Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 silencing inhibits TGFβ1-induced differentiation of fibroblasts in vitro . (A) Quantitative RT-PCR and western blot analysis of α-SMA, fibronectin, and collagen I in HFL1 cells transfected with scrambled shRNA or LTBP2 shRNA followed by PBS or TGF-β1 stimulation. GAPDH was detected as the internal control. ** p < 0.01 vs. scr shRNA, # p < 0.05, ## p < 0.01 vs. scr shRNA + TGFβ1 (B) Representative immunofluorescence staining of α-SMA (red) in HFL1 cells. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. (C) Representative immunofluorescence staining of collagen I (red) in HFL1 cells. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Control, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 silencing suppresses the activation of NF-κB signaling induced by TGFβ1. (A) Representative immunofluorescence staining of phospho-NF-κB p65 (ser536) (red) in HFL1 cells transfected with scrambled shRNA or LTBP2 shRNA followed by PBS or TGF-β1 stimulation. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. (B) Western blot analysis of nuclear NF-κB p65 and nuclear phospho-NF-κB p65 (ser536) in HFL1 cells. Lamin B was detected as the internal control. ** p < 0.01 vs. scr shRNA, # p < 0.05, ## p < 0.01 vs. scr shRNA + TGFβ1. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: Activation Assay, Immunofluorescence, Staining, Transfection, shRNA, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 overexpression-induced lung fibroblast-to-myofibroblast differentiation depends on NF-κB phosphorylation and nuclear translocation in vitro . Western blot analysis of α-SMA, fibronectin, and collagen I in HFL1 cells transfected with LTBP2 cDNA and/or BAY 11-7082 or vector. GAPDH was detected as the internal control. ## p < 0.01 vs. vector. * p < 0.05, ** p < 0.01 vs. OE-LTBP2. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: Over Expression, Phospho-proteomics, Translocation Assay, In Vitro, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Stem Cell Research & Therapy

Article Title: Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy

doi: 10.1186/s13287-016-0463-4

Figure Lengend Snippet: Human umbilical cord-derived mesenchymal stem cell exosomes ( hucMSC-Ex ) prevent deterioration of renal function in vivo. a Representative images of renal histology (200×, scale bar = 50 μm). b The histomorphological score. c Immunohistochemical analysis of PCNA expression in kidney tissues (200×, scale bar = 50 μm). d Percentage of proliferating cell nuclear antigen ( PCNA )-positive cells. e TUNEL assay for apoptotic cells in rat renal tissues (200×, scale bar = 50 μm). f Percentage of apoptotic cells. Serum creatinine ( Cr ) ( g ) and blood urea nitrogen ( BUN ) ( h ) levels were measured at different times after cisplatin injection. Data are expressed as mean ± SD. * P < 0.05, n = 6. Rats treated as mentioned for the in vivo rat model were divided into four groups as follows: 1) control; 2) phosphate-buffered saline ( PBS ); 3) hucMSC-Ex; and 4) human fetal lung fibroblast-1exosomes ( HFL1-Ex ). All animals were sacrificed at 3 days after cisplatin injection

Article Snippet: Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect the apoptotic cells. hucMSCs, HFL1 cells, NRK-52E cells, and the kidney tissues were examined using the TdT-FragEL DNA fragmentation detection kit according to the manufacturer's protocol (Boster, China).

Techniques: Derivative Assay, In Vivo, Immunohistochemical staining, Expressing, TUNEL Assay, Injection, Control, Saline

Journal: Stem Cell Research & Therapy

Article Title: Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy

doi: 10.1186/s13287-016-0463-4

Figure Lengend Snippet: Human umbilical cord-derived mesenchymal stem cell exosomes ( hucMSC-Ex ) inhibit cisplatin-induced apoptosis and secretion of inflammatory cytokines in NRK-52E cells in vitro. a NRK-52E cells were stained with JC-1 dye and observed under fluorescent microscopy (JC-1 produces red fluorescence within the mitochondria as JC-1-aggregates while it emits green fluorescence when it leaks into the cytoplasm as JC-1-monomers; the shift between green and red is proportional to the mitochondrial membrane potential change) (200×, scale bar = 50 μm). b TUNEL assay was performed to detect the apoptotic cells (200×, scale bar = 50 μm). c Percentage of apoptotic cells. ** P < 0.01, n = 3. d The expression of proliferating cell nuclear antigen ( PCNA ), BCL-XL, BCL-2, Bax, Cytochrome-c, cleaved Caspase-3, and GAPDH proteins was evaluated by Western blot. e Immunohistochemical analysis of tumor necrosis factor alpha ( TNF-α ) expression (200×, scale bar = 50 μm). f The expression of interleukin 1-β ( IL1-β ) and GAPDH proteins was evaluated by Western blot. NRK-52E cells treated as mentioned for the in vitro experiment were divided into four groups: 1) control; 2) phosphate-buffered saline ( PBS ); 3) hucMSC-Ex; and 4) human fetal lung fibroblast-1exosomes ( HFL1-Ex )

Article Snippet: Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect the apoptotic cells. hucMSCs, HFL1 cells, NRK-52E cells, and the kidney tissues were examined using the TdT-FragEL DNA fragmentation detection kit according to the manufacturer's protocol (Boster, China).

Techniques: Derivative Assay, In Vitro, Staining, Microscopy, Fluorescence, Membrane, TUNEL Assay, Expressing, Western Blot, Immunohistochemical staining, Control, Saline

Journal: Stem Cell Research & Therapy

Article Title: Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy

doi: 10.1186/s13287-016-0463-4

Figure Lengend Snippet: Human umbilical cord-derived mesenchymal stem cell exosomes ( hucMSC-Ex ) prevent apoptosis and secretion of inflammatory cytokines by activating autophagy in vivo. a Representative images of renal histology (200×, scale bar = 50 μm). b The histomorphological score. c Immunohistochemical analysis of PCNA expression in kidney tissues (200×, scale bar = 50 μm). d Percentage of proliferating cell nuclear antigen ( PCNA )-positive cells. e TUNEL assay for apoptotic cells in rat renal tissues (200×, scale bar = 50 μm). f Percentage of apoptotic cells. g The expression of LC3B, BCL-2, Bax, IL1-β, and GAPDH proteins in the cisplatin-induced rat model were measured by Western blot. The serum inflammatory cytokines ( h ) tumor necrosis factor alpha ( TNF-α ), ( i ) interleukin-1β ( IL1-β ), and ( j ) interleukin-6 ( IL6 ) were measured via Luminex assay. Data are expressed as mean ± SD. * P < 0.05, n = 6. Rats were divided into six groups as follows: 1) control; 2) phosphate-buffered saline ( PBS ); 3) hucMSC-Ex; 4) hucMSC-Ex + 3-methyladenine ( 3MA ); 5) 3MA; and 6) rapamycin ( Rapa )

Article Snippet: Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect the apoptotic cells. hucMSCs, HFL1 cells, NRK-52E cells, and the kidney tissues were examined using the TdT-FragEL DNA fragmentation detection kit according to the manufacturer's protocol (Boster, China).

Techniques: Derivative Assay, In Vivo, Immunohistochemical staining, Expressing, TUNEL Assay, Western Blot, Luminex, Control, Saline

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Surface plasmon resonance analyses of tripartite complexes. A triply diluted concentration series (4,050 to 1.8 nM) of (A) WT or (B) K173A Sbi-III-IV were co-injected with plasma purified FH, recombinant FHR-1, FHR-2, FHR-5, or FH 19−20 . The red response curves were indicative of binding experiment in the absence of Sbi. The co-injection experiments of a fixed analyte concentration in combination with increasing Sbi concentration were depicted by increasingly dark lines. (C) Relative changes of Sbi-III-IV mediated FH (or FHR) binding to C3b. By subtracting the co-injection sensorgram (i.e., Sbi+FH) with the corresponding Sbi binding dataset (Figure ), the changes in FH (or FHRs) binding was deduced (Figure ). Changes in FH (or FHRs) binding were expressed as the relative change, derived from dividing the Sbi mediated binding by the FH (or FHR) only control, using the response-difference values at the equilibrated binding point (173.5 s ). Each sensorgram is representative of two experiments. Relative change curves were fitted using non-linear variable slope (four parameters) function in GraphPad Prism.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: SPR Assay, Concentration Assay, Injection, Purification, Recombinant, Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Functional characterization of tripartite complexes in complement AP regulation. NHS was incubated with Sbi-III-IV, in combination with specified reagents or just buffer, the consumption of AP activity was indicated by the protection of rabbit red blood cell from lysis. (A) Pre-incubation of recombinant FHR-1 or−2 with the presence or absence of Sbi-III-IV. (B) Pre-incubation of recombinant FHR-5 in the presence or absence of Sbi-III-IV. (C) Pre-incubation of recombinant FH 19−20 or FHR-1 1−2 in the presence or absence of Sbi-III-IV. Using an ELISA assay, the ability of FHR-1,−2,−5, FH 19−20 , or FHR-1 1−2 to modulate FH binding to a C3b coated surface was studied in the absence (D) or presence of WT Sbi-III-IV (E) , or K173A Sbi-III-IV (F) . C3 convertase formation in the absence (G) or presence of Sbi-III-IV (H) was assessed by flowing factor B (500 nM) and factor D (100 nM) in the presence of FH (2,000 nM) or FH +FHR-1 (2,000 and 200 nM) or FH+FHR-1&-5 (2,000, 200 and 20 nM) across a surface amine coupled with 500 RU C3b. To form Sbi bound C3 convertase, experiments were conducted in addition of 2,000 nM of Sbi-III-IV. Detailed experimental and data processing procedures are provided in Materials and Methods and Figure . (I) Percentage of intact C3b derived from continuous recording of ANS fluorescence changes between 465 and 475 nm spectrum. Baseline C3b breakdown curve (−) was recorded in the presence of FH and FI, interference caused by the addition of FHR-5 (+) or FHR-5 in combination of Sbi (++) was also examined. The data for FHR-1 and FHR-2 are presented in Figure 3F. Normalized data was depicted in solid lines, simulated breakdown curves were shown as dotted-lines. Each curve represents the mean value of three independent experiments. For (A–F) , the mean and standard deviation for each measurement was calculated; For (G–H) , each sensorgram is representative of two experiments. For (I) , simulated breakdown curves were fitted using one phase exponential decay function in GraphPad Prism.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: Functional Assay, Incubation, Activity Assay, Lysis, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Structural analysis of the Sbi-III-IV:C3d:FHR-1 tripartite complex. SAXS solution structure analysis and EOM modeling of the Sbi-III-IV:C3d:FHR-1 tripartite complex: (A) Left panel, fit of the selected ensemble of conformers to the experimental scattering. Radius of gyration (Rg, middle panel), particle maximum dimension (Dmax, right panel), and distribution histograms of the selected conformers vs. the pool. (B) Kratky plot of the tripartite complex. (C) Examples of rigid body models of the selected conformers corresponding to the histogram peaks. The volume fraction of each species is indicated. The relative positions of C3d, Sbi-III-IV, and FHR-1 in the dimeric tripartite complex are indicated, with C3d in red, Sbi-IV in dark blue, Sbi-III in turquoise and FHR-1 in orange. (D) Schematic representation of the dimeric Sbi-III-IV:C3d:FHR-1 tripartite complex. (E) Comparison of the solutions structure of wild-type Sbi-III-IV:C3d and mutated version Sbi-III-IV(K173A):C3d of the dual complex. Radius of gyration (Rg), particle maximum dimension (Dmax), and distribution histograms of the selected conformers vs. the pool are shown in Figure . (F) Ab initio shape reconstruction shown as gray spheres in comparison to the partial crystal structure Sbi-IV:C3d (2wy8). (G) Examples of rigid body models. Complete set of models as well as flexibility assessment is presented in Figure . C3d in shown red, Sbi-IV in dark blue, and Sbi-III in turquoise.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques:

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Sbi-III-IV is an effective adjuvant in mice. (A) Freshly prepared CD21 −/− mouse serum was mixed with Sbi-III-IV-Ag85b or just Sbi-III-IV. The reaction was stopped at various time points (0, 30, 60, 120 min). Western blot was developed with rabbit anti-C3 at 1/1000 and goat anti-rabbit at 1/2000. C3d is shown as confirmation that C3 has been activated and broken down. (N) is Cr2 −/− serum incubated for 120 min with saline. (B) C57Bl/6 mice (groups of 6) where immunized intraperitoneally with either 2.7 μg Sbi-III-IV-Ag85b protein, 2 μg Ag85b, or 0.7 μg Sbi-III-IV plus 2 μg Ag85b in 150 mM NaCl solution, followed by weekly bleed and boosted (day 28) before terminal bleed at day 49. Serum IgG reactivity to Ag85b was measured over time by ELISA. Sera was diluted 1/50 and the mean absorbance ± SEM of each mouse group is shown. All data has been normalized to the day 0 average of all WT mice. (C) The previous experiment was repeated in C57Bl/6 mice deficient of C3 (C3 −/− )and complement receptor type I and 2 ( Cr2 −/− ). Data is representative of at least 2 repeats ( *** P < 0.001, Student's T -test, GraphPad Prism). (D) Schematic representation of the dimeric Sbi-III-IV:C3d:FHR-1 solution structure providing a nidus for AP C3 convertase generation that overwhelms local complement regulators, leading to the opsonisation of the nearby antigen surface by C3 break-down products that help facilitate the co-ligation of the B cell antigen receptor (BCR) with complement receptor 2 (CR2) thereby lowering the threshold for B cell activation.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Ligation, Activation Assay