Journal: Frontiers in Immunology

Article Title: Single-Cell Characterization of Hepatic CD8 + T Cells in a Murine Model of Primary Biliary Cholangitis

doi: 10.3389/fimmu.2022.860311

Figure Lengend Snippet: Single-cell multi-omics analysis of hepatic immune cells from TG and control mice. (A) Schematic representation of the workflow for single cell preparation, labels for antibodies, cell capture and library preparation. (B) The immune cell subsets identified with a combination of antibodies and genes, include CD19, NK1.1, CD3, CD4, CD8β and Cd8α. (C) Merged t-SNE plots of twelve immune subsets in livers of WT and TG mice. (D) CD8 + T cells from WT and TG mice highlighted with Split t-SNE plots. (E) Percentage of CD8 + T cells in WT and TG mice.

Article Snippet: Cg-Tg (Cd4-TGFBR2)16Flv/J, TG) were initially purchased from the Jackson Laboratory (Bar Harbor, Maine, USA) and housed at the University of California at Davis and then transported to South China University of Technology, Guangzhou, China.

Techniques: Biomarker Discovery, Control

Journal: EBioMedicine

Article Title: Autocrine CTHRC1 activates hepatic stellate cells and promotes liver fibrosis by activating TGF-β signaling

doi: 10.1016/j.ebiom.2019.01.009

Figure Lengend Snippet: CTHRC1 activates both TGF-β and Wnt signaling, while the promotive effect of CTHRC1 on HSC activation is mainly dependent on TGF-β signaling. A. The expression of α-SMA in primary HSCs after 7 days isolated from WT and CTHRC1 −/− mice. B. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein alone, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG. C. Western blotting analysis of phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in five liver tissues of WT or CTHRC1 −/− mice intraperitoneally injected with CCl 4 . GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. D. Phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in primary rat HSCs, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG for 1, 3, 5 days, individually. GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. E and F. Representative immunofluorescence images of α-SMA (green in E, red in F) in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus neutralizing antibodies or inhibitor as follows: TGFBR2 neutralizing antibody or TGF-β receptor inhibitor (E), Wnt5a or Wnt3a neutralizing antibody (F). Nuclei are stained with DAPI (blue). Scale bars, 50 μm. G. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus TGFBR2 neutralizing antibody or TGF-β receptor inhibitor. GAPDH was the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Recombinant CTHRC1 and/or TGFBR2 neutralizing antibody (R&D, AF-241-NA), TGF-βR inhibitor, LY2109761 (SELLECK, S2704) were added in the medium simultaneously.

Techniques: Activation Assay, Expressing, Isolation, Western Blot, Injection, Immunofluorescence, Staining

Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Collagen synthesis. ( A ) Basal collagen synthesis. ( B ) Collagen synthesis after TGF-β (10 nM) stimulation for 24 h. Measurement based on incorporation of [ 3 H]-proline into collagen. Data are mean ± SEM of triplicate determinations. ( C ) Cells treated with or without TGF-β for 24 h were lysed and proteins subjected to immunoblotting using antibodies against TGF-β receptor I and II, phospho-Smad-2, Smad-2, phospho-Smad-3, and Smad-3. GAPDH was used as loading control. * p < 0.05, ** p < 0.01 comparing average of hPSCs with HPaSteC, i-mPSCs for ( A ); ** p < 0.01 comparing control (non-treated) cells with TGF-β treated cells for ( B ). PSC, pancreatic stellate cell; hPSC, human primary PDAC-derived PSC culture; HPaSteC, PSCs from normal human pancreas; i-hPSC, immortalized human PSCs; i-mPSC C2 and C3, immortalized mouse PSCs clone 2 and 3.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Western Blot, Derivative Assay

Journal: Cells

Article Title: Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells

doi: 10.3390/cells8010023

Figure Lengend Snippet: Characteristics of the various pancreatic stellate cell (PSC) cultures.

Article Snippet: Reagents were purchased from the following sources: BODIPY™ 493/503, Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, DMEM F-12 containing GlutaMAX-I, penicillin-streptomycin (Pen-Strep), amphotericin B, trypsin/EDTA, fetal bovine serum (FBS), and Pierce TM BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA); bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), gemcitabine hydrochloride, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), phosphate buffered saline (PBS), and senescence cells histochemical staining kit from Sigma–Aldrich (St. Louis, MO, USA); TGF-β from R&D Systems Europe (Abingdon, UK); human anti-alpha smooth muscle actin (α-SMA; BS66) from Nordic Biosite AB (Taby, Sweden); anti-glial fibrillary acidic protein (GFAP) (GA5), anti-epithelial cell adhesion molecule (EpCAM; VU1D9), anti-vimentin (D21H3), anti-GAPDH and Smad2/3 antibody sampler kit from Cell Signaling Technology (Beverly, MA, USA); anti- TGF-β receptor I (TGF-β RI) and anti-TGF-β receptor II (TGF-β RII) from Abcam (Cambridge, UK); secondary HRP-conjugated antibodies goat anti-mouse and goat anti-rabbit IgG from Bio-Rad Laboratories (Hercules, CA, USA); secondary Alexa Fluor-conjugated antibodies (anti-mouse and anti-rabbit) and DAPI from Jackson ImmunoResearch (West Grove, PA, USA).

Techniques: Isolation, Gradient Centrifugation, Transfection, Expressing, DNA Synthesis, Migration