Journal: Nutrients

Article Title: Prevention of the Pro-Aggressive Effects of Ethanol-Intoxicated Mice by Schisandrin B

doi: 10.3390/nu15081909

Figure Lengend Snippet: Schisandrin B prevents activation of liver inflammasomes in mice consuming ethanol. ( A ) Representative Sirius red-stained histological images showing collagen deposition (red color) in the livers of control, Sch B-treated, ethanol-challenged, and ethanol-challenged + Sch B-treated mice. Scale bar corresponds to 200 μm. Levels of fibrosis were quantified by Ishak score based on the Sirius red-stained slides. ( B ) Representative Western blot images and relative densitometric bar graphs of ( B ) fibrotic markers and ( C ) inflammasome components. Expression levels are standardized by α-tubulin or the pro-form protein levels as appropriate. n = 4 mice. Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking, the membranes were incubated overnight with antibodies against α-tubulin (GeneTex, Irvine, CA, USA), type I collagen (COLA-1; ABclonal, Woburn, MA, USA), type III collagen (COLA-3; ABclonal), fibronectin (GeneTex), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3; Proteintech, Rosemont, IL, USA), caspase-1 (Proteintech), interleukin-1β (IL-1β; Cell Signaling Technology, Danvers, MA, USA), interleukin-18 (IL-18; Proteintech), and gasdermin D (GSDMD; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Staining, Control, Western Blot, Expressing

Journal: Nutrients

Article Title: Prevention of the Pro-Aggressive Effects of Ethanol-Intoxicated Mice by Schisandrin B

doi: 10.3390/nu15081909

Figure Lengend Snippet: Schisandrin B alleviates ethanol-induced liver inflammasome activation and fibrosis. ( A ) Representative Sirius red-stained histological images showing collagen deposition (red color) in the mouse liver. Levels of fibrosis were quantified by Ishak score based on the Sirius red-stained slides. ( B ) Representative Western blot images and relative densitometric bar graphs of ( B ) fibrotic markers and ( C ) inflammasome components. Expression levels are standardized by α-tubulin or the pro-form protein levels as appropriate. n = 4 mice. Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking, the membranes were incubated overnight with antibodies against α-tubulin (GeneTex, Irvine, CA, USA), type I collagen (COLA-1; ABclonal, Woburn, MA, USA), type III collagen (COLA-3; ABclonal), fibronectin (GeneTex), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3; Proteintech, Rosemont, IL, USA), caspase-1 (Proteintech), interleukin-1β (IL-1β; Cell Signaling Technology, Danvers, MA, USA), interleukin-18 (IL-18; Proteintech), and gasdermin D (GSDMD; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Staining, Western Blot, Expressing

Journal: Oncotarget

Article Title: Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells

doi:

Figure Lengend Snippet: A. Immunofluorescence staining. Visceral ASCs (ASCvis) and subcutaneous ASCs (ASCsub) were fixed and stained for α-tubulin, vimentin, pericentrin and DNA. Examples are shown. Scale bar: 20 μm. B. Immunofluorescence staining of cell surface markers in ASCs. Scale bar: 10 μm. C. Multilineage differentiation capability of ASCs. ASCs were incubated with corresponding differentiation medium for 14 or 21 days. Neuronal differentiation (14 days) was verified with lineage-specific staining of Tuj1 and DCX. Adipogenic differentiation (14 days) was assessed by oil red O staining displaying lipid vesicle formation. Osteogenic differentiation (21 days) was examined by Alizarin Red S staining indicating calcium deposits. D. Quantification of the differentiation potential of visceral ASCs by analyzing lineage-specific characteristic marker ( n = 300 cells for each condition). The results are based on three independent experiments with ASCs obtained from three different donors and presented as mean ± SEM ( n = 3).

Article Snippet: The following primary antibodies were used for staining: monoclonal mouse antibody against FITC-conjugated α-tubulin and mouse monoclonal antibody against acetylated α-tubulin (Sigma-Aldrich), rabbit polyclonal antibodies against pericentrin, mouse monoclonal antibody against adiponectin and rabbit monoclonal antibodies against CD90, E-cadherin and N-cadherin (Abcam), mouse monoclonal antibody against DCX, rabbit polyclonal antibody against CD73 and chicken polyclonal antibody against Tuj1 (GeneTex), mouse monoclonal antibody against vimentin (Dako), mouse monoclonal antibody against pHH3 (S10) (Merck Millipore), rabbit polyclonal antibody against phospho-FAK (Y397) (Cell Signaling), rat monoclonal antibody against α-tubulin (Biomol, Hamburg) and mouse monoclonal antibodies against CD14 and CD31 (Biolegend, Fell).

Techniques: Immunofluorescence, Staining, Incubation, Marker

Journal: Oncotarget

Article Title: Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells

doi:

Figure Lengend Snippet: A. The cytokine/chemokine array assay. The factors were measured in the supernatants of visceral ASCs (ASCvis) and subcutaneous ASCs (ASCsub) cultured for 3 days by using a human cytokine antibody array. The six most prominent chemokines/chemokines are demonstrated. The results, relative to the positive control provided by the array, are based on three independent experiments with ASCs obtained from three different donors and presented as mean ± SEM. B. Evaluation of membrane protrusion of ASCs toward MCF-7, MDA-MB-231 and MCF-10A cells. The results are based on three independent experiments with ASCs from three different donors and shown as mean ± SEM. C. Representatives of ASCs homing to breast cancer cell lines. Red arrows indicate membrane protrusions of ASCs toward breast cancer cells. Normal mammary epithelial MCF-10A cells served as negative control. Scale bar: 250 μm. D. Immunofluorescence staining of the migration front between ASCs and MCF-7 or MDA-MB-231 cells. Both cell types were stained for p-FAK, phalloidin, acetylated α-tubulin and DNA. White arrows depict the connections stabilized by acetylated α-tubulin between ASCs and breast cancer cells. Scale bar: 25 μm.

Article Snippet: The following primary antibodies were used for staining: monoclonal mouse antibody against FITC-conjugated α-tubulin and mouse monoclonal antibody against acetylated α-tubulin (Sigma-Aldrich), rabbit polyclonal antibodies against pericentrin, mouse monoclonal antibody against adiponectin and rabbit monoclonal antibodies against CD90, E-cadherin and N-cadherin (Abcam), mouse monoclonal antibody against DCX, rabbit polyclonal antibody against CD73 and chicken polyclonal antibody against Tuj1 (GeneTex), mouse monoclonal antibody against vimentin (Dako), mouse monoclonal antibody against pHH3 (S10) (Merck Millipore), rabbit polyclonal antibody against phospho-FAK (Y397) (Cell Signaling), rat monoclonal antibody against α-tubulin (Biomol, Hamburg) and mouse monoclonal antibodies against CD14 and CD31 (Biolegend, Fell).

Techniques: Cell Culture, Ab Array, Positive Control, Membrane, Negative Control, Immunofluorescence, Staining, Migration

Journal: Oncotarget

Article Title: Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells

doi:

Figure Lengend Snippet: A. Immunofluorescence staining. MCF-7 cells were stained for the epithelial marker E-cadherin, the mesenchymal markers vimentin and N-cadherin, DNA and α-tubulin after 14 days of indirect co-culture with visceral ASCs. Representatives are depicted. Scale bar: 25 μm. B. Western blot analysis. Cellular lysates were prepared from MCF-7 cells, non-treated as control (con), indirectly co-cultivated with visceral ASCs (ASCvis) for 14 days, and co-cultured MCF-7 cells released for 14 and 21 days afterwards. Lysates from untreated fibroblasts, visceral ASCs or subcutaneous ASCs were taken as positive control cells. β-actin served as loading control. C. Cell viability assay. MCF-7 cells indirectly co-cultured with ASCs were seeded in 96-well plates and their viability was evaluated via a CellTiter-Blue ® assay. The results are based on three independent experiments with ASCs obtained from three different donors and presented as mean ± SEM. MCF-7 cells without co-culture served as control (con).

Article Snippet: The following primary antibodies were used for staining: monoclonal mouse antibody against FITC-conjugated α-tubulin and mouse monoclonal antibody against acetylated α-tubulin (Sigma-Aldrich), rabbit polyclonal antibodies against pericentrin, mouse monoclonal antibody against adiponectin and rabbit monoclonal antibodies against CD90, E-cadherin and N-cadherin (Abcam), mouse monoclonal antibody against DCX, rabbit polyclonal antibody against CD73 and chicken polyclonal antibody against Tuj1 (GeneTex), mouse monoclonal antibody against vimentin (Dako), mouse monoclonal antibody against pHH3 (S10) (Merck Millipore), rabbit polyclonal antibody against phospho-FAK (Y397) (Cell Signaling), rat monoclonal antibody against α-tubulin (Biomol, Hamburg) and mouse monoclonal antibodies against CD14 and CD31 (Biolegend, Fell).

Techniques: Immunofluorescence, Staining, Marker, Co-Culture Assay, Western Blot, Control, Cell Culture, Positive Control, Viability Assay, CtB Assay

Journal: PLoS ONE

Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

doi: 10.1371/journal.pone.0114208

Figure Lengend Snippet: A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's t -test. The average nuclear areas observed in 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. B. Cells were infected with N. gonorrhoeae and observed for 24 h by live-cell microscopy. DIC images were captured every 15 min at randomly selected positions, and times spent transitioning from prophase to cytokinesis were measured for 150 mitotic cells. The graph shows mean ± standard deviation values for the time required for progression from prophase to cytokinesis, measured in 3 independent experiments. C. VK2/E6E7 cells were infected with N. gonorrhoeae for 24 h. RNA from control cells and infected cells was isolated and qPCR was performed. Average mRNA levels of MAD1L1 and MAD2L1 from 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. Values were normalized against TUBA1A expression and analyzed using a paired 2-tailed Student's t -test. D. Protein expression in infected cells and control cells was analyzed in western blots using antibodies against of MAD1 and MAD2. GAPDH or α-tubulin was used as loading controls. E. Western blot band intensities were quantified and analyzed with a paired 2-tailed Student's t -test, using GAPDH or α-tubulin expression as controls. Mean expression levels of each protein observed in 3 independent western blots are shown (* p <0.05). Error bars represent S.E.M.

Article Snippet: Polyclonal antibodies against α-tubulin (MBS316320, MyBioSource, 1∶1,000) or GAPDH (G-9545, Sigma-Aldrich, 1∶5,000) were used for normalizing total protein loaded in each well.

Techniques: Bacteria, Staining, Infection, Microscopy, Standard Deviation, Control, Isolation, Expressing, Western Blot