Journal: Scientific reports

Article Title: Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

doi: 10.1038/srep28538

Figure Lengend Snippet: Figure 1. Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. (a) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O2 for 3 days. Hypoxic condition: 37 °C, 20% O2 for 2 days followed by 33 °C, 2% O2 for 1 day. (b) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. (c) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. (d) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. (e) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. (f) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. (g) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.

Article Snippet: In the neutralizing antibody experiment, the conditioned medium was incubated for 90 min at 4 °C with anti-ATM antibody (38 μ g/mL, ab78, Abcam, Cambridge, UK) as a control antibody, anti-TGF-β 1 antibody (38 μ g/mL, ab64715, Abcam), or anti-PDGF-BB antibody (33 μ g/mL, AF-220-NA, R&D Systems, Inc.) and then added to fibroblasts.

Techniques: Comparison, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 1. A, representative gelatin-based zymograms of the effect of hypoxia (H) on MMP-2 and MMP-9 expression by fibroblasts and VSMCs compared with normoxia (N). Treatment of cell culture medium with 20 mMEDTA(24h)abolishedtheexpressionofpro-MMP-2andpro-MMP-9.Arrowsindicatethemigrationposition of purified pro-MMP-2 and pro-MMP-9. B, densitometric analysis of pro-MMP-2 expression by hypoxia in human primary lung fibroblast cell lines (n 8) and the effect of PDGF-BB and the underlying intracellular signaling inhibition. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase, and SB202474 is a negative control for SB203580. C, densitometric analysis of pro-MMP-2 expression by hypoxia in human primary VSMC lines (n 8) and the effect of PDGF-BB and the underlying intracellular signaling pathways. Values are presented as the percentage of control (normoxia 24 h), and each bar represents the mean S.D. of six independent experiments.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Expressing, Cell Culture, Purification, Inhibition, Negative Control, Protein-Protein interactions, Control

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 2. The effect of hypoxia and/or PDGF-BB on TIMP-1 (A) and TIMP-2 (B) by human lung fibroblasts and VSMCs within 48 h was assessed by ELISA. The role of Erk1/2 and p38 MAP kinase on the stimulatory effect of both TIMPs was also determined using PD98059-inhibiting Erk1/2 MAP kinase, SB203580-inhibiting p38 MAP kinase,andSB202474asanegativecontrolforSB203580.Eachbarrepresentsthemean S.D.ofsixindepend- ent experiments, each in triplicate. The control was defined as normoxia (24 h). C, aliquots of cell culture medium from human lung fibroblasts and VSMCs containing the same amount of protein were incubated in the absence () or in the presence () of 4-aminophenylmercuric acetate (APMA; 1 mM, 37 °C, 24 h) and subjected to gelatin zymography. Arrows indicate the migration position of purified pro-MMP-2 and active MMP-2. N, normoxia; H, hypoxia).

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Incubation, Zymography, Migration, Purification

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 3. A, the effect of hypoxia (black bars) and PDGF-BB (striped bars) on MMP-1 expression by fibroblasts and VSMC within 48 h was assessed by ELISA. Each bar represents the mean S.D. of triplicates in six inde- pendent experiments. B, a representative immunoblot demonstrates the role of Erk1/2 and p38 MAP kinase in thehypoxia-inducedexpressionofMMP-1,MMP-13,andhypoxia-induciblefactor-1(HIF-1)aswellasonthe antagonizing effect of PDGF-BB in human lung fibroblasts. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase and SB202474 is a negative control for SB203580. Ab, neutralizing antibody; control t0, protein extract from untreated cells before stimulation. C, hypoxia- and PDGF-BB-induced MMP-13 expression by fibroblasts but not by VSMC within 48 h. Each bar represents the mean S.D. of triplicate determinations from six independent experiments. Similar results were obtained with two additional cell lines.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control, Control

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 4. A, the effect of hypoxia and PDGF-BB on the release of soluble collagen type I within 48 h was determined by ELISA in the cell culture medium obtained from human lung fibroblasts (n 6). Each bar represents the mean S.D. Experiments in each cell line were performed in triplicates. B, the inducing effect of hypoxia and PDGF-BB on collagen type I 1 chain (COL1A1) was confirmed on the mRNA level in three cell lines and the effect of Erk1/2 and p38 MAP kinase was assessed by reverse transcriptase-PCR. -ac- tin gene expression was used as a housekeeping gene, and data are displayed as a representative PCR product analysis. PD98059 inhibits Erk1/2 MAP kinase,SB203580inhibitsp38MAPkinase,andSB202474isanegativecontrol for SB203580.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Reverse Transcription, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 5. A, hypoxia and/or PDGF-BB increased fibroblast numbers significantly within 72 h. Under hypoxic culture condition, PDGF-BB mediates the mitogenic effect that is transmitted via Erk1/2. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase, and SB202474 is a negative control for SB203580. The general inhibitor of MMP activity cis-9-octadecenoyl-N-hydroxylamide oleoyl-N-hydroxylamide (10 mM, OO-Hy) was added 3 h before cells were exposed to any other stimulus. B, hypoxia (black bar) and PDGF-BB up-regulated VSMC proliferation via Erk1/2 and p38 MAP kinase. Purified human soluble collagen type 1 (sol collagen type I (1 mg/ml)) was used to assess its contribution to hypoxia-mediated proliferation. Each bar represents the mean S.D. of triplicates performed in six independent experiments.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Negative Control, Activity Assay, Purification

Journal: Molecular & cellular proteomics : MCP

Article Title: The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions.

doi: 10.1016/j.mcpro.2023.100514

Figure Lengend Snippet: FIG. 3. Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig). A, violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B, representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C, particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering (red, S-EVs; blue, L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D, representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E, representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.

Article Snippet: The EVs were cytometrically characterized following the International Society of Extracellular Vesicles recommendations (MIFlowCytEV, (39)) to identify their enrichment in proteins belonging to the three categories established by MISEV 2018 guidelines (37): CD63 (AntiCD63-FITC, Clone REA1055, Miltenyi Biotec) as “Category 1” protein (Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes); HSP90β (anti-HSP90β-PE, ADI-SPA844PE-050, Enzo Life Sciences) as “Category 2” protein (Cytosolic proteins recovered in EVs), and albumin (Anti-swine Albumin-FITC, CLFAG16140, Cedarlane) as “Category 3” protein (Major components of non-EVs coisolated structures).

Techniques: Isolation, Clinical Proteomics, Protein Concentration, Transmission Assay, Electron Microscopy, Expressing, Cytometry, Control

Journal: Nature Communications

Article Title: Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence

doi: 10.1038/s41467-024-48443-6

Figure Lengend Snippet: A Immunofluorescence of 53BP1 and γH2AX with in situ hybridization of telomeric DNA (TelC) in wt and Tcea1 –/– MEFs. Arrows denote TIFs on telomeres. The graph depicts the mean percentage of cells presenting ≥5 TIFs (n = 3). B pATM protein levels in wt and Tcea1 -/- MEFs whole-cell extracts (n = 3). The graph depicts the total ATM-normalized protein expression levels (n.e.l.). C TRF1 protein levels in whole-cell extracts from wt and Tcea1 –/– MEFs. Fibrillarin was used to normalize protein expression levels (n.e.l., n = 4). D Trf1 mRNA levels in wt and Tcea1 -/- MEFs (n = 3). E ChIP signals of TRF1 protein (shown as percentage of input after IgG normalization) on telomeres of wt and Tcea1 -/- MEFs (n = 5). F Immunofluorescence of TRF1 with in situ hybridization of telomeric DNA (TelC) in wt and Tcea1 –/– MEFs. The graph depicts the mean fluorescence intensity (MFI) of TelC in Tcea1 –/– MEFs and wt controls (n = 4). G OxiDIP signals of 8-oxoG (shown as percentage of input after IgM normalization) on telomeres, GC-rich and AT-rich regions of untreated wt, H 2 O 2 -treated wt and untreated Tcea1 –/– MEFs (n = 3). H Immunofluorescence against 8-oxoG with in situ hybridization of telomeric DNA (TelC) in wt and Tcea1 –/– MEFs. White arrows indicate 8-oxoG signal on telomeres (n = 4). The graph depicts the 8-oxoG mean fluorescence intensity (MFI) on telomeric DNA (TelC) in Tcea1 –/– and wt MEFs. Data analysis was performed using two-tailed Student’s t test. All data are presented as mean values ± SEM. Unless otherwise indicated, n = biologically independent experiments and scale bars are set at 5μm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against 53BP1 (NB100-304, IF: 1:200), ATM (NB100-220, WB: 1:500) and goat anti-mouse IgM 550 (NB120-9167R, IF: 1:200) were from Novus Biologicals.

Techniques: Immunofluorescence, In Situ Hybridization, Expressing, Fluorescence, Two Tailed Test