Journal: Infection and Immunity

Article Title: Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

doi:

Figure Lengend Snippet: [Ca 2+ ] i response in BAMs to LPS. (A) Trace showing the response to 1 ng of LPS per ml in the presence of 5% FBS ( n = 50 cells). (B) Representative trace showing no elevation of [Ca 2+ ] i even up to 1 μg of LPS per ml in the absence of FBS ( n = 50 cells). Subsequent addition of 5% FBS resulted in an elevation of [Ca 2+ ] i . (C) Integrated [Ca 2+ ] i response of BAMs to LPS (1 or 100 ng/ml) ( n = 129 cells) and the response to 100 ng of LPS per ml in nominally Ca 2+ -free HBSS ( n = 62 cells). Note that there is no significant difference in the responses to the two concentrations of LPS. However, the response in nominally Ca 2+ -free HBSS is significantly attenuated. The asterisk denotes statistical significance ( P

Article Snippet: DMEM, HBSS, antibiotics, and FBS were purchased from GIBCO/BRL (Grand Island, N.Y.).

Techniques:

Journal: Korean Journal of Urology

Article Title: Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

doi: 10.4111/kju.2010.51.8.572

Figure Lengend Snippet: Effect of different carbon monoxide (CO) concentrations (1%, 2%, and 5%) on relaxation in Phe-precontracted corpus cavernosum smooth muscle before exposure to electrical field stimulation. HBSS: Hank's balanced salt solution.

Article Snippet: 10x Hank's balanced salt solution (HBSS) was obtained from Invitrogen Corporation (Grand Island, NY, USA).

Techniques:

Journal: Korean Journal of Urology

Article Title: Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

doi: 10.4111/kju.2010.51.8.572

Figure Lengend Snippet: Effect of carbon monoxide (5%) on corpus cavernosum smooth muscle (CCSM) relaxation induced by electrical field stimulation (EFS). Rat CCSM was phenylephrine-precontracted and responded to EFS (0.5 to 32 Hz, 0.2 ms duration). HBSS: Hank's balanced salt solution.

Article Snippet: 10x Hank's balanced salt solution (HBSS) was obtained from Invitrogen Corporation (Grand Island, NY, USA).

Techniques: Mass Spectrometry

Journal: Korean Journal of Urology

Article Title: Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

doi: 10.4111/kju.2010.51.8.572

Figure Lengend Snippet: Effect of nitric oxide synthase inhibitor, N G -nitro-L-arginine, on rat corpus cavernosum smooth muscle relaxation induced by electrical field stimulation in the presence of carbon monoxide (5%). CO: carbon monoxide, HBSS: Hank's balanced salt solution, L-NOARG: N G -nitro-L-arginine, EFS: electrical field stimulation.

Article Snippet: 10x Hank's balanced salt solution (HBSS) was obtained from Invitrogen Corporation (Grand Island, NY, USA).

Techniques:

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

Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

doi: 10.4049/jimmunol.1001822

Figure Lengend Snippet: Impaired calcium flux in Atg7-deficient T cells (A). Calcium influx in autophagy-deficient T cells upon TCR engagement. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 and then stained with7-AAD, anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE antibodies. Cells were resuspended in HBSS buffer containing 1.26 mM CaCl 2 . The Indo-1 loaded cells were stimulated with biotin-anti-CD3 (5 μg/ml) and biotin-anti-CD4 (1 μg/ml) antibodies for analysis of CD4 + T cells (or biotin-anti-CD8 antibody for CD8 + T cells) for 1 min. After establishing a base line, the cells were crosslinked with 25 μg/ml streptavidin and the stimulation was indicated by the arrow in the figure (same in other calcium flux figures). Intracellular Ca 2+ kinetics were expressed as the ratio of emission at 405 nm to that at 510 nm. Graphs represent calcium influx in live naïve 7-AAD - CD44 low CD4 + or 7-AAD - CD44 low CD8 + cells. This experiment was repeated three times independently. (B). Phosphorylation of p38, ERK, and PLCγ1 in autophagy-deficient T cells upon TCR stimulation. Purified naïve CD44 low T lymphocytes were stimulated with biotin-labeled anti-CD3 (5 μg/ml), biotin-labeled anti-CD4 (1 μg/ml) and biotin-labeled anti-CD8 (1 μg/ml) for 1 min and crosslinked with streptavidin (25 μg/ml) for 1, 1.5, 3, 5, or 10 min. Phosphorylated proteins were detected with anti-phosphorylated p38, ERK, or PLCγ1 and visualized using Alexa Fluor 680- or IRDye 800-conjugated anti-species antibodies. Numbers represent the ratios of intensity of the target molecule bands to intensity of actin bands. These Western blots analysis were repeated five times using purified T cells from different pairs of wildtype and Atg7 f/f Lck-Cre mice. (C). IkBα degradation in autophagy-deficient T cells after CD3/CD28 stimulation. T cells were stimulated with anti-CD3 (5μg/ml) and anti-CD28 (2μg/ml) antibodies overnight and whole cell lysates were subjected to Western blot analysis. Numbers represent the ratios of intensity of the IkBα bands to intensity of actin bands. (D). Increased calcium store in Atg7-deficient T cells. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 in Ca 2+ -free HBSS buffer. Cells were stained with anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE, and resuspended in calcium-free HBSS containing 1 mM EGTA. A total of 1×10 6 cells were incubated in calcium-free conditions with biotin-anti-CD3 and either biotin-anti-CD4 or biotin-anti-CD8 antibodies for 1 min. After establishing a base line using flow cytometry, Ca 2+ -free streptavidin was added to crosslink anti-CD3 and anti-CD4 (or anti-CD8) antibodies (upper two panels). Above Indo-1 loaded cells were also stimulated with 1μM thapsigargin (TG) in calcium-free HBSS containing 1 mM EGTA to measure the calcium store in ER (lower two panels). The kinetic changes in [Ca 2+ ] i were visualized as the ratio of emission at 405 nm to that at 510 nm over a period of 7 min. All cells were gated on CD44 low and 7-AAD negative cells. Above experiments were repeated three times independently.

Article Snippet: Lymph nodes cells from Atg7f/f or Atg7f/f Lck-Cre mice were incubated with 5 μM Indo-1 (Invitrogen) for 30 min in HBSS buffer (Invitrogen).

Techniques: Mouse Assay, Staining, Purification, Labeling, Western Blot, Incubation, Flow Cytometry, Cytometry

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

Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

doi: 10.4049/jimmunol.1001822

Figure Lengend Snippet: The SERCA pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T cells (A). The expression of SERCA2 was analyzed by Western blot (left panel). Numbers represent ratios of the intensity of SERCA2 bands to intensity of actin bands. This Western blot analysis was repeated six times using purified T cells from different pairs of wildtype and Atg7-deficient mice splenocytes. Normalized Western blot intensities were quantified and showed in the right panel figure (mean ± SD, *p=0.006). (B). Thapsigargin-induced calcium influx in Atg7-deficient T cells. Lymph node cells from Atg7 f/f and Atg7 f/f Lck-Cre mice were loaded with Indo-1, suspended in HBSS containing 1.26 mM CaCl 2 and stimulated with different concentrations of thapsigargin. Samples were gated on naïve CD44 low CD4 + 7-AAD - or CD44 low CD8 + 7-AAD - cells. Dashed lines represent Atg7 f/f cells and solid lines represent Atg7 f/f Lck-Cre cells. This experiment was repeated three times. (C). Analyzing the calcium influx through CRAC channel in autophagy-deficient T cells by flow cytometry. Lymph node cells from Atg7 f/f Lck-Cre and Atg7 f/f mice were loaded with Indo-1 and cell surfaces were stained with anti-CD4-PE, anti-CD44-FITC and 7-AAD. The Indo-1 loaded cells were resuspended in HBSS containing 1 mM EGTA and 1 mM EDTA but without Ca 2+ and Mg 2+ . After establishing a base level of fluorescence intensity ratio of 405 nm to 510 nm on flow cytometry for 1 min, the Indo-1 loaded cells were stimulated with 1 μM thapsigargin (TG) and the events were collected over a period of 7 min. Then the flow cytometry was paused and TG stimulated cells were spun down. The Ca 2+ -free buffer was removed and replaced with HBSS containing 1.26 mM Ca 2+ . The TG stimulated cells were washed once and then resuspended in HBSS with 1.26 mM Ca 2+ . The flow cytometry was resumed and more events were recorded for another 7 min period. The profiles were gated on CD4 + CD44 LOW 7-AAD - cell population. This experiment was repeated three times independently.

Article Snippet: Lymph nodes cells from Atg7f/f or Atg7f/f Lck-Cre mice were incubated with 5 μM Indo-1 (Invitrogen) for 30 min in HBSS buffer (Invitrogen).

Techniques: Expressing, Western Blot, Purification, Mouse Assay, Flow Cytometry, Cytometry, Staining, Fluorescence

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

Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

doi: 10.4049/jimmunol.1001822

Figure Lengend Snippet: Impaired calcium flux in Atg7-deficient T cells stimulated with ionomycin (A). Calcium influx in autophagy-deficient T cells upon ionomycin stimulation. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 and stimulated with 13 nM ionomycin. Samples were gated on naïve CD44 low CD4 + 7-AAD - or CD44 low CD8 + 7-AAD - cells. Dashed lines represent Atg7 f/f T cells and solid lines represent Atg7 f/f Lck-Cre T cells. This experiment was repeated four times. (B). Indo-1-loaded cells were stimulated with 60 nM ionomycin in calcium-free HBSS containing 1 mM EGTA, and [Ca 2+ ] i were analyzed over a period of 5 min. Dashed lines represent Atg7 f/f cells and solid lines represent Atg7 f/f Lck-Cre cells. This experiment was repeated three times. (C). The integrated increase in Ca 2+ in Atg7 f/f or Atg7 f/f Lck-Cre CD4 + T cells over a period of 5 min. Cells were loaded with Fura-2 and stimulated with 13 nM ionomycin. The integrated Ca 2+ increase was calculated as described in Materials and Methods for sixty individual cells. The beginning of calcium influx was defined as the timepoint at which the first 5% increase in fluorescence intensity occurred. Thirty frames were collected over a period of 5 min after the beginning of calcium influx for calculation of the absolute integrated Ca 2+ increase for each individual cell (p=6.19×10 -46 ). (D). Kinetics of Ca 2+ influx in Atg7-deficient and wildtype T cells. Sixty cells were synchronized by defining the beginning of calcium flux as the timepoint at which the first 5% of cells displayed an increased fluorescence intensity ratio. Images obtained during the 5 min period after initiation of calcium influx were used to analyze kinetic changes in calcium. (E). Length of time to reach the peak level of Ca 2+ influx in each individual cell. Calcium influx was calculated in 60 cells for each group (p=6.13×10 -9 ).

Article Snippet: Lymph nodes cells from Atg7f/f or Atg7f/f Lck-Cre mice were incubated with 5 μM Indo-1 (Invitrogen) for 30 min in HBSS buffer (Invitrogen).

Techniques: Mouse Assay, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice

doi: 10.2147/IJN.S168556

Figure Lengend Snippet: p5RHH-JNK2 siRNA NPs reduce thrombotic risk, restore endothelial integrity, and decrease necrotic plaque area and macrophages. Notes: ( A ) Shorter occlusion times for untreated ApoE −/− mice indicate more aggressive clotting (ie, heightened thrombotic risk) compared to mice treated with p5RHH-JNK2 siRNA NPs (n=6), which manifest prolonged occlusion times compared to HBSS control (n=5, P =0.02). ( B ) 19 F MRS demonstrates significantly less perfluorocarbon (CE volume) NP accumulation in ApoE −/− mice after p5RHH-JNK2 siRNA NP treatment (n=8) compared to those treated with HBSS (n=7) ( P =0.003), confirming restoration of endothelial barrier integrity that now prohibits passive permeation of perfluorocarbon NPs. ( C and D ) Representative aorta sections demonstrate that necrotic plaque area is reduced in p5RHH-JNK2 siRNA NP-treated animals ( D ) (n=8) compared to those treated with HBSS ( C ) (n=4, bar 100 μm). ( E ) Necrotic plaque areas from mice treated with p5RHH-JNK2 siRNA NPs are reduced compared to mice receiving HBSS ( P =0.004). ( F and G ) Representative immunofluorescence stains demonstrate significantly fewer Moma-positive cells in atherosclerotic plaque from ApoE −/− mice treated with p5RHH-JNK2 siRNA NPs ( G ) (n=36) compared to HBSS control mice ( F ) (n=17, P

Article Snippet: For in vitro applications, p5RHH-siRNA nanoparticles were formulated by mixing p5RHH and siRNA in Hanks’ Balanced Salt solution (HBSS) (14025-092; Thermo Fisher Scientific) and incubated at 37°C for 40 minutes, followed by albumin stabilization, as previously reported.

Techniques: Mouse Assay, Coagulation, Immunofluorescence

Journal: International Journal of Nanomedicine

Article Title: Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice

doi: 10.2147/IJN.S168556

Figure Lengend Snippet: p5RHH-JNK2 siRNA NPs neither suppress systemic immune-cell function nor induce innate/adaptive immunoresponses. Notes: ( A ) After seven sequential doses of p5RHH-siRNA NPs over 3.5 weeks, spleens were extracted and splenocytes enumerated 24 hours after the last dose. ApoE −/- mice treated with p5RHH-JNK2 NPs (n=4) exhibit significantly fewer splenocytes compared to HBSS control (n=3) ( P =0.02). ( B ) Spleen sizes of mice treated with p5RHH-JNK2 siRNA NPs (n=19) are significantly smaller than from mice with HBSS treatment (n=12, P =0.001) and approximate spleen sizes of control C57BL/6 mice (n=9, P =0.511; one-way ANOVA followed with Scheffé post hoc test). ( C ) Distribution of splenic immune-cell subpopulations was not affected by the p5RHH-JNK2 siRNA NP treatment (n=4) compared to HBSS control (n=3; FoxP3 + p5RHH-JNK2 siRNA NP treatment, n=6; HBBS, n=5). ( D ) Splenic CD4 + T cells stimulated with anti-CD3 monoclonal antibody responded normally (HBSS, n=5; p5RHH-JNK2 siRNA NPs, n=6). ( E ) C3a assay indicates that p5RHH-JNK2 siRNA NPs (n=5) do not activate complement (innate immune response) compared with DOTAP NPs known to activate strongly (n=5). C3a level of the mice treated with the p5RHH-JNK2 siRNA NPs is significantly smaller than those of mice treated with DOTAP NP ( P

Article Snippet: For in vitro applications, p5RHH-siRNA nanoparticles were formulated by mixing p5RHH and siRNA in Hanks’ Balanced Salt solution (HBSS) (14025-092; Thermo Fisher Scientific) and incubated at 37°C for 40 minutes, followed by albumin stabilization, as previously reported.

Techniques: Cell Function Assay, Mouse Assay

Journal: International Journal of Nanomedicine

Article Title: Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice

doi: 10.2147/IJN.S168556

Figure Lengend Snippet: p5RHH-JNK2 siRNA NPs reduce inflammatory signaling in atherosclerotic plaque. Notes: ( A and B ) Representative confocal images of immunofluorescence stains exhibit reduced nuclear localization of p65 in atherosclerotic plaques from treated ApoE −/− mice ( B ) (n=10) compared to HBSS control ( A ) (n=16, P

Article Snippet: For in vitro applications, p5RHH-siRNA nanoparticles were formulated by mixing p5RHH and siRNA in Hanks’ Balanced Salt solution (HBSS) (14025-092; Thermo Fisher Scientific) and incubated at 37°C for 40 minutes, followed by albumin stabilization, as previously reported.

Techniques: Immunofluorescence, Mouse Assay

Journal: International Journal of Nanomedicine

Article Title: Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice

doi: 10.2147/IJN.S168556

Figure Lengend Snippet: p5RHH-JNK2 siRNA NPs selectively inhibit JNK2 expression at both mRNA and protein levels in aorta from ApoE −/− mice on a Western diet. Notes: ( A ) RT 2 -PCR results demonstrate the JNK2-mRNA knockdown by p5RHH-JNK2 siRNA NPs (n=7) compared to HBSS control (n=6, P =0.044) and p5RHH-scrambled siRNA NPs (n=6, P =0.027; one-way ANOVA followed with Scheffé post hoc test). ( B ) JNK1 mRNA expression is not affected by p5RHH-JNK2 siRNA NP treatment (n=6) compared to HBSS control (n=6). ( C and D ) Western blot results illustrate JNK2 protein knockdown by p5RHH-JNK2 siRNA NPs (n=5) compared to HBSS control (n=6, P =0.042; unpaired two-sided Student’s t -test). GAPDH was used as internal control. Data presented in dot plots with means ± SE. * P

Article Snippet: For in vitro applications, p5RHH-siRNA nanoparticles were formulated by mixing p5RHH and siRNA in Hanks’ Balanced Salt solution (HBSS) (14025-092; Thermo Fisher Scientific) and incubated at 37°C for 40 minutes, followed by albumin stabilization, as previously reported.

Techniques: Expressing, Mouse Assay, Western Blot, Polymerase Chain Reaction

Journal: Blood Advances

Article Title: Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia

doi: 10.1182/bloodadvances.2016000877

Figure Lengend Snippet: DLS analysis of PF4/UFH and PF4/polyP 130 complexes . (A) PF4/UFH complexes. PF4 (50 µg/mL) was incubated with a range of concentrations of UFH (0.1 U/mL, 1 U/mL, and 10 U/m) in HBSS for 30 minutes at RT to create theoretically net positively charged, neutral, and negatively charged complexes. The resultant complexes were analyzed for size and distribution by DLS. Complexes formed with 10 U/m heparin were too unstable for analysis. The data shown here and all subsequent figures are representative of 2 to 3 independent experiments. (B) PF4/polyP 130 complexes. The same experiment was performed with PF4 incubated with 2 µM, 20 µM, and 200 µM polyP 130 , respectively. (C) Intrinsic stability of PF4/UFH and PF4/polyP 130 complexes. PF4 (5 µg/mL) was incubated with optimal antigenic concentrations of UFH (0.1 U/mL) or polyP 130 (3 µM) was incubated for 30 minutes at RT and the complexes were analyzed using DLS analysis 30 minutes or 96 hours later. Essentially identical protection was seen at 120 hours. (D) Binding of KKO to PF4/polyP 130 complexes. Complexes composed of PF4 (5 µg/mL) and polyP 130 (3 µM) were incubated for 1 hour with buffer alone or with KKO (1.6-16 µg/mL at RT before DLS analysis). (E) Effect of KKO on the intrinsic stability of PF4/polyP 130 complexes. Complexes were formed between PF4 (5 µg/mL) and polyP 130 (3 µM) as in panel D; DLS analysis was performed 1 and 120 hours later. (F) Effect of KKO on the susceptibility of PF4/polyP 130 complexes to phosphatases. Complexes were formed between PF4 (5 µg/mL) and polyP 130 (3 µM) as described in panel D. The complexes were then incubated with KKO (5 µg) or buffer for 30 minutes at RT; CIP (200 units) or buffer was added for 1 hour and the DLS analysis was repeated. d, diameter.

Article Snippet: The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO).

Techniques: Incubation, Binding Assay

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: Effects of bortezomib on OATP1B3-mediated transport. (A) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. control (CTL) in HEK293-OATP1B3 cells without any pre-incubation (Co-incubation). (B) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in HEK293-OATP1B3 cells pretreated with bortezomib (Btz) vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in human SCH pretreated with bortezomib (Btz) (50 and 250 nM, 7 h) vs. vehicle CTL. After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. Model-estimated fold change and associated SE of OATP1B3-mediated [ 3 H]pitavastatin (1 μM, 1 min) (D) and [ 3 H]E 2 17βG accumulation (1 μM, 2 min) (E) in bortezomib pretreatment vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). In D and E, HEK293-OATP1B3 and HEK293-Mock cells were pretreated with vehicle control (CTL) or bortezomib at the indi cated concentrations and time. After washing with the HBSS buffer, OATP1B3-mediated [ 3 H]pitavastatin (D) and [ 3 H]E 2 17βG accumulation (E) was determined by subtracting the values determined in the HEK293-Mock cells from those in HEK293-OATP1B3 cells. (F) Model-estimated fold change and associated SE in [ 3 H]CCK-8 accumulation (1 μM, 3 min) vs. CTL. Cells were pre-incubated with bortezomib-free (CTL) or 50 nM bortezomib-containing media for 2 h. At the end of pre-incubation, the culture medium was removed. After washing, CTL- and bortezomib-pretreated cells were cultured in bortezomib-free medium for the indicated time duration. [ 3 H]CCK-8 (1 μM, 3 min) accumulation was determined at the indicated time points after washing three times ( n = 3 in triplicate). A generalized linear mixed model was fit to the data in A-F as described in the “Materials and Methods” (n = 3 for A, D-F; n = 6 for B; n = 5 for C; all experiments were performed in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p

Article Snippet: Materials Unlabeled cholecystokinin-8 (CCK-8), estradiol 17β-D-glucuronide (E2 17βG), IGEPAL (NP-40), Hanks' Balanced Salt Solution (HBSS), dexamethasone, dimethyl sulfoxide (DMSO), Triton X-100, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), N-ethylmaleimide, trypsin-EDTA solution, antibiotic antimycotic solution, Dulbecco's Phosphate-Buffered Saline (DPBS), bromosulfophthalein (BSP), bovine serum albumin (BSA), and epoxomicin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: CCK-8 Assay, CTL Assay, Incubation, Concentration Assay, Cell Culture

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: Effects of bortezomib on OATP1B1-mediated [ 3 H]pitavastatin and [ 3 H]E 2 17βG transport in HEK293-OATP1B1 cells and on [ 3 H]pitavastatin accumulation in human SCH. HEK293-OATP1B1 cells were seeded at a density of 1.2 x 10 5 cells/well in a 24-well plate and were cultured to confluence. Human SCH were cultured as described in the “Materials and Methods”. (A) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle control (CTL) in HEK293-OATP1B1 cells (Co-incubation). (B) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) vs. vehicle control (CTL) at each indicated time and concentration (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]pitavastatin accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle CTL in HEK293-OATP1B1 cells (Co-incubation). (D) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation (1 μM, 2 min) vs. vehicle CTL treatment in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) for the indicated times and at the indicated concentrations (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]E 2 17βG accumulation was determined in the absence of bortezomib. (E) Model-estimated fold change and associated SE of [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in human SCH pre-incubated with bortezomib (50 nM for 7 h) or co-incubated with positive control bromosulfophthalein (BSP) (100 μM) vs. vehicle CTL. Fold changes and SEs were estimated by linear mixed effects models, as described in the “Data Analysis” section (n = 3 hepatocyte donors in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p

Article Snippet: Materials Unlabeled cholecystokinin-8 (CCK-8), estradiol 17β-D-glucuronide (E2 17βG), IGEPAL (NP-40), Hanks' Balanced Salt Solution (HBSS), dexamethasone, dimethyl sulfoxide (DMSO), Triton X-100, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), N-ethylmaleimide, trypsin-EDTA solution, antibiotic antimycotic solution, Dulbecco's Phosphate-Buffered Saline (DPBS), bromosulfophthalein (BSP), bovine serum albumin (BSA), and epoxomicin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, CTL Assay, Incubation, Concentration Assay, Positive Control

Journal: Autophagy

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

doi: 10.1080/15548627.2015.1124223

Figure Lengend Snippet: The LRS of SQSTM1 and TBC1D25 is essential for phagophore localization, but not sufficient for their degradation. (A) Schematic representation of the LRS-swapping mutants of TBC1D25 and SQSTM1, named TBC1D25-(SQT-LRS) and SQSTM1-(TBC-LRS), respectively. The asterisks indicate the residues that are highly conserved in many LRSs. The WA mutant contains a Trp-to-Ala (WA) mutation (shaded background), which disrupts binding activity to LC3 homologs. (B) Differing LC3 homolog binding activity of TBC1D25, TBC1D25-W136A (designated as WA), and TBC1D25-(SQT-LRS) (designated as SQT) as revealed by GST affinity isolation assays. COS-7 cell lysates expressing EGFP-TBC1D25, EGFP-TBC1D25-WA, or EGFP-TBC1D25-(SQT-LRS) were incubated with GST-LC3, GST-GABARAP, or GST-GABARAPL2 that had been coupled with glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with HRP-conjugated anti-GFP antibody (top panel) and HRP-conjugated anti-GST antibody (bottom panel). The asterisks indicate the nonspecific bands of the anti-GFP antibody. (C) sqstm1 -KO MEF cells stably expressing FLAG-SQSTM1, FLAG-SQSTM1-WA, FLAG-SQSTM1-(TBC-LRS) (FLAG-SQSTM1; left panels), FLAG-TBC1D25, FLAG-TBC1D25-WA, or FLAG-TBC1D25-(SQT-LRS) (FLAG-TBC1D25; right panels) were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 20 μM BafA1. Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panels) and anti-ACTB antibody (bottom panels). The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Article Snippet: To achieve starvation, MEF cells were washed once with HBSS (Sigma-Aldrich, H9269) and transferred to HBSS.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Isolation, Expressing, Incubation, Stable Transfection, Cell Culture, FLAG-tag

Journal: Autophagy

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

doi: 10.1080/15548627.2015.1124223

Figure Lengend Snippet: Colocalization of TBC1D25-chimera mutants with LC3. sqstm1 -KO MEF cells stably expressing EGFP-TBC1D25ΔN, EGFP-TBC1D25ΔN-WA, EGFP-PB1-TBC1D25ΔN, EGFP-PB1-TBC1D25ΔN-WA, EGFP-TBC1D25ΔN-UBA, EGFP-TBC1D25ΔN-WA-UBA, EGFP-PB1-TBC1D25ΔN-UBA, or EGFP-PB1-TBC1D25ΔN-WA-UBA were cultured for 1 h in DMEM (nutrient-rich; far left column) or HBSS (starvation; right 3 columns). Note that cytoplasmic dots were often observed in the cells expressing an TBC1D25 mutant with the LRS that contained a PB1 domain, i.e., EGFP-PB1-TBC1D25ΔN and EGFP-PB1-TBC1D25ΔN-UBA, even under nutrient-rich conditions, whereas no dots were observed with other TBC1D25 mutants under nutrient-rich conditions. Scale bars: 10 μm.

Article Snippet: To achieve starvation, MEF cells were washed once with HBSS (Sigma-Aldrich, H9269) and transferred to HBSS.

Techniques: Stable Transfection, Expressing, Cell Culture, Mutagenesis

Journal: Autophagy

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

doi: 10.1080/15548627.2015.1124223

Figure Lengend Snippet: TBC1D25 is not a substrate of starvation-induced autophagy. (A) Domain organization of TBC1D25 and sequestosome-1 (SQSTM1) and sequence alignment of the PB1-like domain of TBC1D25 and PB1 domain of SQSTM1. The N-terminal domains of TBC1D25 and SQSTM1 were aligned by using the HHpred software program (see also Fig. S2). Amino acid residues in the sequences that are identical and similar are shown against a black background and a shaded background, respectively. (B) MEF cells stably expressing EGFP-TBC1D25 were cultured for 1 h under nutrient-rich conditions (far left column) or starved conditions (right 3 columns). Note that EGFP-TBC1D25 and endogenous SQSTM1 colocalized well with LC3 dots under starved conditions. Scale bars: 10 μm. (C) The TBC1D25 protein level in both control MEF cells and sqstm1 -KO MEF cells was unaltered by starvation. Control MEF cells and sqstm1 -KO MEF cells were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 100 μM E64d and 100 μg/ml pepstatin A, and their cell lysates were analyzed by immunoblotting with anti-TBC1D25 antibody (top panel), anti-SQSTM1 antibody (middle panel), and anti-ACTB antibody (bottom panel). Note that SQSTM1, but not TBC1D25, was degraded by starvation. The asterisk indicates the nonspecific band of the anti-SQSTM1 antibody. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Article Snippet: To achieve starvation, MEF cells were washed once with HBSS (Sigma-Aldrich, H9269) and transferred to HBSS.

Techniques: Sequencing, Software, Stable Transfection, Expressing, Cell Culture

Journal: Autophagy

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

doi: 10.1080/15548627.2015.1124223

Figure Lengend Snippet: Colocalization of FM-fusion proteins of TBC1D25ΔN mutants with LC3. sqstm1 -KO MEF cells stably expressing EGFP-tagged SQSTM1, FM1-TBC1D25ΔN, FM2-TBC1D25ΔN, or FM4-TBC1D25ΔN were cultured for 1 h in HBSS (starved conditions) with (monomer conditions) or without 1 μM D/D solubilizer (oligomerization conditions). Note that all of the FM-fusion proteins colocalized well with LC3 dots under starved conditions irrespective of the presence of the D/D solubilizer. Scale bars: 10 μm.

Article Snippet: To achieve starvation, MEF cells were washed once with HBSS (Sigma-Aldrich, H9269) and transferred to HBSS.

Techniques: Stable Transfection, Expressing, Cell Culture

Journal: Autophagy

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

doi: 10.1080/15548627.2015.1124223

Figure Lengend Snippet: The PB1 domain of SQSTM1 is necessary for starvation-induced degradation of TBC1D25-chimera mutants. (A) sqstm1 -KO MEF cells stably expressing the indicated FLAG-tagged SQSTM1 (wild-type and WA) and TBC1D25 chimera mutants were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation). Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panel) and anti-ACTB antibody (bottom panel). The levels of PB1-TBC1D25ΔN expression (lanes 9 and 10) and PB1-TBC1D25ΔN-UBA expression (lanes 17 and 18) were much lower than the levels of expression of other TBC1D25 mutants, because both of these proteins were degraded by basal autophagy even under nutrient-rich conditions (see Fig. S3B). (B) Quantification of the band intensity of FLAG-tagged proteins shown in the top panel in A . The bars represent the means and SE of data from 3 independent experiments. **, P

Article Snippet: To achieve starvation, MEF cells were washed once with HBSS (Sigma-Aldrich, H9269) and transferred to HBSS.

Techniques: Stable Transfection, Expressing, Cell Culture, FLAG-tag

Journal: Autophagy

Article Title: The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

doi: 10.1080/15548627.2016.1140293

Figure Lengend Snippet: ULK-mediated phosphorylation of ATG14 Ser29 is upregulated under autophagy-inducing conditions. (A) ATG14 Ser29 phosphorylation is induced by nutrient starvation, rapamycin and Torin1. HEK293T cells were incubated in HBSS medium deprived of amino acids or RPMI medium deprived of glucose, or treated with rapamycin (100 nM) or Torin1 (250 nM) in full DMEM medium during the indicated periods of time. The phosphorylation state at Ser29 and the total amount of endogenous ATG14 in cell lysate were analyzed by WB. (B, C) Starvation-induced phosphorylation of ATG14 Ser29 depends on ULK1 and ULK2. MEFs (B) and HCT116 cells (C) with the indicated gene deficiency were incubated in either DMEM (full) or EBSS (starv.) for 30 min. (D) Starvation-induced phosphorylation of ATG14 Ser29 depends on MTORC1. HEK293T cells were stably transduced by the indicated shRNA. The phosphorylation states of ATG14 Ser29 and RPS6KB1 Thr398 and the amounts of the indicated proteins were analyzed by WB. (E) MTORC1-dependent phosphorylation of ATG14 have an effect on ATG14 Ser29 phosphorylation. ATG14 KO HCT116 cells reconstituted with MYC-tagged WT ATG14 or the ATG14[4SA,TA] mutant were incubated with either DMEM or EBSS for 30 min. (F) Quantitative analysis of the effect of the ATG14[4SA,TA] mutation on ATG14 Ser29 phosphorylation. Values are relative to the phosphorylation of WT ATG14 at starvation. Data are represented as mean ± SEM (*, P

Article Snippet: For nutrient starvation, we used either HBSS (Life Technologies, 24020117) or EBSS (Sigma, E2888) supplemented with dialzed fetal bovine serum (Life Technologies, 26400044).

Techniques: Incubation, Western Blot, Stable Transfection, shRNA, Mutagenesis

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Amyloidogenic V122I but not non-amyloidogenic T119M TTR induces the generation of superoxide species and caspase 3/7 activation in human cardiomyocytes ( a ) AC16 cells were treated with V122I TTR, T119M TTR, Antimycin A (positive control) or HBSS (vehicle control) for 24 h. The insults were then removed and the cells loaded with the superoxide-specific reagent DHE as detailed in the experimental section. Red fluorescence produced by DHE oxidation is shown. ( b ) AC16 cells were treated with V122I TTR, T119M TTR or, HBSS for 6 h at 37°C. Cell lysates were prepared as detailed in the experimental section and were added to Ac-DEVD-AFC caspase 3/7 substrate. Fluorescence intensity generated by cleavage of the substrate was measured. Asterisks in both panels indicate statistical significance with P

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Activation Assay, Positive Control, Fluorescence, Produced, Generated

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Native V122I TTR competes with f-V122I TTR for the same interaction sites in human cardiomyocytes ( a ) AC16 cells were co-incubated with f-V122I TTR (320 nM) and increasing concentrations of amyloidogenic V122I TTR or non-amyloidogenic T119M TTR for 3 h at 4°C. The fluorescence associated with the cells was measured and normalized for total protein content. The data were normalized by the fluorescence from the samples incubated with f-V122I TTR only (100%). Filled triangles, V122I TTR; open circles, T119M TTR. ( b ) AC16 cells were co-incubated with f-V122I (600 nM) and equimolar amounts of unlabelled V122I TTR, T119M TTR, V122I stabilized with a small molecule (V122I-SM) or HBSS. Fluorescence associated with the cells was measured, normalized by total protein content and plotted as percentage with respect to the fluorescence of cells that had been incubated with f-V122I and HBSS only. The data show that similarly to T119M TTR, V122I-SM is unable to efficiently displace cell-associated f-V122I, whereas under the same conditions V122I TTR displaces more than 50% of f-V122I TTR.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, Fluorescence

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Differential interaction of the amyloidogenic V122I TTR and the non-amyloidogenic T119M TTR with the human cardiac cell line AC16 AC16 cells were treated with trypsin (or HBSS as control) to remove membrane-associated proteins. V122I and T119M TTR were then incubated with the cells for 4 h at 4°C or 37°C. Cell lysates were prepared and analysed by SDS—PAGE (15% gel) and Western blot using an anti-TTR and anti-actin antibodies. The lower panel shows densitometry analysis (absorbance) of total cell-associated TTR normalized to actin. Error bars represent S.D. of the biological duplicates. The percentage of TTR/actin ratio in cells pre-treated with trypsin (trypsin +) with respect to control cells (trypsin −) is shown.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, SDS Page, Western Blot

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

Article Title: Mechanisms of acute toxicity in NKG2D Chimeric Antigen Receptor T cell treated mice

doi: 10.4049/jimmunol.1600769

Figure Lengend Snippet: Determination of maximum tolerated dose (MTD) for NKG2D CAR T cells (a) overview of approach to determine MTD for single and multi-dose treatment with murine NKG2D CAR T cells. (b) The health score for mice injected with 2 x 10 7 wild type NKG2D T cells (WT) or NKG2D CAR T cells (CH) in non-tumor bearing (upper) and tumor-bearing (lower) mice is shown. Each symbol represents an individual mouse at the time of euthanasia. (c–j) The percent change in initial weight in the days following injection of NKG2D CAR T cells, wild type NKG2D T cells, or HBSS is plotted for 3 to 28 d after T cell injection. Average values for each group are shown (n=10/group). Mice were treated with a single dose of 5 x10 6 T cells (c, d), 10 7 T cells (e, f), or 2 x 10 7 T cells (g, h). Mice were RMA tumor bearing (c, e, g) or non-tumor bearing (d, f, h). (i) RMA tumor-bearing mice (RMA cells i.v. day -5) were injected with 10 7 T cells or HBSS on days 0 and 7. (j) Non-tumor bearing mice were injected with 10 7 T cells or HBSS on days 0, 7 and 14. Data are from two independent experiments (n=10/group). A Student’s t-test was used to compare differences between groups using a two-tailed test assuming unequal variance. An * indicates significant differences compared to WT; * p

Article Snippet: RMA-RG tumor cells (105 cells) in 0.4 mL HBSS (Hyclone) were injected i.v. into C57BL/6 (B6) mice on day 0.

Techniques: Mouse Assay, Injection, Two Tailed Test

Journal: Neurogenetics

Article Title: Loss of mitochondrial ClpP, Lonp1, and Tfam triggers transcriptional induction of Rnf213, a susceptibility factor for moyamoya disease

doi: 10.1007/s10048-020-00609-2

Figure Lengend Snippet: RT-qPCR analyses of wild-type MEF, human neuroblastoma SH-SY5Y cells, and human umbilical vein endothelial cells (HUVEC) for the expression of Rnf213 after exposure to different stress situations. Rnf213 transcript in a MEF, b SH-SY5Y, and c HUVEC cells after serum starvation (DMEM, Dulbecco’s modified Eagle medium; FCS, fetal calf serum; HBSS, Hank’s balanced salt solution; CTRL, untreated control) for indicated times. Rnf213 transcript in d MEF, e SH-SY5Y, and f HUVEC cells is quantified after application of the pathogenic dsRNA analog Poly(I:C) for 16. Rnf213 transcript in g MEF, h SH-SY5Y, and i HUVEC cells is quantified after incubation with the bacterial cell wall component Lipopolysaccharide (LPS) for 24 h. R nf213 transcript in j MEF, k SH-SY5Y, and l HUVEC cells is quantified after incubation with murine or human interferon gamma (IFNG). The Y -axis of each plot shows the ratio of a transcript of interest versus mouse Tbp or human HPRT1 as loading control. The bar graphs show mean and standard error of the mean (SEM), illustrating the significances with asterisks (Trend T 0.05

Article Snippet: Starvation time course of SH-SH5Y cells, HUVEC and MEFCells were grown in 6-well plates and medium was switched to HBSS medium (Gibco) without FCS, to subject them to starvation conditions and cells were collected at indicated time points and stored for RNA extraction.

Techniques: Quantitative RT-PCR, Expressing, Modification, Incubation

Journal: PLoS ONE

Article Title: Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer

doi: 10.1371/journal.pone.0157762

Figure Lengend Snippet: In Vivo Tumor Growth Inhibition. Male nude, athymic mice, 4 weeks of age, were subcutaneously inoculated with BxPC-3 cells (2x10 6 ) embedded in 50:50 HBSS:Matrigel. When tumors were palpable (~1 week), mice were tagged and randomly separated into 3 groups. Mice in group 1 (n = 6) received PBS (vehicle). Group 2 (n = 6) received free paclitaxel (6.7uM) in PBS, while group 3 (n = 5) received equimolar amounts of paclitaxel conjugated to antibody (fluorescent ADC, 100ug α-CEA-680-PTX, 6.7uM effective paclitaxel). Each mouse received a total of five doses of the respective treatment, spaced 3 days apart, via retro-orbital injection. Arrows indicate treatment days (days 3, 6, 9, 12, and 15). Tumor size was measured every 3 days using digital calipers, and tumor volume was calculated with the modified ellipsoid formula (v = ½(length × width 2 )). Each data point represents average tumor volume (n = 5–6 mice). and error bars denote standard error.

Article Snippet: Tumor induction After a one week acclimatization period, mice were subcutaneously inoculated with 2x106 BxPC-3 cells embedded in 100uL of 50:50 HBSS:Matrigel (Corning Incorporated, #354262, Corning NY).

Techniques: In Vivo, Inhibition, Mouse Assay, Injection, Modification