Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 1. Smad protein phosphorylation by TGF-1 stimulation in LX2, PSC, and MEF cells. LX2, PSC, and MEF cells were serum-starved overnight, then treated with different concentrations of TGF-1 for 45 min. Antibodies against p-Smad1/5 (Ser-463/465), p-Smad2 (Ser-465/467), and p-Smad3 (Ser-423/425) were used. Both Smad1/5 and Smad2/3 were phosphorylated by TGF-1 stimulation in all of the cells. An asterisk (*) indicates the nonspecific band.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 2. The effects of eliminating or overexpressing NRP-1 on Smad protein phosphorylation and the Smad target gene expression. A, eliminating NRP-1 up-regulated Smad1/5 phosphorylation and down-regulated Smad2/3 phosphorylation. NRP-1 was knocked down by siRNA in LX2 and PSC. For MEF cells, wt MEF, and NRP-1/ MEF cells were used. All the cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. An asterisk (*) indicates the nonspecific band. B, overexpress NRP-1 in NRP-1/ MEF cells down-regulated Smad1/5 phosphorylation and up-regulated Smad2/3 phosphorylation. NRP-1/ MEF cells were infected with NRP-1 encoding retrovirus. LacZ retrovirus was used as control. After 36 h, cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. C–E, effect of knocking down NRP-1 on the Smad proteins transcriptional activity in the cells measured by the luciferase promoter assay. 5 103/well LX2 cells were plated into a 96-well plate transfected with NRP-1 siRNA for 24–30 h, then serum-starved overnight. The cells were then transfected with the corresponding luciferase promoter plasmids together with pRL-TKRenilla luciferase plasmid astheinternalcontrol.Onehourafterthetransfection,TGF-1wasaddedtothecorrespondingwelltothefinalconcentrationof10ng/ml.Fireflyluciferaseand Renilla luciferase activities were performed. C, Luc-Id-1 promoter. D, Luc-ARE, co-transfected with FAST. E, Luc-SBE. F, knocking down NRP-1 affected the Smad protein responding gene expression. Cells were transfected with NRP-1 siRNA for 24–30 h in the complete medium, then serum-starved overnight. 10 ng/ml TGF-1 was added to the cells for 24 h. In response to TGF-1 stimulation, Id-1 protein (the Smad1-responding gene) expression was more highly up-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1. In response to TGF-1 stimulation, -SMA and PAI-1 protein (the Smad3-responding gene) expression was more highly down-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Targeted Gene Expression, Infection, Control, Activity Assay, Luciferase, Promoter Assay, Transfection, Plasmid Preparation, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 3. NRP-1 interacted with TGF- receptors. A, NRP-1 bound to TGF-RII. LX2 cells grown in the complete medium were made for cell lysate, and immunoprecipitation was performed using anti-NRP-1 antibody and West- ernblotforTGF-RII.ControlantibodywithcelllysateandNRP-1antibodywithoutcelllysatewereusedasnegative controls.B,TGF-1stimulationdidnotincreasetheassociationbetweenNRP-1andTGF-RII.LX2cellswereserum- starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 15, and 45 min. The cell lysate was made, and immu- noprecipitation was performed using anti-NRP-1 antibody and Western blot for TGF-RII. C, siRNA knocked down TGF-RII eliminated the effect of NRP-1 on Smad protein phosphorylation. LX2 cells were transfected with control, NRP-1, and TGF-RII siRNA alone or combined, as indicated in the figure. 24–30 h later, cells were serum-starved overnight. 10 ng/ml TGF-1 was added to the indicated plates for 45 min, and the cell lysate was subjected to Western blot analysis. An asterisk (*) indicates the nonspecific band. D, TGF-RI (ALK5) inhibitors eliminated both Smad1/5 and Smad2/3 protein phosphorylation on TGF-1 stimulation. LX2 cells were serum-starved overnight. TGF-RI inhibitors SB431542 (10 M) and ALK5 Inhibitor (10 M) were added to the cells 30 min before TGF-1 stimulation. An asterisk (*) indicates the nonspecific band. E, NRP-1 associated with TGF-RI (ALK5) by TGF-1 stimulation. 293T cells were transfected with NRP-1 and Flag-ALK5-expressing plasmids. Cells were serum-starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 5, and 15 min. The cell lysate was made, and immunoprecipi- tation was performed using anti-NRP-1 antibody and Western blot for Flag tag. F, knockdown TGF-RII eliminated theassociationbetweenNRP-1andTGF-RI(ALK5)inthepresentofTGF-stimulation.293Tcellsweretransfected with TGF-RII siRNA, and the following experiments were preformed as described in panel E.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Control, Expressing, FLAG-tag, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 8. The schematic illustration of NRP-1 function in regulating TGF- signaling. TGF- induced both Smad1/5/8 and Smad2/3 phosphorylation in the fibroblast cells. Without NRP-1 (left panel), Smad1/5/8 is more phosphorylated and Smad2/3 less phosphorylated, and the corresponding gene expression controlled by the Smad proteins (e.g. Smad1/Id-1, Smad3/a-SMA, PAI-1) caused the cell to enter a less activated state (more quiescent). With NRP-1 (right panel), Smad1/5/8 is less phosphorylated, and Smad2/3 is more phosphorylated, and the corresponding gene expression controlled by the Smad proteins caused the cell to enter a more activated state (less quiescent).

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Gene Expression

Journal: ACS Omega

Article Title: Respiratory Syncytial Virus F Protein Adjuvanted with a Carbomer-Based Adjuvant Induces Protective Humoral Response in Mice

doi: 10.1021/acsomega.5c08819

Figure Lengend Snippet: Formulation of RSV-F and RSV-G with adjuvant elicited a humoral response. BALB/c mice ( n = 5 per group; # indicates mortality due to agonistic behavior due to which n is 4 in the group) were intranasally immunized using 5 μg of RSV-F and RSV-G with 5% Adjuplex or 20 μg CpG ODN or both. Mice receiving only RSV-F and RSV-G served as the control group. Blood samples were collected on day 0, day 21 (prebooster), day 35 (postbooster), and day 111. Mice were challenged with 10 6 PFU/mL of RSV-A2 strain on day 111, and blood samples were collected 4 days later (day 116) for analysis. RSV-F- and RSV-G-specific IgG titers were measured by ELISA. End point titers were plotted using the definition of maximum dilution rate which was the OD value minus the background value (greater than or equal to 0.1). (a) Schematic showing immunization and infection timeline. (b) Total IgG antibody response by RSV-F ELISA in immunized BALB/c mice serum samples. (c) Total IgG antibody response by RSV-G ELISA in immunized BALB/c mice serum samples. Serum dilutions used in ELISA for different groups are indicated in Table S3 . Samples with no detectable antibodies in the first dilution used were assigned a value of first dilution/2. Samples with no detectable antibodies in 1:50 dilution [lower limit of quantitation (LLOQ)] were assigned a value of LLOQ/2. (d) Neutralizing antibodies against RSV in immunized BALB/c on day 21. (e) Neutralizing antibodies against RSV in immunized BALB/c on day 35. (f) Neutralizing antibodies against RSV in immunized BALB/c on day 111. (g) Neutralizing antibodies against RSV in immunized BALB/c on day 116 in serum samples. Serum dilutions used in the FRNT assay are provided in Supplementary Table S4 . NT 50 below 50 was considered as the LLOQ and indicated as the dotted line. Samples below LLOQ were assigned a value of LLOQ/2. (h) Immunized and control BALB/c mice were challenged with 10 6 PFU of RSV-A2. Four days post challenge, animals were sacrificed and lungs were harvested, and viral loads were determined by the plaque assay. PFU per gram weight of lungs below 80 was considered as LOD and were assigned values of LOD/2. Data are represented as geometric mean with 95% confidence interval (CI). ELISA data were analyzed by comparing the means of control group (RSV-F+RSV-G group) with every other group using two-way ANOVA or mixed-effects model with Dunnet’s multiple comparisons test with a single pooled variance. Plaque assay data were analyzed by the nonparametric, Kruskal–Wallis test with Dunn’s multiple comparison correction.

Article Snippet: The permeabilization buffer was removed, and 50 μL of primary mouse anti-RSV-F monoclonal antibody at 1:1000 dilution (Bio-Rad# MCA490) was added and incubated for 1 h at RT, followed by HRP-conjugated anti-mouse IgG secondary antibody at 1:1000 dilution (Invitrogen, Cat. no. A16072) for 1 h. Cells were washed twice with PBS and incubated with TrueBlue substrate (KPL Inc. # 5510-0030) for 10 min and washed once with PBS.

Techniques: Formulation, Adjuvant, Control, Enzyme-linked Immunosorbent Assay, Infection, Quantitation Assay, Plaque Assay, Comparison

Journal: ACS Omega

Article Title: Respiratory Syncytial Virus F Protein Adjuvanted with a Carbomer-Based Adjuvant Induces Protective Humoral Response in Mice

doi: 10.1021/acsomega.5c08819

Figure Lengend Snippet: RSV-F-Adjuplex elicited an enhanced immune response compared to the RSV-G-Adjuplex. BALB/c mice ( n = 5 per group; # indicates mortality due to agonistic behavior due to which n is 4 in the groups) were intranasally immunized twice using RSV-F and RSV-G with 5% Adjuplex and 20 μg CpG ODN or both. Mice receiving only Adjuplex and CpG ODN served as the control group. (a) Total IgG response by RSV-F ELISA in immunized BALB/c mice serum samples. (b) Total IgG response by RSV-G ELISA in immunized BALB/c mice serum samples. Serum dilutions used in ELISA for different groups are indicated in Supplementary Tables S5 and S6 . Samples with no detectable antibodies in the first dilution used were assigned a value of first dilution/2. Samples with no detectable antibodies in a 1:50 dilution (LLOQ) were assigned a value of LLOQ/2. (c) Neutralizing antibodies against RSV in immunized BALB/c on day 21. (d) Neutralizing antibodies against RSV in immunized BALB/c on day 35. (e) Neutralizing antibodies against RSV in immunized BALB/c on day 111. (f) Neutralizing antibodies against RSV in immunized BALB/c on day 116 in serum samples. Serum dilutions used in the FRNT assay are provided in Table S7 . (g) Immunized and control BALB/c mice were challenged with 10 6 PFU of RSV-A2. Four days post challenge, lungs were harvested, and viral loads was determined by the plaque assay. Data are represented as geometric mean with 95% CI. ELISA data were analyzed by comparing the means of control group (Adjuplex + CpG ODN) with every other group using two-way ANOVA or mixed-effects model with Dunnet’s multiple comparisons test, with a single pooled variance. Plaque assay data were analyzed by the nonparametric, Kruskal–Wallis test with Dunn’s multiple comparison correction.

Article Snippet: The permeabilization buffer was removed, and 50 μL of primary mouse anti-RSV-F monoclonal antibody at 1:1000 dilution (Bio-Rad# MCA490) was added and incubated for 1 h at RT, followed by HRP-conjugated anti-mouse IgG secondary antibody at 1:1000 dilution (Invitrogen, Cat. no. A16072) for 1 h. Cells were washed twice with PBS and incubated with TrueBlue substrate (KPL Inc. # 5510-0030) for 10 min and washed once with PBS.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Plaque Assay, Comparison

Journal: ACS Omega

Article Title: Respiratory Syncytial Virus F Protein Adjuvanted with a Carbomer-Based Adjuvant Induces Protective Humoral Response in Mice

doi: 10.1021/acsomega.5c08819

Figure Lengend Snippet: Histopathology analysis of lungs from immunized mice after virus challenge. (a–k) Indicated groups of BALB/c mice ( n = 5 per group; # indicates mortality due to agonistic behavior due to which n is 4 in the respective groups) were intranasally immunized with two doses using RSV-F and RSV-G with 5% Adjuplex and 20 μg CpG O DN or both. Mice receiving only Adjuplex and CpG ODN served as the control group for statistical analysis. Lungs collected 4 days after challenge with RSV A2 were fixed, sectioned, and stained with hematoxylin and eosin and were scored for inflammation in bronchioles, near veins (vascular), and alveoli.

Article Snippet: The permeabilization buffer was removed, and 50 μL of primary mouse anti-RSV-F monoclonal antibody at 1:1000 dilution (Bio-Rad# MCA490) was added and incubated for 1 h at RT, followed by HRP-conjugated anti-mouse IgG secondary antibody at 1:1000 dilution (Invitrogen, Cat. no. A16072) for 1 h. Cells were washed twice with PBS and incubated with TrueBlue substrate (KPL Inc. # 5510-0030) for 10 min and washed once with PBS.

Techniques: Histopathology, Virus, Control, Staining

Journal: ACS Omega

Article Title: Respiratory Syncytial Virus F Protein Adjuvanted with a Carbomer-Based Adjuvant Induces Protective Humoral Response in Mice

doi: 10.1021/acsomega.5c08819

Figure Lengend Snippet: Combination of RSV-F and G with adjuvants show reduced pathology in lungs. (a) Hemorrhage. (b) Immune cell infiltration. (c) Peribronchiolitis. (d) Perivasculitis. (e) Interstitial pneumonia. (f) Alveolitis. Lung histology scores represent the average of 4–5 mice per group. Pathology was scored as follows: 0, not observed; 1, minimal; 2, slight; 3, moderate; 4, marked; and 5, severe. Images were acquired at 10× and 20× magnification. Error bars represent mean with SD. P values were estimated by Kruskal–Wallis test with Dunn’s multiple comparisons test for correction. Only P values that were < 0.05 are indicated.

Article Snippet: The permeabilization buffer was removed, and 50 μL of primary mouse anti-RSV-F monoclonal antibody at 1:1000 dilution (Bio-Rad# MCA490) was added and incubated for 1 h at RT, followed by HRP-conjugated anti-mouse IgG secondary antibody at 1:1000 dilution (Invitrogen, Cat. no. A16072) for 1 h. Cells were washed twice with PBS and incubated with TrueBlue substrate (KPL Inc. # 5510-0030) for 10 min and washed once with PBS.

Techniques:

Journal: ACS Omega

Article Title: Respiratory Syncytial Virus F Protein Adjuvanted with a Carbomer-Based Adjuvant Induces Protective Humoral Response in Mice

doi: 10.1021/acsomega.5c08819

Figure Lengend Snippet: Adjuplex induces enhanced mucosal IgA response against RSV-F/G. BALB/c mice ( n = 5 per group) were challenged intranasally with RSV A2 on day 111 post immunization, and lungs were harvested on day 116 homogenized. (a) Total RSV-F-specific IgA antibody response was measured by RSV-F-ELISA in lung homogenates of immunized BALB/c mice. (b) RSV-G-specific IgA antibody response measured by RSV-G-ELISA in lung homogenates of immunized BALB/c mice. End point dilution titers are plotted. Error bars indicate geometric mean with 95% CI. P values were estimated by Kruskal–Wallis test with Dunn’s multiple comparisons test for correction. Only P values that were <0.05 are indicated. # indicates mortality due to agonistic behavior due to which n is 4 in the respective groups.

Article Snippet: The permeabilization buffer was removed, and 50 μL of primary mouse anti-RSV-F monoclonal antibody at 1:1000 dilution (Bio-Rad# MCA490) was added and incubated for 1 h at RT, followed by HRP-conjugated anti-mouse IgG secondary antibody at 1:1000 dilution (Invitrogen, Cat. no. A16072) for 1 h. Cells were washed twice with PBS and incubated with TrueBlue substrate (KPL Inc. # 5510-0030) for 10 min and washed once with PBS.

Techniques: Enzyme-linked Immunosorbent Assay