-19

Figure Lengend Snippet: HIV + exosomes enhance KSHV infection in an EGFR-dependent fashion. (A) KSHV infection in OKF6/TERT2 cells treated with exosomes from Jurkat or J1.1 cells (4 × 10 9 exosomes/ml) with or without cetuximab (20 μg/ml). GFP + cells were detected by flow cytometry. Data (mean ± SD) represent those from one independent experiment out of three repeats. no KSHV, no KSHV infection control; Ctrl, no exosome treatment control. *, P < 0.05. (B) OKF6/TERT2 cells were pretreated with J1.1 and Jurkat cell exosomes (4 × 10 9 ml) in the presence or absence of cetuximab (Cet) or AG1478 (2 μm) for 30 min, followed by KSHV infection for 20 h for total protein extraction and immunoblotting of LANA and ORF K8. Tubulin was used as a loading control. (C) Flow cytometry of GFP + HOECs treated with exosomes isolated from the culture supernatants of Jurkat cells or J1.1 cells or with EGF (10 ng/ml) in the presence or absence of cetuximab (20 μg/ml) ( n = 3). *, P < 0.05. (D) OKF6/TERT2 cells were treated with EGF (10 ng/ml) for 10 min or remained untreated, followed by KSHV infection for 2 h. ORF65 (red) was detected by immunofluorescent staining. Representative images are shown. (E) Oral buccal mucosal tissue cultures (MatTek Inc.) were treated with J1.1 or Jurkat cell exosomes (4 × 10 9 /ml) in the presence or absence of cetuximab (20 μg/ml), followed by KSHV infection for 20 h. Cells were stained for LANA and GFP. Arrowheads, LANA; green, GFP; blue, nuclei. The lower left image represents the zoomed-in box of each merge photo to detail cellular expression of GFP and LANA.

Article Snippet: The antibodies used in immunoblotting and immunofluorescence microscopy were CD9 (clone TS9, catalog number 10626D), CD81 (clone M38, catalog number MA1-10290), and rabbit polyclonal claudin 1 (catalog number 51-9000) antibodies from Thermo Fisher Scientific; CD63 (clone NKI/C3, catalog number NBP2-32819) and KSHV K8 (clone 8C12G10G1, catalog number NB-100-1086) antibodies from Novus Biologicals; pY1175-EGFR (catalog number 05-483), EGFR (catalog number 4267), phopho-p38 (catalog number 9211), p38 (clone D13E1, catalog number 8690), and phospho-p38 (catalog number 9211) antibodies from Cell Signaling Technology; KSHV LANA antibody (clone LN53, catalog number MABE1109) from Millipore; and GFP antibody (catalog number 13970) from Abcam.

Techniques: Infection, Flow Cytometry, Control, Protein Extraction, Western Blot, Isolation, Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 1. Leiomodin 1 mRNA and protein expression analysis. A, RT-PCR analysis of Lmod1, Lmod2, and Lmod3 in the indicated adult mouse tissues. Note the similar mRNA expression pattern between Lmod1 and Cnn1, a well known SMC-restricted gene (43). B, quantitative RT-PCR of Lmod1 mRNA in the same tissues as A. Expression levels are displayed as normalized fold increases over brain (set to 1). C, Western blot of LMOD1 protein expression in mouse tissues and in 3T3 cells transfected either with an empty control plasmid or an expression plasmid carrying Lmod1 (top panel). LMOD1 protein expression following preabsorption of the LMOD1 antibody with LMOD1 antigen (middle panel). D, endogenous LMOD1 protein expression in PAC1 SMCs following siRNA knock- downofLMOD1.E,immunohistochemistryofLMOD1proteininaorta(panelsaande),bladder(panelsbandf),brain(panelscandg),andskeletalmuscle(panels d and h) using either the LMOD1 antibody (panels a–d) or a nonimmune, isotype-matched IgG control (panels e–h). The bar in panel h is 30 m for all panels. The data shown in all of the panels are representative of three independent experiments. Arrows in panels c and d indicate LMOD1 positive blood vessels.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 2. LMOD1 protein expression in developing mouse embryos. Immunohistochemistry of LMOD1 protein expression (red stain) in sagittal sections of E9.5 (A), E13.5 (C and D), and E15.5 (E and F) mouse embryos. An isotype-matched IgG control antibody shows no staining of an E9.5 embryo (B). Similar lack of staining was seen with the IgG control applied to E13.5 and E15.5 embryo sections (not shown). ao, aorta; br, bronchiole; bv, blood vessel; he, heart; in, intestine; li, liver; lu, lung; st, stomach. The bars in A and B repre- sent 100 m, and the bars in C–F represent 500 m.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Immunohistochemistry, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 3. Functional analysis of human LMOD1 and LMOD2 promoters. A, VISTA plot indicating nucleotide sequence homology between human (Hsa), mouse (Mmu) and rat (Rno) LMOD1 over a 50-kb genomic interval. The x axis represents the human base sequence, and the y axis indicates the percentage of homology between Hsa versus Mmu (top plot) and Hsa versus Rno (bottom plot). The bent arrow at the top represents the inferred transcription start site. Light teal peaks represent untranslated regions; dark blue peaks represent protein-coding exons; and pink peaks are the conserved nonprotein coding sequences. Sequence logos of two CArG-like elements near LMOD1 are shown below the VISTA plots and illustrate the conservation of each CArG box across six vertebrate species. B and C, LMOD1 and LMOD2 promoter activity in PAC1 SMCs transfected either with SRF-VP16 (B) or MYOCD_v3 (C). The promoter activity reported here and below is a ratio between luciferase and Renilla normalized to that of the pGL3 basic plasmid set to 1. Similar SRF/MYOCD-mediated promoter activity was observed in at least three independent studies.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, Sequencing, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 4. SRF binding to LMOD1 CArG elements. 32P-Labeled double- stranded oligonucleotides containing CArG elements from CNN1 and LMOD1 promoter CArG Far and CArG Near were incubated with the in vitro translated (IVT) SRF in the presence of SRF or MEF2A antibody or a 100-fold molar excess ofunlabeledwildtype(WT)ormutant(Mut)oligonucleotide.SRF-CArGnucle- oprotein complex (SRF) and supershift (SS) are indicated with arrows. Expo- sure times for each EMSA were 11 h (for CNN1) or 64 h (for LMOD1).

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Binding Assay, Labeling, Incubation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 5. Functional analysis of LMOD1 CArG elements in vitro. A, sche- maticofthewildtype(WT)LMOD1promoterandvariousCArGmutants.Band C, each indicated LMOD1 promoter construct was transfected into PAC-1 SMCs in the presence of SRF-VP16 (B) or MYOCD_v3 (C), and luciferase activity was measured. D, COS-7 cells were similarly transfected with WT or double CArG mutant LMOD1 promoter and either MYOCD_v3, MRTF-A, or MRTF-B. The results shown were reproduced in one independent experiment. *, p 0.001; **, p 0.01.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, In Vitro, Construct, Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 6. LMOD1 expression in SRF-deficient cells and tissues. RT-PCR analysis of Lmod1 and Srf mRNA either in PAC-1 SMC transduced with shEGFP or shSRF for 72 h (A) or indicated tissues from adult mice carrying homofloxed Srf alleles and the tamoxifen (Tmx)-inducible Myh11-Cre driver (34) (B). Note the reduction in both Srf and Lmod1 mRNA in tissues derived from mice treated with Tmx (to activate Cre recombinase and effect excision of the Srf gene) versus the vehicle control (Oil). The latter results were extended by comparing protein expression of SRF (C, panels a and b) versus LMOD1 (C, panels c and d) in similar mice treated either with Tmx (C, panels b and d) or sunflower oil (C, panels a and c). The bar in panel d of C is 30 m for all panels. The images in A and B were inverted in Adobe Photoshop so as to better indicate the bands in each gel.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Derivative Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 8. LMOD1 promoter activity in transgenic mice. Sagittal E12.5 day mouse embryos stained either with -galactosidase to assess LMOD1 promoter activity (A–C and E–G) or an antibody to LMOD1 (D) or control IgG (H). Shown are three WT LMOD1 promoter mice (A–C) and three double CArG mutant LMOD1 promoter mice (E–G). The thick arrows indicate the heart, and the thinner arrows point to aorta and vessels of head. The bar in H is 1 mm for all panels. Eight of 25 wild type founders displayed promoter activity in cardiac muscle, and five of 25 showed promoter activity in vascular tissue. In contrast none of the 10 CArG mutant founders showed LMOD1 promoter activity in cardiac muscle or vascular tissue.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Activity Assay, Transgenic Assay, Staining, Control, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 7. LMOD1 mRNA and protein expression in human cells overexpressing MYOCD. A, the indicated human cell lines were transduced with equal amounts of adenovirus carrying MYOCD or LacZ and endogenous LMOD1 and LMOD2 mRNA expression assessed by RT-PCR. B, same experiment as in A only LMOD1 and MYOCD protein expression were determined using Western blotting. This result was reproduced in an independent experiment.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: A Novel Localized Tracing Technique to Explore Intra-Amygdala Functional and Structural Connectivity Patterns as Mediators of Individual Variability in Stress Response

doi: 10.1101/2022.11.29.517584

Figure Lengend Snippet: (A) Example triple-channel image of the amygdala region (red=mCherry, green=GFP, magenta=cFos). Top inset (pink box and outline) shows examples of a structural projection neuron (open pink arrow, GFP + /cFos - ) and a functional projection neuron (closed pink arrow, GFP + /cFos + ). Bottom inset (yellow box and outline) shows examples of a starter neuron (closed yellow arrow, GFP + /mCherry + ) and a projection neuron (open yellow arrow (GFP + /mCherry - ). (B) Map of all identified starter cells (GFP + /mCherry + ). (C) Map of all identified structural (GFP + /cFos - ) and functional (stress-activated) projection (GFP + /cFos + ) neurons. (D) Correlations between starter cell count in each region and SI ratio with FDR-corrected and non-FDR-corrected significance threshold indicated (solid horizontal line, Pearson R = 0.835, p = 0.00263; dashed horizontal line, Pearson R = 0.632, p = 0.05; respectively). Starter cell counts did not predict behavioral phenotype in any identified brain region. (E) Correlations between structural projection neuron (GFP + /cFos - ) count (striped) and functional projection neuron (GFP + /cFos + ) count (solid) in each region and SI ratio with FDR-corrected and non-FDR-corrected significance threshold indicated (solid horizontal line, Pearson R = 0.749, p = 0.0125; dashed horizontal line, Pearson R = 0.632, p = 0.05; respectively). No significant correlations identified. (F) Correlations between overall number of activated cells per brain region and SI ratios indicate (G) MeA and (H) PAA activation positively predict social approach (Pearson R = 0.851, p = 0.00181 and Pearson R = 0.751, p = 0.0123, respectively).

Article Snippet: Sections were serially stained with rabbit anti-cFos (9F6) primary monoclonal antibody (Cell Signaling, catalog number: 2250S, lot number: 12, 1:2000 in 0.3% PBST + 5% Normal Goat Serum + 1% BSA + 0.2% Cold Water Fish Skin Gelatin block solution) and Alexa Fluor-conjugated AffiniPure goat anti-rabbit secondary antibody (Alexa Fluor-647, Sigma, code number: 111-605-033, lot number: 157045, 1:500 in 0.3% PBS-T).

Techniques: Functional Assay, Cell Counting, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 3. Functional analysis of human LMOD1 and LMOD2 promoters. A, VISTA plot indicating nucleotide sequence homology between human (Hsa), mouse (Mmu) and rat (Rno) LMOD1 over a 50-kb genomic interval. The x axis represents the human base sequence, and the y axis indicates the percentage of homology between Hsa versus Mmu (top plot) and Hsa versus Rno (bottom plot). The bent arrow at the top represents the inferred transcription start site. Light teal peaks represent untranslated regions; dark blue peaks represent protein-coding exons; and pink peaks are the conserved nonprotein coding sequences. Sequence logos of two CArG-like elements near LMOD1 are shown below the VISTA plots and illustrate the conservation of each CArG box across six vertebrate species. B and C, LMOD1 and LMOD2 promoter activity in PAC1 SMCs transfected either with SRF-VP16 (B) or MYOCD_v3 (C). The promoter activity reported here and below is a ratio between luciferase and Renilla normalized to that of the pGL3 basic plasmid set to 1. Similar SRF/MYOCD-mediated promoter activity was observed in at least three independent studies.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, Sequencing, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 4. SRF binding to LMOD1 CArG elements. 32P-Labeled double- stranded oligonucleotides containing CArG elements from CNN1 and LMOD1 promoter CArG Far and CArG Near were incubated with the in vitro translated (IVT) SRF in the presence of SRF or MEF2A antibody or a 100-fold molar excess ofunlabeledwildtype(WT)ormutant(Mut)oligonucleotide.SRF-CArGnucle- oprotein complex (SRF) and supershift (SS) are indicated with arrows. Expo- sure times for each EMSA were 11 h (for CNN1) or 64 h (for LMOD1).

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Binding Assay, Labeling, Incubation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 5. Functional analysis of LMOD1 CArG elements in vitro. A, sche- maticofthewildtype(WT)LMOD1promoterandvariousCArGmutants.Band C, each indicated LMOD1 promoter construct was transfected into PAC-1 SMCs in the presence of SRF-VP16 (B) or MYOCD_v3 (C), and luciferase activity was measured. D, COS-7 cells were similarly transfected with WT or double CArG mutant LMOD1 promoter and either MYOCD_v3, MRTF-A, or MRTF-B. The results shown were reproduced in one independent experiment. *, p 0.001; **, p 0.01.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, In Vitro, Construct, Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 6. LMOD1 expression in SRF-deficient cells and tissues. RT-PCR analysis of Lmod1 and Srf mRNA either in PAC-1 SMC transduced with shEGFP or shSRF for 72 h (A) or indicated tissues from adult mice carrying homofloxed Srf alleles and the tamoxifen (Tmx)-inducible Myh11-Cre driver (34) (B). Note the reduction in both Srf and Lmod1 mRNA in tissues derived from mice treated with Tmx (to activate Cre recombinase and effect excision of the Srf gene) versus the vehicle control (Oil). The latter results were extended by comparing protein expression of SRF (C, panels a and b) versus LMOD1 (C, panels c and d) in similar mice treated either with Tmx (C, panels b and d) or sunflower oil (C, panels a and c). The bar in panel d of C is 30 m for all panels. The images in A and B were inverted in Adobe Photoshop so as to better indicate the bands in each gel.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Derivative Assay, Control

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Metastasis immune pressure positively selects TIM3 + metastatic cells (A) Experimental design using immunocompetent (IC) Balb/c mice and immunodeficient (ID) NOD scid gamma (NSG) mice for the assessment of metastatic immune pressure. (B) Principal-component analysis (PCA) of the RNA-seq from EpRas cells isolated from three organ (lung, liver, and brain) metastatic samples in IC and ID hosts. (C) Unsupervised hierarchical clustering from lung, liver and brain metastasis ( Z score) in ID and IC hosts ( n = 3 IC and n = 3 ID independent biological replicates). (D) Gene ontology enrichment analysis of top 50 upregulated genes in all organs from IC mice. (E) GSEA of indicated gene lists with the ranked gene expression list of IC vs. ID in all organ samples. (F) Volcano plot of gene expression in all organs comparing IC and ID mice samples ( n = 3 independent biological replicates). (G) Dot plot representing TIM3 expression of human breast cancer cell lines from the CCLE. Primary tumor-derived cells (gray) and metastasis-derived cells (pink). (H) Dot plot representing TIM3 protein levels measured by flow cytometry of mouse breast cancer cell lines with metastatic potential in experimental models. Color code as (G). (I) TIM3 immunofluorescence of liver tissue metastasis derived from 4T07 intracardiac injection in ID and IC mice. Representative image of TIM3 immunofluorescence. Scale bars, 100 μm. Dashed line delineates metastasis tissue. Boxplot quantification of TIM3 staining mean fluorescent intensity in from 5 independent mice ( n = 5 independent biological replicates). Data represent mean ± SEM. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by unpaired Student’s t test. Also see and .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: RNA Sequencing, Isolation, Gene Expression, Expressing, Derivative Assay, Flow Cytometry, Immunofluorescence, Injection, Staining

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 drives breast cancer metastasis (A) Kaplan-Meier survival plot after intracardiac injection of 4T07 control cells versus Tim3 -KD cells in ID (NSG) and IC (Balb/c) mice with the indicated conditions. Statistical analysis using Log rank (Mantel-Cox) test. (B) Relative photon flux BLI quantification of metastatic organs at day 16 after i.c. injection of 4T07 Ctrl and Tim3 -KD cells. Data represents mean + SEM; dots represent independent biological replicates. (C) Relative photon flux BLI quantification of whole-body metastasis of 4T07 Ctrl and Tim3 -KD in IC mice. Data represents mean + SEM. n = 22 independent biological replicates. (D) Hematoxylin-eosin staining of metastatic livers from unlabeled 4T07-Ctrl and - Tim3 -KD cells. Arrows indicate metastatic lesions. (E) Quantification of micro- and macro-metastatic lesions from (D). (F) Mammary fat pad (MFP) injection of 4T1-Ctrl and - Tim3 -KD cells in Balb/c mice. Data represents tumor growth by mean + SEM of n = 16 independent biological replicates. (G) Incidence of spontaneous metastasis at day 40 after primary tumor resection (day 20) of 4T1-Crtl and 4T1- Tim3 -KD MFP injected mice. Individual BLI images from upper body. n = 8 Ctrl and n = 6 KD mice followed after resection. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Chi-square test. (H) Representative immunofluorescence of breast tumor cell marker Mamaglobulin-1 (MGB1) in red and TIM3 in green in human breast cancer tissue. Scale bars, 30 μm. Representative immunohistochemistry image of TIM3 showing tumor-epithelial cell staining. Dash line delineates the tumor areas. Scale bars, 100 μm. (I) Percentage of tumor TIM3-positive samples from primary (P) and metastatic (M) matched clinical samples (ConvertHER cohort). TIM3 score percentage in primary and paired-metastatic samples (right panel). n = 75 for each condition P and M. Data represented as mean ± SEM. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test in (B), (C), (E), and (I). Also see .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: Injection, Control, Staining, Immunofluorescence, Marker, Immunohistochemistry, Two Tailed Test

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 + MICs display Stemness/EMT-like features and β-catenin activation (A) Unsupervised hierarchical clustering heatmap of the indicated conditions from 4T07 metastases RNA-seq analysis ( n = 2 independent biological replicates). (B) GSEA from (A) experiment comparing Ctrl and Tim3 -KD ( n = 2 independent biological replicates). (C) Gene ontology integration of the metastasis immunoediting RNA-seq from A and the Tim3 -KD metastasis RNA-seq (A). (D) GSEA ranked list Ctrl and Tim3 -KD 4T07 tumors (lung and liver metastases) interrogated with the indicated EMT-like and stem-like gene signatures. (E) Tissue immunofluorescence representative image: for N-cadherin (red) and vimentin (green) in Ctrl and Tim3 -KD metastatic livers. Dash line delineates the metastatic tissue. Scale bars, 100 μm. (F) Tumorsphere quantification at day 5 after seeding 500 4T07 cells of indicated conditions ( n = 3 independent biological replicates). Data represented as mean ± SEM. (G) MFP injection and limiting dilution assay (LDA) of 4T07 Ctrl and Tim3 -KD cells. Table represents serial dilution injections and tumor take rate. Tumor-initiating cell (TIC) frequency calculated by ELDA software shown in red. p value by Pearson’s Chi-squared two-tailed test. (H) Proximity ligation assay showing the interaction (red dots) of P85 and TIM3 in 4T07 Ctrl and Tim3 -KD cells. Quantification of the interactions per area. Data represented as mean ± SEM. (I) Immunofluorescence of active β-catenin (ABC) in 4T07-Ctrl and Tim3 -KD cells. Quantification of the nuclear staining of ABC. (J) Tumorsphere quantification of 4T07 Control, Tim3 -KD, and Tim3 -overexpression upon 20 μM dose of β-catenin inhibitor. (K) Rho correlation of TIM3 mRNA levels and the Reactome β-Catenin signaling signature in 237 TNBC patients TCGA. R 2 and p value are shown. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test in (F) and (H); unpaired Student’s t test in (I). Also see .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: Activation Assay, RNA Sequencing, Immunofluorescence, Injection, Limiting Dilution Assay, Serial Dilution, Software, Two Tailed Test, Proximity Ligation Assay, Staining, Control, Over Expression

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 spatiotemporal dynamics in metastasis (A) Dual reporter system designed to track bulk tumor metastasis (Firefly luciferase [Fluc]-eGFP) and Tim3 promoter activity (mCherry-Nanoluciferase [Nluc]). (B) Representative BLI images of Fluc and Nluc signal in IC mice (Balb/c) upon i.c. injection of 4T07 cells experimental metastasis. BLI monitorization of metastatic growth: metastasis curve (blue line) and Tim3 reporter curve (orange line). Bar plot shows the BLI signal ratio of NLuc/FLuc representing the intensity of the Tim3 reporter signal versus the overall bulk tumor metastasis at micrometastasis and macrometastasis time points. n = 8 mice. (C) BLI ratio of NLuc/FLuc of liver metastasis. n = 8 mice. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by one-tailed Student’s t test. (D) BLI ratio curve of NLuc/FLuc metastasis along days of experiment. Each line represents individual mice. (E) Flow cytometry of 4T07- Tim3 reporter positivity measured by mCherry intensity of liver metastatic digested tissues from ID and IC at micrometastasis and macrometastasis time points. Representative plot of n = 3 individual mice per time point. (F) IF images from liver micrometastasis and macrometastasis. Luciferase (red) and TIM3 (pink) stainings. Dash line delineates the metastatic tissue. Scale bars, 100 μm. (G) Schematic representation of the lineage tracing system introduced in 4T07 cells (see for details). Tim3 - cancer cells have red fluorescence of dsRed. Tim3 + cancer cells have green fluorescence of eGFP. (H) In vitro lineage tracing test of Tim3 + and Tim3 - 4T07 cells. Induction by 4-hydroxy-tamoxifen (4-OHT) O/N at 1 μM. Flow cytometry plots represent dsRed and GFP positive events in no induced cells (top) and 4-OHT induced cells (bottom). (I) Metastasis lineage tracing using 4T07 cells after intracardiac injection in IC mice. Short TAM induction during the first 3 days of metastatic seeding (see ). Bar plots quantifications of all lesions (113) present in livers of 6 independent experiments. Representative immunofluorescence images of liver sections showing TIM3 + (green) and TIM3 - (red) metastasis in (I) and (J). (J) Long-term TAM induction during 21 days after intracardiac injection of 4T07 cells in IC and ID mice. Bar plots quantifications of all lesions (61 IC and 37 ID) present in livers of 4 independent experiments. Also see .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: Luciferase, Activity Assay, Injection, One-tailed Test, Flow Cytometry, Fluorescence, In Vitro, Immunofluorescence

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 + MICs induce an immunosuppressive environment during micrometastasis (A) Single cell RNA-seq uniform manifold approximation and projection (UMAP) of CD45 + immune cells isolated from liver metastasis after 4T07 Ctrl and Tim3 -KD i.c. injection. UMAP representation of: Ctrl (red) and Tim3 -KD (blue) in healthy liver adjacent tissue (AT), liver micrometastasis and liver macrometastasis. (B) scRNA-seq bubble plot showing average expression (color) and percentage expression (size) of different genes defining lymphoid annotated cell compartments across metastatic conditions. Average expression legend is shared among populations. Percentage expression is relative within each immune population type. (C) Idem B for the myeloid compartment. (D) scRNA-seq cell fraction of the lymphoid cell compartment in micrometastatic samples from 4T07 Ctrl and Tim3 -KD conditions. (E) Flow cytometry analysis of CD4 + T cells, CD8 + T cells and γδ T cells from liver micrometastasis from 4T07-Ctrl and 4T07- Tim3 -KD. Data represents mean ± SEM, n = 11 independent biological replicates in (E), (F), and (G). (F) Flow cytometry of cytotoxic γδ T cells (GZMB + ) and immunosuppressive (IL17 + ) γδ T cells. Dot plot represents fold change of IL-17 and GZMB γδ T cell populations in TIM3+ (Ctrl) vs. TIM3- ( Tim3 -KD) micrometastasis. (G) Flow cytometry of cytotoxic CD8 + T cells (GZMB + , CD69 + ) and exhausted T cells (PD1 + ) populations. Dot plot represents fold change of PD1, CD69, and GZMB positivity in TIM3 + (Ctrl) vs. TIM3 - ( Tim3 -KD) micrometastasis. Statistical significance respect to the Ctrl; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by unpaired Student’s t test in (E), (F), and (G). Also see , , and .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: RNA Sequencing, Isolation, Injection, Expressing, Flow Cytometry

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Functional metastasis assessment of γδ T cells, CD8 T cells, and IL-1β in TIM3-mediated immunosuppression (A) Blocking antibody scheme; intraperitoneal administration and doses indicated. Seven days before 4T07 tumor cell i.c. injection and, weekly reminder of 250 μg of antibody during the experiment. (B) Representative BLI images of 4T07 whole body metastasis for the indicated immune blocking conditions. (C) Boxplot quantification of liver metastasis at micro (day 3) and macro (day 14) time points upon IgG2b, CD8, γδ TCR, and double (CD8, γδ TCR) cell depletion. Each dot represents independent mice. (D) Boxplot quantification of whole-body metastasis at micro (day 3) and macro (day 14) time points upon IgG2b, CD8, γδ TCR, and double (CD8, γδ TCR) cell depletion. Each dot represents independent mice. (E) In vitro proliferation and co-culture of γδ T cells with 4T07 tumor cells and flow cytometry. See for details. (F) Flow cytometry quantification of IL-17 γδ T cell levels after co-culture in non-treated (NT) conditions and upon anti-IL1β treatment. Data represents mean ± SEM, n = 6 (NT) and n = 4 (anti-IL1β) independent biological replicates. (G) Whole-body metastasis assays after systemic delivery of 4T07-IL-1β - KO cells compared to 4T07 TIM3 + (Ctrl) cells. Representative BLI images of metastasis at the indicated conditions. BLI metastasis growth curves. Data represents mean ± SEM, n = 10 independent mice. (H) Liver metastatic lesions at day 3 of metastatic seeding of 4T07-IL-1β-KO cells compared to 4T07 TIM3 + (Ctrl) cells. (I) Representative BLI images of ex vivo metastatic livers at day 14 after i.c. injection of 4T07-Tim3 + (Ctrl) and 4T07-IL-1β-KO cells. Statistical significance respect to the Ctrl; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test in (C), (D), (F), and (G); unpaired Student’s t test in (H). Also see .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: Functional Assay, Blocking Assay, Injection, In Vitro, Co-Culture Assay, Flow Cytometry, Ex Vivo, Two Tailed Test

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Clinical and preclinical evaluation of TIM3 blockade for breast cancer metastasis (A) Disease free survival (DFS) and overall survival (OS) Kaplan-Meier curves of IHC epithelial-tumor TIM3 + and TIM3 - breast cancer primary tumor samples. Data obtained from Tissue microarrays (TMAs) with 257 breast cancer primary tumors from all subtypes. Statistical significance calculated by Log Rank (Mantel-Cox) for Chi-square and p value. (B) DFS Kaplan-Meier curves of IHC intratumoral tumor infiltrating lymphocytes (TILs) TIM3 + and TIM3 - breast cancer primary tumor samples. TMAs with 252 breast cancer primary tumors from all subtypes. Statistical significance calculated by Log Rank (Mantel-Cox) for Chi-square and p value. (C) Violin plot of TIM3 score in patients stratified by relapse ( n = 42) and non-relapse ( n = 215) from breast cancer primary tumor samples. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test. (D) Multivariate Cox regression analysis of TIM3 IHC from previous samples including p value and hazard ratio (HR) with confidence interval. Factor names: TIM3 in tumor epithelial cells (eTIM3), intratumoral TILs (iTIM3), and fibroblasts (fTIM3). Estrogen-receptor positivity (ER), HER2 positivity (HER2), patient stage-II BC (pT2), patient stage-III BC (pT3), patient lymph node-1 (pN1), patient lymph node-2 (pN2), and tumor infiltrating lymphocytes (TILs). (E) Intraportal 4T07 cell injection and anti-TIM3 treatment. Representative images of liver metastasis of anti-IgG2a and anti-TIM3 conditions. Boxplots representing liver metastatic growth by BLI measurements during early time points of the experiment. Each dot represents an individual mouse. (F) Spontaneous metastasis assay using 4T1 MFP injection in Balb/c mice. At 4 × 4 mm size, anti-Tim3 treatment starts. On the left, the graph represents the primary tumor volume, and the scheme of neoadjuvant/adjuvant treatment regime of TIM3-blockade therapy (250 μg). On the right, bar plot quantification of spontaneous metastatic incidence at day 40 and take rate of metastasis incidence. Met (metastasis detection) or No-Met (no metastasis detection). n = 14 and n = 15 mice per condition. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Chi-square test. (G) Graphical abstract of TIM3 + tumor cells from early seeding to macrometastasis in the liver. Also see .

Article Snippet: IF: TIM3 Monoclonal antibody , Proteintech , Cat#: 60355-1-Ig; RRID: AB_2881464.

Techniques: Two Tailed Test, Injection, Adjuvant

Journal:

Article Title: Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins

doi: 10.1128/JVI.76.17.8910-8919.2002

Figure Lengend Snippet: Schematic diagram of the functional domains of the E1A 243- and 289-aa proteins and their interactions with cellular transcription factors. The Ad5 E1A gene contains two exons and encodes the 243- and 289-aa proteins mainly through alternative splicing. The functional domains of the E1A proteins are indicated as NTR, CR1, CR2, and CR3. Those domains responsible for interaction with the cellular transcription factors STAT, p300/CBP, p53, pRb, TBP, and ATFs are also indicated.

Article Snippet: Serial frozen sections from each lung were blocked and incubated at 4°C overnight with anti-Ad5 E1A primary rabbit antibody (13S-5; Santa Cruz) and visualized by biotinylated anti-rabbit secondary antibody and ABC staining (Santa Cruz).

Techniques: Functional Assay

Journal:

Article Title: Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins

doi: 10.1128/JVI.76.17.8910-8919.2002

Figure Lengend Snippet: The SPCE1A transgene and its expression in transgenic mice. (A) The 3.7k-SPC/Ad5 E1A constructor used for generation of the transgenic mice and two transcripts, 12S and 13S mRNAs, resulted from alternative splicing. (B) Southern blot analyses of tail genomic DNAs from A30 and A97 founder (F0) mice. A 5-kb HindIII-digested band (lanes H) represents an intact SPCE1A transgene in the A97 founder. A rearranged 3.5-kb band or the other larger HindIII-digested bands also contain an intact E1A transgene in the A30 founder, as confirmed by PCR. The EcoRI-digested bands (lanes E) indicate the existence of one or two insertion sites in the A97 or A30 founders. The preferential expression of the E1A transgene in the lungs of A97 and A30 mice was determined at the level of mRNA by RT-PCR (C) and at the level of protein by immunoblotting (D), and it seems that the levels of transgene products in female mice (labeled F) were higher than those in male mice (labeled M). (E) The preferential expression of each E1A protein was quantified by determination of relative levels of the 289- and 243-aa proteins on immune blots in a densitometer (n = 4). (F) The E1A proteins were localized in the nuclei of pulmonary epithelial cells of alveoli, bronchiole, and trachea in both transgenic lines by immunostaining frozen lung sections with anti-Ad5 E1A antibodies, but the proteins were not found in nontransgenic littermates (Non-Tg). Scale bar, 8 μm.

Article Snippet: Serial frozen sections from each lung were blocked and incubated at 4°C overnight with anti-Ad5 E1A primary rabbit antibody (13S-5; Santa Cruz) and visualized by biotinylated anti-rabbit secondary antibody and ABC staining (Santa Cruz).

Techniques: Expressing, Transgenic Assay, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Western Blot, Labeling, Immunostaining

Journal:

Article Title: Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins

doi: 10.1128/JVI.76.17.8910-8919.2002

Figure Lengend Snippet: Phenotypic differences of A97 and A30 mice. (A) In comparison with the normal appearance of A97 mice and nontransgenic littermates (Non-Tg), 3-month-old A30 mice showed a frail phenotype with signs of respiratory distress, swollen nose, enlarged cervical lymph nodes, and a shortened life span. The lung histological sections were stained with hematoxylin and eosin in panels B, C, and E. (B) Thickened alveolar septa and marked accumulation of mucus in airspace and airway in the lungs of 5-week-old A97 mice, a mild preinflammatory response in the lungs of 5-week-old A30 mice, and normal appearance of the lungs of 5-week-old nontransgenic littermates. (C) Normal lungs in 12-month-old nontransgenic littermates, nodular lesions of lymphocyte-like cells in the lungs of 12-month-old A97 mice, and an inflammatory response or dysplastic changes in the lungs of 12-month-old A30 mice. Scale bars, 40 μm. (D) Embryonic lethality caused by the expression of the Ad5 E1A transgene during embryonic development was examined by dissection of A97 F2 embryos at 15.5 days postcoitum. Some transgenic embryos were underdeveloped compared with those of nontransgenic littermates. (E) Nodular lesions of lymphocyte-like cells developed in the lungs of A97 mice at 16 months of age.

Article Snippet: Serial frozen sections from each lung were blocked and incubated at 4°C overnight with anti-Ad5 E1A primary rabbit antibody (13S-5; Santa Cruz) and visualized by biotinylated anti-rabbit secondary antibody and ABC staining (Santa Cruz).

Techniques: Staining, Expressing, Dissection, Transgenic Assay

Journal:

Article Title: Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins

doi: 10.1128/JVI.76.17.8910-8919.2002

Figure Lengend Snippet: Distinct effects of the E1A proteins on cell proliferation and proapoptotic injury. (A) The cell proliferation induced by expression of the E1A transgene was detected by BrdU incorporation, and BrdU-labeled nuclei (arrows) present in proliferating epithelial cells in the paraffin-embedded lung sections of 3-month-old transgenic mice are indicated. Increased BrdU labeling index of the epithelial cells appeared in the alveolar region in A97 mice. Original magnification, ×200. (B) The apoptosis induced by expression of the E1A transgene was detected by TUNEL DNA fragmentation assay with frozen lung sections. TUNEL-labeled positive cells (arrows) were seen in apoptotic epithelial cells of 3-month-old transgenic mice. Elevated numbers of TUNEL-labeled cells and cells exhibiting nucleus condensation or fragmentation were seen in the alveolar and bronchiolar regions in A30 mice, and most proapoptotic cells were undergoing an early stage of apoptosis. Original magnification, ×200. (C) The levels of BrdU-labeled positive cells were quantified for 3-month-old A97 and A30 mice and nontransgenic littermates (n = 3). (D) The ratio of cells undergoing proapoptosis from the total lung cells was quantified based on TUNEL DNA fragmentation assay and nucleus condensation or fragmentation analyses for 3-month-old A97 and A30 mice and nontransgenic littermates (n = 3). (E) The expression of the E1A transgene stimulated expression of p53, and the levels of p53 in the lung lysates of 3-month-old nontransgenic littermates and A30 and A97 mice were detected by immunoblotting assay with anti-mouse p53 antibody. Non-Tg, nontransgenic.

Article Snippet: Serial frozen sections from each lung were blocked and incubated at 4°C overnight with anti-Ad5 E1A primary rabbit antibody (13S-5; Santa Cruz) and visualized by biotinylated anti-rabbit secondary antibody and ABC staining (Santa Cruz).

Techniques: Expressing, BrdU Incorporation Assay, Labeling, Transgenic Assay, TUNEL Assay, DNA Fragmentation Assay, Western Blot

Journal:

Article Title: Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins

doi: 10.1128/JVI.76.17.8910-8919.2002

Figure Lengend Snippet: p53-independent apoptosis pathways. (A) The expression of the E1A transgene induced TNF-α-mediated proapoptotic injury and/or proinflammation, and the levels of TNF-α in the blood of 3-month-old nontransgenic, A30, and A97 mice were quantified by TNF-α enzyme-linked immunosorbent assay (n = 2 to 4). (B) The expression of the E1A transgene induced Fas/FasL-mediated proapoptosis. The levels of Fas molecules in the lung lysates of 3-month-old nontransgenic (Non-Tg), A30, and A97 mice were detected by immunoblotting assay with anti-mouse Fas antibody. (C) The expression of FasL in the epithelial cells of bronchiole and alveoli of 3-month-old nontransgenic, A30, and A97 mice was detected by immunohistochemistry with anti-mouse FasL antibody. (D) The expression of E1A induced cell-mediated immune apoptosis, which was evaluated by cytometric analysis (FACS) (Table ​(Table1).1). The FACS results were confirmed by staining NK cells and macrophages with anti-Mac-1 antibody and by staining T cells with anti-CD8+ antibody in the paraffin-embedded lung sections of 3-month-old mice (arrows). The small windows in the top right corners show stained cells in blood vessels. (E) The ratio of the cells with Mac-1 mark or CD8+ mark was quantified (n = 3) to confirm that cell-mediated immune apoptotic injury occurred in the lungs of 3-month-old mice. Scale bars, 8 μm.

Article Snippet: Serial frozen sections from each lung were blocked and incubated at 4°C overnight with anti-Ad5 E1A primary rabbit antibody (13S-5; Santa Cruz) and visualized by biotinylated anti-rabbit secondary antibody and ABC staining (Santa Cruz).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways

doi: 10.3389/fimmu.2022.884399

Figure Lengend Snippet: Small molecule ligands bind to the TYK2 JH2 pseudokinase domain. (A) Domain structural illustration of TYK2 and schematic model of cytokine-mediated receptor/TYK2 activation: TYK2 protein contains 4 components: the kinase domain (JH1), the pseudokinase domain (JH2), the FERM (Four-point-one protein, Ezrin, Radixin, Moesin) domain, and the Src homology (SH2) domain; In the quiescent state, TYK2 kinase domain (JH1) activity is autoinhibited by its pseudokinase JH2 domain through intermolecular regulation. Upon cytokine (e.g., IL12, IL23, IFNα) stimulation, TYK2/STAT pathway is initiated by extracellular binding of cytokines with their cognate receptors, which results in apposition of receptor-associated TYK2 and triggering their enzymatic activity. Activated TYK2 further mediates phosphorylation of STATs on their tyrosine residues, leading to the cascade of down-stream cytokine signaling, including STAT dimerization, nuclear translocation, DNA binding and target gene induction. Small molecule TYK2 JH2 ligands block TYK2-associated cytokine signaling by binding to the TYK2 pseudokinase domain and allosterically inhibiting its kinase activities. This prevents TYK2 from phosphorylating STATs and other substrates, so that cytokine-induced intracellular signals cannot be transduced; (B) Model of the TYK2 pseudokinase domain bound to TYK2 ligands, SHR2396, 2178 and 9332. TYK2 pseudokinase domain residues corresponding to those of protein kinases normally involved in catalytic machinery are shown in stick; (C) Chemical structure of small molecule TYK2 JH2 ligands and JAK kinase inhibitors described in this study [SHR2178, 2396, 8751, 9332, 2915, 3290, 3110, 3591, Deucravacitinib (BMS986165, TYK2 pseudokinase ligand), Ropsacitinib (PF647, TYK2-IN-8, TYK2 kinase inhibitor), Brepocitinib (PF841, TYK2/JAK1 inhibitor) and Tofacitinib (JAK1/JAK3 inhibitor)].

Article Snippet: The cells were treated with serially diluted compounds for 1 hour and stimulated with 80 ng/mL human recombinant IFNα (R&D Systems) for 15 minutes.

Techniques: Activation Assay, Activity Assay, Binding Assay, Phospho-proteomics, Translocation Assay, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways

doi: 10.3389/fimmu.2022.884399

Figure Lengend Snippet: Small molecule TYK2 pseudokinase ligands block IFNα- and IL23- mediated TYK2/STAT phosphorylation in human T cells. (A, B) Representative dose-response curves of TYK2 JH2 ligands, SHR8751, 2396 and 9332 on IFNα-induced phosphorylation (Tyr701) of STAT1 (A) and IL23-induced phosphorylation (Tyr705) of STAT3 (B) in Kit225 cells. Cells were seeded in 384-well plate at 1×10 5 cells/well and treated with serially diluted compounds (30 μM, 1:3) for 1 hour and stimulated with human recombinant IFNα or IL23 for 20 minutes. The treated cells were then lysed and cellular phosphorylated-STAT1 (Tyr701) or STAT3 (Tyr705) levels were measured by AlphaLISA; Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. Dose response curves were generated to determine the concentration required to suppress 50% of cellular response (IC 50 ) as derived by non-linear regression analysis using GraphPad Prism. Confirmatory studies were performed for the compounds. The figures are representative of three independent experiments with duplicates performed for each experiment unless stated otherwise. The average IC 50 values and standard deviations of each compound from three independent experiments are calculated and summarized in the table. (C, D) Suppression of STAT5 phosphorylation in human CD3+ T cells by a TYK2 JH2 ligand, SHR1759. After 1hour incubation with serially diluted compounds, human whole blood samples were stimulated with recombinant human IFNα for 15 minutes and then lysed for removal of red blood cells, fixed, and permeabilized. White blood cells were stained with anti-CD3 FITC antibody, AF647 anti-Stat5 (pY694) antibody and later analyzed by flow cytometry. (C) Representative histogram of AF647 showed that IFNα-induced phosphorylation of STAT5 (Tyr694) in CD3+ T cells of human whole blood was suppressed by SHR1759 in a dose-dependent manner; (D) After gating on the CD3-positive population, the phosphorylation of cellular STAT5 in each treatment condition was quantitated by median fluorescence intensity (MFI). Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. Each bar represents the % inhibition at indicated compound concentration. Confirmatory studies were performed for the compounds. The figures are representative of two independent experiments with duplicates performed for each experiment.

Article Snippet: The cells were treated with serially diluted compounds for 1 hour and stimulated with 80 ng/mL human recombinant IFNα (R&D Systems) for 15 minutes.

Techniques: Blocking Assay, Phospho-proteomics, Recombinant, Inhibition, Comparison, Control, Generated, Concentration Assay, Derivative Assay, Incubation, Staining, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways

doi: 10.3389/fimmu.2022.884399

Figure Lengend Snippet: TYK2 pseudokinase ligands demonstrate significant functional activity in suppressing IL12- or IFNα- mediated TYK2/STAT pathways in human PBMCs and whole blood. (A) Dose dependent response of TYK2 JH2 ligands, SHR2915, 0936, 1039 and 1213 on IL12-induced IFNγ production in human PBMCs. 1.0 × 10 6 /ml PBMCs were treated by serially diluted (30 μM, 1:3) compounds and incubated with hIL12 for overnight in complete RPMI. The cultured supernatant was collected for detection of IFNγ secretion using AlphaLISA method. (B-D) Dose dependent effect of TYK2 JH2 ligands, SHR8751 and 9332 (B) , SHR2915 and Ropsacitinib (C) and SHR0936 (D) on IFNα-stimulated CXCL10 production in human whole blood. Blood samples were incubated with serially diluted compounds (30 μM, 1:3) and stimulated with recombinant human IFNα overnight. The plasma was collected for detection of CXCL10 production by AlphaLISA method. Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. IC 50 values were derived by non-linear regression analysis using GraphPad Prism. The figures are representative of the experiments on specimens from three different donors with duplicates performed for each donor. The average IC 50 values and standard deviations of each compound from experiments on three donors are calculated and summarized in the table.

Article Snippet: The cells were treated with serially diluted compounds for 1 hour and stimulated with 80 ng/mL human recombinant IFNα (R&D Systems) for 15 minutes.

Techniques: Functional Assay, Activity Assay, Incubation, Cell Culture, Recombinant, Clinical Proteomics, Inhibition, Comparison, Control, Derivative Assay