Journal: Journal of Veterinary Science

Article Title: In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

doi: 10.4142/jvs.2013.14.2.125

Figure Lengend Snippet: The effect of MEL on IL-10 (A), TGF-β (B), and IFN-γ (C) production. The results are expressed as a percentage of CD25 high CD4+, CD25 low CD4+, and CD25-CD4+ cells expressing IL-10, TGF-β, or IFN-γ. Typical cytograms (D) illustrating IFN-γ expression in particular lymphocyte subpopulations are shown. Results are presented as the mean ± SE (n = 18) of three independent experiments (each one using six animals). * p < 0.01, MEL-treated cells versus the control cells.

Article Snippet: After extracellular staining (as described above) and fixation (200 μL 2% paraformaldehyde in Dulbecco's PBS per sample for 15 min on ice; both reagents were purchased from Sigma-Aldrich, USA), the cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin and 0.05% (w/v) NaN 3 in Hanks' balanced salt solution (HBSS; all from Sigma-Aldrich, USA)] and stained with APC (allophycocyanin) -conjugated mouse anti-TGF-β mAb (1 : 20, 1D11, IgG1, this antibody reacts with human, mouse, and bovine TGF-β1 and TGF-β2; R&D Systems, USA) for 45 min at RT in the dark.

Techniques: Expressing, Control

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Stress‐Induced Reorganization of Proteasomes Through Diffusion and Cytoskeleton‐Dependent Mechanisms

doi: 10.1002/smll.202506260

Figure Lengend Snippet: Proteotoxic stress induces major redistribution of proteasome particles. A) Cartoon representation of CRISPR‐edited cells expressing subunit of the RP (PSMD14, left ) or CP (PSMB2, right ) in tandem with FP at the C‐terminus. B) A typical PSMD14‐eGFP cell reconstructed in 3D from HILO imaging with 100 nm step size z‐stack before ( left ) and 24 hr after ( right ) incubation with aS aggregates (Alexa647 partial labeling). The inset shows a 2D image with a scale bar = 5 µm. C) HILO imaging of individual proteasomal particles in PSMD14‐mEos cells expressing an ER marker, eGFP‐Sec61 (white), at rest versus after 24 hrs incubation with aS‐Alexa647 (fire‐LUT). Cells were imaged using SMLM (see Experimental Section). Reconstructed RPs localized to the nucleus (blue), overlapping with the ER (red), or the cytosol (green) are color‐coded. Scale bars = 1 µm. D) Calculated relative abundance of PSMD14‐mEos found in each subcellular compartment before and after aS aggregate incubation presented in box‐plots ( n = 35 and n = 31 cells, respectively). E) Immunofluorescence labeling and SMLM imaging of a typical HEK293A cell for CP (red) and RP (green) enabled calculation of capped versus uncapped proteasome CP ratios. Scale bars represent 5 µm and 100 nm (inset). Inset with cartoon representations of singly‐ and doubly capped holoenzymes, are shown ( right ). F) Relative ratios of free CP and RP versus singly‐ and doubly capped holoenzymes in HEK293A are shown in pie charts. Mean ± standard deviation from 22 cells are presented to two decimal places. G–H) HEK293A cells were incubated with aS aggregates for 24 hrs ( n = 33 cells) and labeled as in E, for fractional quantification of (G) CP and (H) RP. Box plots show that incubation of aggregates does not induce any significant changes between the relative ratios of free particles and holoenzymes. This is in line with observations made in differentiated SH‐SY5Y neuroblastoma cells (Figure , Supporting Information). Statistical significance was calculated using Student's t ‐test, where no significant difference (n.s.) p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Cell lysates (20 μg) were resolved on gels and transferred to 0.45 μm PVDF membranes (Bio‐Rad) using Trans–Blot semi‐dry Transfer System at 25 V, 2.5 A for 15 min. Membranes were then blocked in 5% BSA in Tris‐Buffered Saline with 0.05% Tween 20 (TBS‐T) for 1 hr at 4 °C, and incubated with primary antibody for PSMD14 (MA5‐35818, Thermo Fisher) or PSMB2 (sc‐58410, Santa Cruz) overnight at 1:1 000 dilution.

Techniques: CRISPR, Expressing, Imaging, Incubation, Labeling, Marker, Immunofluorescence, Standard Deviation

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Stress‐Induced Reorganization of Proteasomes Through Diffusion and Cytoskeleton‐Dependent Mechanisms

doi: 10.1002/smll.202506260

Figure Lengend Snippet: Membrane potential modulates proteasome localization. A) Unpatched and patched PSMD14‐eGFP (top) and PSMB2‐eGFP (bottom) cells are imaged in TIRF mode with snapshots shown from various time points (four panels of t1‐t2 as in B and C), where the normalized fluorescence intensity is colored (see Experimental Section for image processing and bleach correction). Three PSMB2‐eGFP cells are visible in the bottom field of view, with only the cell on the left under patch clamp. The patch clamp position is marked with an “×” (red) on the cell ( P ) in panel t1 . Regions of the patched cells used for intensity plots are marked in panel t1 as dotted lines. Intensity changes in control cells C1 and C2 are shown in C. See also Video (Supporting Information). B) Changes in the normalized fluorescence signal are shown over time for the PSMD14‐eGFP cell in A. Solid black lines represent signals after applying a filter in the frequency dimension to remove modulations smaller than the oscillation in potential. The clamped potentials applied are indicated on the top and oscillated between +100 mV (red) and −100 mV (blue) every 30 s (see also Video , Supporting Information). C) Changes in normalized fluorescence signal over time in PSMB2‐eGFP cells are shown for the patched cell and two control cells shown in A. D) Cells expressing eGFP‐USP21 did not show any change in fluorescence intensity following changes in membrane potential induced by a patch clamp (see also Video , Supporting Information). E) Depolymerization of microtubules and actin filaments affects voltage‐induced proteasome re‐localization. PSMB2‐eGFP cells were treated with 5 µ m Colcemid or Latrunculin A (LatA) for 20 min at 37 °C and washed with cell media before mounted for TIRF imaging. Fluorescence intensity traces of the cells are plotted over time, and the applied potential at each time indicated. PSMB2‐eGFP cells treated with DMSO or ethanol (EtOH) vehicle controls showed similar results (see Video , Supporting Information). F) Repeated measurements of patch clamp applied on PSMB2‐eGFP cells treated with ( from left ) DMSO (mean rate with SD = 0.031 ± 0.009 nFI s −1 , n = 4), EtOH (0.032 ± 0.004 nFI s −1 , n = 4), Colcemid (0.0001 ± 0.0003 nFI s −1 , n = 4), LatA (−0.004 ± 0.006 nFI s −1 , n = 5). PSMD14‐eGFP cells were also treated with Colcemid (−0.0002 ± 0.0002 nFI s −1 , n = 4) and LatA (0.006 ± 0.009 nFI s −1 , n = 8). All images are contrast‐adjusted for optimal representation of the difference in intensity between hyper‐ and depolarized states.

Article Snippet: Cell lysates (20 μg) were resolved on gels and transferred to 0.45 μm PVDF membranes (Bio‐Rad) using Trans–Blot semi‐dry Transfer System at 25 V, 2.5 A for 15 min. Membranes were then blocked in 5% BSA in Tris‐Buffered Saline with 0.05% Tween 20 (TBS‐T) for 1 hr at 4 °C, and incubated with primary antibody for PSMD14 (MA5‐35818, Thermo Fisher) or PSMB2 (sc‐58410, Santa Cruz) overnight at 1:1 000 dilution.

Techniques: Membrane, Fluorescence, Patch Clamp, Control, Expressing, Imaging

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Stress‐Induced Reorganization of Proteasomes Through Diffusion and Cytoskeleton‐Dependent Mechanisms

doi: 10.1002/smll.202506260

Figure Lengend Snippet: Mean‐squared displacement (MSD) analysis of SPT data. Individual tracks recorded from PSMB2‐eGFP or PSMD14‐eGFP molecules were classified as A) free (stochastic) diffusion, B) confined diffusion, or C) active transport. D) The fraction of molecules in each cell that was best described as free diffusion, confined diffusion, or active transport are plotted from resting cells expressing PSMD14‐eGFP ( n = 18 cells and 33 380 tracks), and cells incubated with aS aggregates ( n = 14 cells and 25 679 tracks). E) Similarly, resting cells expressing PSMB2‐eGFP ( n = 11 cells and 35 312 tracks) and after incubation with aS aggregates ( n = 10 cells and 14 158 tracks) were also characterized by MSD analysis. F) Schematic model comparing the global proteasome reorganization of cells at rest, and G) under proteotoxic stress. Here, proteasomes in resting cells are predominantly localized to the nucleus (green shading). Cells under proteotoxic stress re‐balance their proteasome distribution, and can form TAADs (dark green shading). Individual proteasomes are increasingly confined (red tracks) in cells under proteotoxic stress compared to resting cells, while the fraction of proteasomes that can freely diffuse (black tracks) are significantly reduced. Active transport (blue tracks) of RP and CP show slight increases and decreases, respectively, during proteotoxic stress.

Article Snippet: Cell lysates (20 μg) were resolved on gels and transferred to 0.45 μm PVDF membranes (Bio‐Rad) using Trans–Blot semi‐dry Transfer System at 25 V, 2.5 A for 15 min. Membranes were then blocked in 5% BSA in Tris‐Buffered Saline with 0.05% Tween 20 (TBS‐T) for 1 hr at 4 °C, and incubated with primary antibody for PSMD14 (MA5‐35818, Thermo Fisher) or PSMB2 (sc‐58410, Santa Cruz) overnight at 1:1 000 dilution.

Techniques: Diffusion-based Assay, Expressing, Incubation

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: TGF-β levels are elevated in melioidosis patients. (A) On admission, TGF-β levels in plasma of patients with culture-proven melioidosis (n = 33) were elevated compared to healthy controls (n = 30). (B) Plasma levels of TGF-β decreased in melioidosis patients 2 weeks after successful antibiotic treatment (n = 6). *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Increased expression of TGF-β in lung tissue of wild-type mice infected with B. pseudomallei. Elevated pulmonary TGF-β levels were observed in mice (n = 8) 72 h after intranasal infection with 7.5 × 102 CFU of B. pseudomallei compared to 24 h after infection. ***, P < 0.001.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Decreased expression of phosphorylated Smad2 in mice receiving anti-TGF-β treatment. TGF-β signaling was evaluated by quantifying the total levels of Smad2 and Smad3 and their phosphorylation in lung tissue by Western blot 48 h after intranasal infection with B. pseudomallei. The left 4 lanes show the expression of Smad2 (A) and Smad3 (B) in lung tissue of control mice and the right 4 lanes of mice that received anti-TGF-β treatment (2 × 200 μg TGFβ antibody). The bar graph shows the ratio of expressed phosphorylated Smad/total Smad corrected for actin loading levels. (C) A significant decrease in p-Smad2 is demonstrated in mice that were treated with anti-TGF-β antibody. *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Western Blot, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Cytokine profile during experimental melioidosis in mice treated with control antibody or anti-TGF-β a

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Less organ damage in mice receiving anti-TGF-β treatment. Levels of (A) BUN, (B) creatinine, (C) AST, and (D) ALT were measured in B. pseudomallei-infected mice (n = 8/group) that were treated with anti-TGF-β antibody and their controls at 72 h as parameters for organ damage. Expression of AST and BUN were decreased, demonstrating less organ damage. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection, Expressing

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Anti-TGF-β treatment results in diminished bacterial growth during experimental melioidosis. Mice (n = 8/group) were treated with anti-TGF-β antibody or control IgG antibody and inoculated with 7.5 × 102 CFU of B. pseudomallei intranasally. Bacterial numbers were quantified 72 h postinfection. Less growth was seen in the lungs (A) and spleens (B) of mice treated with anti-TGF-β antibody. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: No influence on survival of anti-TGF-β treatment during murine melioidosis. Mice (n = 12/group) received either 2× 200 μg control antibody (black squares) or 2× 200 μg anti-TGF-β antibody (white squares) and were infected with 7.5 × 102 CFU, a lethal dose, of B. pseudomallei intranasally, after which they were followed until death. Mortality was assessed every 4 h. No difference in survival was seen.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection