Journal: Nature Communications

Article Title: Pancreatic tumor eradication via selective Pin1 inhibition in cancer-associated fibroblasts and T lymphocytes engagement

doi: 10.1038/s41467-022-31928-7

Figure Lengend Snippet: a Establishment and validation of CAFs via high expressions of both FAP-α and α-SMA. b Cellular uptake of Cy5-labeled formulations on Pan02 cells, NIH-3T3 cells, and CAFs, measured by flow cytometry ( n = 3 biologically independent experiments; error bars, mean ± SD). c Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, imaged by fluorescent microscopy. Scale bars are 20 μm. Three independent repetitions got similar results. d Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, quantified by flow cytometry ( n = 6 biologically independent samples; error bars, mean ± SD). Cytotoxicity on Pan02 cells ( e ) and CAFs ( f ) ( n = 3 biologically independent experiments; error bars, mean ± SD). g Western blotting detection of Pin1 and relative proteins from Pan02 cells after treating with PBS, AG17724 (0.5 μM), or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. h Quantitative analysis of the western blotting results from Pan02 cells (Three independent repetitions got similar results.). i Western blotting detection of Pin1 and relative proteins from CAFs treated with PBS, AG17724, or antiCAFs-DMS after treating with PBS, AG17724 (0.5 μM) or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. j Quantitative analysis of the western blotting results from CAFs (Three independent repetitions got similar results.). One-way analysis of variance followed by Turkey posttests. Source data are provided as a Source Data file.

Article Snippet: Anti-FAP-α antibody (MAB9727-100, 0.25 μg/mL as working concentration.) and anti-phospho-CDC2/CDK1 (Y15) antibody (AF888-SP, 0.2 μg/mL as working concentration.) were purchased from R&D SYSTEMS.

Techniques: Biomarker Discovery, Labeling, Flow Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

doi: 10.1101/491894

Figure Lengend Snippet: A-D) and I-L) Heatmaps of single cell SMAD4/2 translocation (A-B and I-J) and ctgf expression (C-D and K-L) upon 5nM TGF-β stimulation. Data is represented both in absolute luminescence levels (A,C and I,K) and intensities normalized on the maximal intensity for each trace (B,D and J,L). E) and M) Population average of SMAD4/2 translocation and ctgf expression. F and N) Single cell distribution of peak timing for SMAD4/2 translocation (green) and ctgf expression (blue). SMAD4: Mean = 55 min, CV=0.33; SMAD2: Mean = 66 min, CV= 0.35. G and O) Single-cell distribution of nuclear to cytoplasmic ratio at translocation peak for SMAD4 (G) and SMAD2 (O). H and P) Single-cell distribution of ctgf expression peak levels for iS4 (H) and iS2 (P) cell lines.

Article Snippet: The GT:ctgf, iS4, iS2 NIH-3T3 cell lines and HEK 293T cells (ATCC) were cultured in DMEM (Thermofisher; 41966029), supplemented with 10% fetal bovine serum (Thermofisher, 10270106) and 1% penicillin/streptomycin (BioConcept, 4-01F00H), at 37°C and 5% CO 2 .

Techniques: Translocation Assay, Expressing

Journal: bioRxiv

Article Title: Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

doi: 10.1101/491894

Figure Lengend Snippet: A) Schematics of analog and digital responses of individual cells. White filled circles represent the unresponsive single-cells while blue shades represent the response strength. B) Stimulation dose-response of TGF-β-induced SMAD4 signaling and ctgf response in single cells. At time t=0, cells were treated with the indicated concentrations of TGF-β and representative traces for active single cells are shown. Left: traces of nuclear to cytoplasmic signal difference in SMAD4; right: ctgf responses in the corresponding single cells. C-D) Nuclear/cytoplasmic ratio change from stimulation to the peak and ctgf response peak upon treatment with different doses of TGF-β in the iS4 (C) and iS2 (D) cell lines. E-F) Time lag between TGF-β stimulation and peak of nuclear/cytoplasmic SMAD4/2 ratio (left), between TGF-β stimulation and ctgf response initiation (middle) and peak (right), in the iS4 (E) and iS2 (F) cell lines.

Article Snippet: The GT:ctgf, iS4, iS2 NIH-3T3 cell lines and HEK 293T cells (ATCC) were cultured in DMEM (Thermofisher; 41966029), supplemented with 10% fetal bovine serum (Thermofisher, 10270106) and 1% penicillin/streptomycin (BioConcept, 4-01F00H), at 37°C and 5% CO 2 .

Techniques:

Journal: bioRxiv

Article Title: Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

doi: 10.1101/491894

Figure Lengend Snippet: A) Single cell ctgf responses after stimulation with different doses of TGF-β, clustered into transient and sustained classes using k-means clustering (n transient = 1666; n sustained = 427). Heat maps for ctgf traces belonging to transient (upper panel) and sustained (lower panel) are shown using absolute (left) and normalized levels of ctgf responses. B) Silhouette plot of cells sorted according to ctgf expression dynamics. C) Fraction of cells responding in a transient or sustained manner at different concentrations of dox in the iS2 (left) and iS4 (right) cell lines. (*p<0.001, **p<10e-6; Chi-squared test). D) Average SMAD4/2 expression level during the 12 hours following 5nM TGF-β stimulation, belonging to the transient and the sustained classes from samples treated with 2 ng/ml and 10 ng/ml doxycycline (*p<0.05; t-test).

Article Snippet: The GT:ctgf, iS4, iS2 NIH-3T3 cell lines and HEK 293T cells (ATCC) were cultured in DMEM (Thermofisher; 41966029), supplemented with 10% fetal bovine serum (Thermofisher, 10270106) and 1% penicillin/streptomycin (BioConcept, 4-01F00H), at 37°C and 5% CO 2 .

Techniques: Expressing