chicken igy isotype  (R&D Systems)

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: The promotion of hair follicle regeneration after wound healing is dependent on TLR2. ( A ) Representative confocal images of Nile red-labeled (sebaceous gland) wild-type (WT) telogen hair follicles co-immunostained for MPO showing complete co-localization of MPO to the sebaceous gland. The isotype control panel shows the images of hair follicles stained with MPO isotype control antibody. Scale bars are 20 μm. ( B ) Representative confocal images of hair follicles from old vs young mice stained for MPO. Scale bars are 10 μm. ( C ) Quantification of MPO fluorescent intensity in B showing significantly less MPO in hair follicles from older mice. N=6 for each group. ( D ) Representative microphotographs of human hair follicle stem cells (HFSCs) pre-treated with 10 µg/ml MAb-mTLR2 or DMSO and co-cultured with/without 2.5 µM of CEP. Representative images from at least three independent assays are shown. Scale bar 50 µm. ( E ) Bar graphs show increased proliferation of HFSC in the presence of TLR2 endogenous ligand CEP compared to control, which was abolished in the presence of TLR2 blocking antibody. N=6 independent experiments. ( F ) Bar graphs show increased proliferation of human hair follicle dermal papilla cells incubated with 5 µM of CEP compared to the control. N=9 independent experiments. ( G ) Representative confocal images of Ki67 immunostaining of dorsal skin adjacent to wound of CEP-treated WT bone marrow transplanted WT and TLR2 KO mice. Scale bars are 50 μm. ( H ) Quantitative results showed increased Ki67 intensity in hair follicles around wounds of CEP-treated WT bone marrow transplanted WT mice with no differences in TLR2 KO with WT bone marrow. N=4 per group. ( I ) Representative photographs of dorsal skin (upper panels), inner skin flaps (middle panels), and representative confocal images of Ki67 immunostaining (lower panels) of vehicle- or CEP-treated WT or TLR2 KO skin. Scale bars are 1 mm for dorsal skin, 500 μm for skin flaps, and 50 μm for confocal images. ( J ) Bar graph showing quantification of hair follicle numbers of vehicle or CEP-treated skin from I. N=5 per group. ( K ) Bar graph showing quantification of Ki67 fluorescent intensity of Ki67 staining of vehicle or CEP-treated skin from I. N=4 per group. Unpaired two-tailed t-test ( C ), or non-parametric Mann-Whitney test ( H, J, K ), or Kruskal-Wallis test with Dunn’s multiple comparisons test ( F ), or one-way ANOVA with Tukey’s multiple comparisons test ( E ) was used to determine statistical differences. All bar graphs are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: Antibody , Chicken IgY Isotype Control , Novus Biologicals , Cat.# AB-101-C RRID: AB_354263 , According to immune antibody concentration.

Techniques: Labeling, Control, Staining, Cell Culture, Blocking Assay, Incubation, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet:

Article Snippet: Antibody , Chicken IgY Isotype Control , Novus Biologicals , Cat.# AB-101-C RRID: AB_354263 , According to immune antibody concentration.

Techniques: Activity Assay, Knock-In, Sequencing, Isolation, Blocking Assay, Control, Concentration Assay, Staining, Membrane, Recombinant, Software

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: A, Immunofluorescence analyses (IFA) were performed on DV-infected K562. K562 cells were infected at M.O.I. 10 and fixed at 3 d.p.i. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. B, K562 cells were mock-infected, treated with UV-irradiated DV (UV-DV) at a M.O.I. 10, infected with DV (M.O.I. 10), or treated with 5 µg/ml of LPS. The nuclear and cytosolic fractions were harvested at 3 d.p.i. and Western blot analysis was performed to detect for HMGB1. The band intensities of HMGB1 of the mock-infected, UV-DV treated, DV-infected and LPS-stimulated nuclear fractions were expressed as a ratio to its respective cytosolic fraction. The housekeeping proteins TFIID and actin were included as loading controls for the nuclear and cytosolic fractions, respectively. C, K562 cells were infected with DV at M.O.I. of 1. Cell culture supernatants from DV-infected K562 were harvested at 3 d.p.i. and concentrated for the detection of HMGB1 via Western blot. D, DV-infected PBM cells infected at M.O.I. 1 and the cells fixed at 3 d.p.i. for IFA. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. E, PBM were infected with DV at a M.O.I. of 1 and Western blot analysis of HMGB1 in the cell culture supernatants of DV-infected PBM was performed. The band intensities of HMGB1 from DV-infected sample and LPS-treated cells at day 1 p.i. were assigned to a value of 1. The relative fold difference in the intensities of DV-infected samples and LPS-treated cells from day 2- to 5 p.i. was measured in relation to the band intensity its respective treatment group at day 1 p.i.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: Immunofluorescence, Infection, Staining, Irradiation, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: A, cell viability assay of K562 or PBM cells treated with 1, 3 and 5 mM of EP for 3 days. The percentages of viable K562 and PBM cells were calculated in relation to its untreated control groups. B, immunofluorescence analysis of EP-treated DV-infected K562 and PBM cells to visualize the sub-cellular localization of HMGB1 (stained green) in these cells. DV antigens were stained red and the cell nuclei were stained blue. C, K562 and PBM cells were infected with DV (M.O.I. of 1) and the cells were treated with EP at 0 hr p.i. Cell culture supernatants of DV-infected K562 and PBM cells were harvested 3 d.p.i. and concentrated for the detection of HMGB1 by Western blotting. The fold intensity of DV-infected K562 and PBM cells without EP treatment were assigned with a value of 1. The relative fold difference in the HMGB1 intensities of EP-treated DV-infected samples, mock-infected and LPS-treated cells were compared in relation to that of the DV-infected K562 or PBM cells. D, amount of HMGB1 in the cell culture supernantants was quantified using ELISA. * denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in K562 cell culture supernatants to that of the DV-infected K562 cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in PBM cell culture supernatants to that of the DV-infected PBM cells. E, plaque assays were conducted on the cell culture supernatants of DV-infected EP-treated K562 cells at 3 d.p.i.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: Viability Assay, Immunofluorescence, Infection, Staining, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: A, HUVEC was incubated with 50, 100 or 150 nM of rHMGB1 for 5 days and cell viability assay was performed to assess for cytotoxicity induced by rHMGB1 on HUVEC. The percentage of the viable cells from the rHMGB1 treated groups was calculated in relation to the untreated negative control cells. B, 50, 100 and 150 nM of rHMGB1 was added to the confluent HUVEC monolayer to observe for the presence of vascular leakage (indicated by a lowered TEER value) in the endothelial cells. The TEER values of the rHMGB1-treated monolayer were expressed as percentage to the untreated negative control cells. TNF-α, a known inducer of vascular leakage was added to HUVEC monolayer was used as a positive control. C, cell culture supernatant of K562 mock- or DV-infected and EP-treated was collected 3 d.p.i. and added to HUVEC monolayer for 5 days. The TEER of the HUVEC monolayer was measured and the values of the monolayer treated with mock-infected, DV-infected and EP-treated K562 cell culture supernatants were expressed as percentage to HUVEC treated with K562 cell culture medium RPMI-1640 in the absence of any treatment of viral infection. D, mock- or DV-infected or LPS-treated K562 cell culture supernatant was added to HUVEC monolayer for 5 days with chicken anti-HMGB1 neutralizing antibody (IgHMGB1) or chicken HMGB1 control chicken IgY (IgCtrl). The TEER of the supernatant-treated cells was expressed as percentage to HUVEC treated with control RPMI-1640 cell culture medium.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: Incubation, Viability Assay, Negative Control, Positive Control, Cell Culture, Infection

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: A, K562 cells were transfected with 25, 50 or 100 nM of siRNA for 3 days and cell viability assay was performed to evaluation of the cytotoxicity of siRNA transfection on K562 cells. The percentage of the viable transfected cells was expressed as a percentage to that of the non-transfected cells. B, siRNA (concentration of 25 to 100 nM) targeting against PCAF was used to transfect K562 cells. The transfected K562 cells were subjected to DV infection 48 hours post-transfection. The cell lysates and culture supernatants were harvested at 1 d.p.i. and Western blot analysis was then performed to detect for the presence of PCAF and HMGB1, respectively. The PCAF band intensities of the siRNA transfected K562 cells were calculated in relation to the non-transfected mock-infected cells (assigned to a value of 1). Similarly, the band intensities of HMGB1 of the transfected cell culture supernatants were measured in relation to the band intensity of non-transfected DV-infected cell culture media (assigned to value of 1). Actin was used as a control to ensure equal loading. C, ELISA was performed to quantify the amount of HMGB1 released into the supernatants from siRNA transfected (0, 25, 50 or 100 nM of siRNA) and DV-infected K562 cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in siRNA transfected K562 cell culture supernatants to that of the non-siRNA transfected K562 cells. D, K562 cells were transfected with 0, 25, 50 or 100 nM of siRNA for 48 hours before infected with DV. The supernatants were harvested at 1 d.p.i. and plaque assay was performed to quantify the viral yield.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: Transfection, Viability Assay, Concentration Assay, Infection, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Plaque Assay

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: A, K562 cells were transfected with individual plasmid encoding for DV capsid protein or GFP and stable cell line K562-Capsid and K562-GFP were obtained, respectively. IFA and Western blot were performed on the cell lysate to detect for the expression of capsid-GFP fusion protein (39 kDa) and GFP (27 kDa). The cell nuclei were stained blue with DAPI (i) and actin was used as a control to ensure equal loading of the cell lysate (ii). Cell culture supernatants of the stable cell line were harvested and concentrated to probe for the presence of HMGB1 using Western blot. B, stable cell line K562-Capsid was subjected to EP treatment and 3 days post EP treatment, the cell culture were harvested and concentrated to detect for the presence of HMGB1 using Western blot. The band intensities of HMGB1 of the EP treated cell culture supernatants were measured in relation to the band intensity of untreated control cell culture media (assigned to value of 1). C, siRNA was employed to knockdown PCAF protein in the K562-Capsid cells and cell viability assay was performed to evaluate for cytotoxicity upon siRNA transfection. D, K562-Capisd was transfected with siRNA to knockdown PCAF protein and both cell lysate and cell culture supernatants were collected 72 hrs post-transfection. Western blot was performed on the cell lysate and cell culture supernatants to detect for the PCAF and HMGB1, respectively. The band intensities of PCAF protein from the siRNA-transfected cells were compared in relation to the band intensity of untransfected control cell lysate. Actin was used as a control to ensure equal loading of total protein. The band intensities of HMGB1 of siRNA transfected cell culture media was measured in relation to the band intensity of K562-Capsid cells without siRNA. E, the amount of HMGB1 released into the cell culture supernatants from K562-Capsid cells treated with EP or transfected with 0, 25, 50 or 100 nM of siRNA were measured using ELISA. * denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in EP-treated K562-Capsid cell culture supernatants to that of the untreated K562-Capsid cells. ⧫ denotes p-value <0.05 for T-tests comparing the mean amount of HMGB1 detected in siRNA transfected K562-Capsid cell culture supernatants to that of the non-siRNA transfected K562-Capsid cells.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: Transfection, Plasmid Preparation, Stable Transfection, Western Blot, Expressing, Staining, Cell Culture, Viability Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

doi: 10.1371/journal.pone.0041932

Figure Lengend Snippet: DV enters the monocytes and uncoat. The capsid protein enters the nucleus and triggers the acetylation of HMGB1 by PCAF complex. HMGB1 then translocates from the nucleus to cytoplasm, a process that can be inhibited by EP. HMGB1 gets released from the monocytes and binds to RAGE receptors on endothelial cells, triggering a signalling pathway which leads to the lost of vascular integrity. The increased in vascular leakage can be repressed by HMGB1-neutraling antibody or anti-RAGE antibody.

Article Snippet: Chicken anti-HMGB1 neutralizing antibody or chicken HMGB1 control chicken IgY (IBL International, Hamburg, Germany) was added to the confluent monolayer to achieve a final concentration of 100 µg/ml.

Techniques: