Journal: PLoS ONE

Article Title: CXCR4 Is Dispensable for T Cell Egress from Chronically Inflamed Skin via the Afferent Lymph

doi: 10.1371/journal.pone.0095626

Figure Lengend Snippet: Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.

Article Snippet: Sterile-filtered AMD3100 in PBS (Sigma-Aldrich) or PBS alone (Invitrogen) was loaded into the osmotic pumps before equilibration in sterile PBS at 37°C for 12 h according to the manufacturer’s instructions.

Techniques: Inhibition, Chemotaxis Assay, Mouse Assay, Labeling, Flow Cytometry, Cytometry

Journal:

Article Title: LidA, a Translocated Substrate of the Legionella pneumophila Type IV Secretion System, Interferes with the Early Secretory Pathway

doi: 10.1128/IAI.73.7.4370-4380.2005

Figure Lengend Snippet: Membrane association of LidA after translocation into host cells. (A) L. pneumophila (WT) and an isogenic dotA mutant ( dotA ), both expressing GFP, were grown to postexponential phase and resuspended in PBS containing either 1% Triton X-100, 1% CHAPS,

Article Snippet: Sixty microliters of postexponential L. pneumophila expressing green fluorescent protein (GFP) was washed once and resuspended in 5 ml of phosphate-buffered saline (PBS) containing either 1% Triton X-100 (Bio-Rad), 1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; Sigma}, 1% octylglucoside (Calbiochem), or 1% purified digitonin (Calbiochem) and incubated for 20 min at room temperature with intermittent vortexing.

Techniques: Translocation Assay, Mutagenesis, Expressing

Journal: Scientific Reports

Article Title: Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

doi: 10.1038/s41598-017-02599-y

Figure Lengend Snippet: The implementation of size-exclusion chromatography to remove Optiprep remnants from EV samples. ( a ) Optiprep density gradients were loaded with PBS to obtain blank EV-density fractions (F1) and with CCM to obtain EV-containing fractions (F2). EVs were retrieved by pelleting at 100,000 g (P1) or by size-exclusion chromatography (S1). Quantity of Optiprep in S1 and P1 was calculated using DC Protein assay. SEC was performed on F2 and consecutive SEC fractions (S2) were analyzed by NTA and protein analysis for GFP and CD9. ( b ) Comparative graph of NTA from S2 (connected line, marked area = SD) and Optiprep quantification of S1 (three differently colored bars representing three independent gradients, SD indicated). ( c ) Protein analysis of S2 by Western blot analysis for GFP and CD9 quantification by TRIFic CD9 assay. Original immunostaining results are shown in Supplementary Fig. 10 . Abbreviations: PBS: phosphate buffered saline. CCM: concentrated conditioned medium. GFP: green fluorescent protein. EV: extracellular vesicle. WB: Western blot. NTA: Nanoparticle tracking analysis. SEC: size-exclusion chromatography.

Article Snippet: A dilution series of 0 to 10% of Optiprep in PBS was obtained as a standard curve for performing absorbance readings with DC Protein assay (Bio-Rad, Hercules, California, USA), according to manufacturer’s instructions.

Techniques: Size-exclusion Chromatography, DC Protein Assay, Western Blot, Immunostaining

Journal: Angewandte Chemie (International ed. in English)

Article Title: Controlled Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides

doi: 10.1002/anie.201800387

Figure Lengend Snippet: Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) AcOSu, PBS (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .

Article Snippet: The compounds 26 – 28 were chemically N-acetylated with N -acetyl succinimide (AcOSu) in PBS buffer to give 29 – 31 , respectively, after purification by BioRad P4 size exclusion column chromatography.

Techniques: Papanicolaou Stain

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways

doi: 10.1186/s13046-018-0731-5

Figure Lengend Snippet: Metformin inhibits tumor growth in an RPMI8266 xenograft mouse model. a NOD/SCID mice received metformin or PBS by oral gavage every day after subcutaneous implantation of RPMI8226 cells. Tumor sizes were monitored every 3 days. b Body weight of mice was monitored every 3 days. c Immunohistochemical analysis of tumors from the metformin and control groups showing upregulation of AMPK and downregulation of mTOR and Ki-67. Expression levels were scored semi- quantitatively as the percentage of positive cells according to the following system: +,

Article Snippet: Reagents Metformin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) as a stock solution of 1 M. Primary antibodies for specific detection of p-AMPK (Thr172), AMPK, Phospho-Tuberin/TSC2 (Ser1387), p-mTOR (Ser2481), p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K, p-4EBP1 (Thr37/46), 4EBP1, p-AKT (Ser473), and AKT, as well as horseradish peroxidase (HRP)-conjugated anti-rabbit secondary detection antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Mouse Assay, Immunohistochemistry, Expressing

Journal: Autophagy

Article Title: MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint

doi: 10.1080/15548627.2019.1606647

Figure Lengend Snippet: MTORC1 activation by MHY1485 or genetic deletion of Tsc1 suppresses chondrocyte autophagy and promotes chondrocyte apoptosis and TMJ cartilage loss in UAC animals. (a) Fifty μl, 100 nM MHY1485 or equal volume PBS was injected into the TMJ region of mice with UAC surgery from 0 wkto 4 wk. Protein extracts were prepared from the condylar cartilage for western blotting for the expression of p-MTOR, p-RPS6, p-EIF2AK3, CASP12, DDIT3, BECN1 and LC3B-I/II. (b) Quantitative data of (a). (c) Sagittal central sections of the condylar cartilage of each group were subjected to Safranin O staining. IHC staining of p-EIF2AK3, CASP12, and DDIT3. Black scale bar, 100 μm. IF staining for autophagosome and lysosome location. LC3B-II, green; LAMP2, red; DAPI, blue. White scale bar: 10 μm. (d-g) Quantitative data of (C). (h) Deletion of Tsc1 in chondrocytes. Mice were subjected to UAC or sham surgery for 3 wk and injected with TM as described in the Materials and Methods. IF staining: sagittal central sections of the TMJ were stained with antibody against p-RPS6 (S235/236). p-RPS6, red; DAPI, blue. White dots, superficial of condylar cartilage; Green dots, the border between cartilage and subchondral bone. Safranin O staining. IHC staining of p-EIF2AK3. TUNEL staining. White dots, superficial of condylar cartilage; Blue dots, the border between cartilage and subchondral bone. Scale bar: 100 μm. (i-l) Quantitative data for (H). Results are expressed as mean ± standard deviation. IHC staining: N = 6 for rat, N = 5 for mice. Western blotting and qPCR analysis: N = 3. * P

Article Snippet: Fifty μl of 100 nM rapamycin (Selleck, S1039) or 100 nM MHY1485 (Selleck, S7811) were diluted in PBS (Cell Signaling Technology, 9872) and injected locally into the right or left TMJ areas of the injection groups every other day.

Techniques: Activation Assay, Injection, Mouse Assay, Western Blot, Expressing, Staining, Immunohistochemistry, TUNEL Assay, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint

doi: 10.1080/15548627.2019.1606647

Figure Lengend Snippet: MTORC1 inactivation promotes chondrocyte autophagy, suppresses chondrocyte apoptosis and prevents cartilage loss under UAC. (a and b) Injection of 100 nM MTORC1 inhibitor rapamycin (50 μl, Rapa group) or equal volume PBS (vehicle group) in TMJ region of UAC rats from 8 to 12 wk every other day. The samples were harvested after 4 and 12 wk’ injection. Condylar cartilage was prepared for protein extracts for western blotting and its quantification. (c) Sagittal central sections of the condylar cartilage were subjected to safranin O staining for proteoglycans and the positive areas in each group at 12 wk. IHC staining for expression of the p-EIF2AK3, CASP12 and DDIT3. Black scale bar: 100 μm. IF staining for detecting the autophagosome and lysosome location. LC3B-II, green; LAMP2, red; DAPI, blue. White scale bar: 10 μm. (d-g) Quantitative data of cartilage area and p-EIF2AK3-, CASP12- and DDIT3-positive cells in each group are shown. (h) Cartilage-specific deletion of MTORC1 in chondrocytes. Mice were subjected to UAC or sham surgery for 7 wk and injected with TM as described in the Materials and Methods. IF staining: sagittal central sections of the TMJ were stained with antibody against p-RPS6 (S235/236). p-RPS6, red; DAPI, blue. White dots, superficial of condylar cartilage; Green dots, the border between cartilage and subchondral bone. Safranin O (San O) staining. IHC staining of p-EIF2AK3. TUNEL staining. White dots, superficial of condylar cartilage; Blue dots, the border between cartilage and subchondral bone. Scale bar: 100 μm. (i-l) Quantification for (h). Results are expressed as mean ± standard deviation. Safranin O, TUNEL and IHC staining: N = 6 rats, N = 5 mice. Western blotting and qPCR analysis: N = 3. * P

Article Snippet: Fifty μl of 100 nM rapamycin (Selleck, S1039) or 100 nM MHY1485 (Selleck, S7811) were diluted in PBS (Cell Signaling Technology, 9872) and injected locally into the right or left TMJ areas of the injection groups every other day.

Techniques: Injection, Western Blot, Staining, Immunohistochemistry, Expressing, Mouse Assay, TUNEL Assay, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Regenerative Therapy

Article Title: Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media

doi: 10.1016/j.reth.2016.12.002

Figure Lengend Snippet: Detection of fetal hNSCs and hiPSC-derived hNSCs. (A) Supernatants from Ff-I01 hiPSC cultures were serially diluted with PBS and analyzed by GlycoStem-HP. (B) Cell culture supernatants of fetal hNSCs and hiPSC-derived hNSCs were serially diluted with PBS and analyzed by GlycoStem-HP. Absorbance at OD450-620 was measured. Data are shown as the mean ± SD of triplicate samples. (C) hiPSC-derived hNSCs were stained with Tra-1-60 and SSEA4 and analyzed by flow cytometry.

Article Snippet: 2.5 GlycoStem-HP Biotin-labeled rBC2LCN (15 ng) diluted in PBS (Takara) was immobilized on streptavidin-coated plates (SUMITOMO BAKELITE, BS-X7603) at room temperature for 1 h. After washing 5 times with 200 μL of wash buffer (PBS containing 0.1% Triton X-100), 50 μL of cell culture media were allowed to react at room temperature for 1 h. After washing, 50 μL of HRP-labeled R-10G was overlaid for 1 h at room temperature.

Techniques: Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry