Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Histopathological analysis of lungs from infected WT and PLG-KO mice inoculated with A/PR/8/34 virus (day 3 post-infection) or A/Netherlands/602/09 virus (day 5 post-infection). Thin sections of lungs obtained from infected and uninfected WT and PLG-KO mice (as indicated) were stained with hematoxilin end eosin (HE) to evaluate histopathological changes. Note the marked infiltration of inflammatory cells in the lungs of infected WT mice, which was largely absent in the lungs of PLG-KO mice. The results shown are representative for two-three mice for both groups. Immunohistochemistry (IHC) using a monoclonal antibody for the influenza A virus nucleoprotein was used to detect virus-infected cells. Cells positive for the presence of viral antigen stained red. (B) Cytokine levels in BAL were assessed by ELISA on the indicated days post inoculation of WT (black bars) and PLG-KO mice (white bars) with IAV A/PR/8/34 or A/Netherlands/602/09. Data represent mean ± s.e.m. of 3–6 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Levels of Plasminogen, Active Plasmin, FDP, D-dimer and Fibrinogen, were determined by ELISA in the BAL of A/PR/8/34 infected or uninfected (−) C57BL/6 mice after the indicated days post-inoculation. Markers were also evaluated in the BAL of WT or PLG-KO mice infected with A/Netherlands/602/09 virus. Data represent mean ± s.e.m of n = 3–6 mice per group. (B) Western blot analysis for the detection of fibrinogen and FDP in the lungs of IAV-infected mice on the indicated days post inoculation (representative of n = 3). kDa: apparent molecular weight. n = mice per group. (C) Presence of fibrinogen was assessed in the blood of mice treated or not with Ancrod by ELISA (left panel) or Western blot analysis (right panel). The results represent the mean values ± s.e.m from 3 individual animals per group for the ELISA. The western blot analysis is representative for results of 3 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Virus, Western Blot, Molecular Weight

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Survival and weight loss of mice treated with Ancrod (open symbols, n = 11) or not (closed symbols, n = 11) after infection with IAV A/PR/8/34 (squares) or uninfected mice (diamonds, n = 5). Weight loss data represent weight average ± s.e.m of the above indicated number of mice. (B) Cytokines levels in the BAL were measured by ELISA after A/PR/8/34 infection of WT and PLG-KO (KO) mice treated with Ancrod (white bars) or not (black bars). Data represent mean ± s.e.m. of n = 4 mice per group. (C) Survival rate (left panels) and weight loss (right panels) of WT (squares) and PLG-KO (triangles) mice treated with Ancrod (open symbols) or not (closed symbols) after intranasal inoculation with IAV A/PR/8/34 (n = 8–10 mice per group). Weight loss data represent weight average ± s.e.m of the above indicated number of mice.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

doi: 10.4049/jimmunol.1901131

Figure Lengend Snippet: FIGURE 3. Differential effects of ROS scavenging by NAC and VAS2870. B cells were stimulated with or without CpG-ODN and anti-RP105 as described in the legend of Fig. 1. When indicated, the cells were pretreated for 30 min with NAC (20 mM) or VAS (2.5 mM) prior to stimulation. (A and B) Supernatants were collected after 96 h of stimulation, and IgG (A) and IgM (B) levels were determined by ELISA. Each dot represents the value from one donor, and the horizontal lines represent median values. (A) n = 12 or 6 (VAS2870) and (B) n = 16 or 6 (VAS2870). *p # 0.03. (C and D) The fraction of IgG+ and CD27+ cells was determined by flow cytometry using Brilliant Violet–conjugated Abs directed against IgG and CD27 after 96 h of stimulation. Where indicated, cells were pretreated for 30 min with 20 mM NAC (n = 4, mean 6 SEM). (E and F) Activation of the cells was determined by analyses of CD80 expression (E) or by activation of NF-kB as revealed by the phosphorylation of IkB (F). (E) The fraction of CD80+ cells was determined by flow cytometry using FITC-conjugated Ab directed against CD80 after 24 h of stimulation. Upper panel, Graphs from one representative of six flow cytometry analyses. Lower panel, The horizontal lines represent median values, the boxes represent 50% of measured values, and the whiskers represent 6 1.5 interquartile range (n = 6). *p # 0.05 (Wilcoxon matched-pairs signed rank test). (F) Western blot analyses of p-IkB were performed after 30 min of stimulation. One of two reproducible blots are shown, using calnexin as a loading control. (G) At the indicated times after stimulation, proliferation was measured by the CellTiter-Glo assay. The luminescence values obtained were normalized against the values at time 0 and are presented as mean values (n = 4). (H–J) Ninety-six hours after stimulation, ELISPOT analyses of IgG and IgM were performed on equal numbers of cells. (H) The numbers of IgG- or IgM-producing cells are indicated as the number of spots per 1000 cells. (I) The amount of Ig produced per cell is indicated by the average spot sizes. (H and J) Each dot represents the value from one blood donor, and the horizontal lines represent median values (n = 9). *p # 0.03 (Wilcoxon matched-pairs signed rank test). (J) One representative plate from nine ELISPOT experiments.

Article Snippet: ELISPOTwas performed according to the manufacturer’s recommendations using kits from ImmunoSpot/Cellular Technology Limited: human IgG and IgM singlecolor ELISPOT (hIgG-SCE-1M/2 and hIgM-SCE-1M/2).

Techniques: Enzyme-linked Immunosorbent Assay, Cytometry, Activation Assay, Expressing, Phospho-proteomics, Western Blot, Control, Glo Assay, Enzyme-linked Immunospot, Produced

Journal: Photodiagnosis and photodynamic therapy

Article Title: Effect of Photofrin-mediated photocytotoxicity on a panel of human pancreatic cancer cells

doi: 10.1016/j.pdpdt.2012.11.001

Figure Lengend Snippet: Clonogenic assay. Poorly differentiated human pancreatic cancer cell lines were subjected to Photofrin PDT at the following drug and light doses — Mia PaCa-2: 5 μg/ml and 3 J/cm2; MPanc-96: 5 μg/ml and 3 J/cm2 and PL-45: 5 μg/ml and 6 J/cm2. Control (drug only) and treated cells were grown in a fresh medium immediately after PDT. Clonogenic assay was examined after two weeks. There was no colony formation in Photofrin PDT groups.

Article Snippet: The moderately well to poorly differentiated human pancreatic cancer cell lines (BxPc-3, HPAF-II, Mia PaCa-2, MPanc-96, PANC-1 and PL-45) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 medium (HyClone Laboratories, Inc., Logan, UT) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc.) and antibiotics.

Techniques: Clonogenic Assay, Control

Journal: Photodiagnosis and photodynamic therapy

Article Title: Effect of Photofrin-mediated photocytotoxicity on a panel of human pancreatic cancer cells

doi: 10.1016/j.pdpdt.2012.11.001

Figure Lengend Snippet: Flow cytometric assay. PDT-induced apoptosis was detected by PI staining and flow cytometric analysis at 24 h after PDT. Pancreatic cancer cell lines were subjected to Photofrin PDT at the following drug and light doses respectively — BxPc-3: 2 μg/ml and 3 J/cm2; HPAF-II: 5 μg/ml and 3 J/cm2; Mia PaCa-2: 5 μg/ml and 3 J/cm2; MPanc-96: 2 μg/ml and 3 J/cm2; Panc-1: 2 μg/ml and 3 J/cm2 and PL-45: 5 μg/ml and 6 J/cm2. Representative light and drug controls are shown on the top panel. The percentage of apoptotic cells is indicated.

Article Snippet: The moderately well to poorly differentiated human pancreatic cancer cell lines (BxPc-3, HPAF-II, Mia PaCa-2, MPanc-96, PANC-1 and PL-45) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 medium (HyClone Laboratories, Inc., Logan, UT) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc.) and antibiotics.

Techniques: Flow Cytometry, Staining

Journal: Photodiagnosis and photodynamic therapy

Article Title: Effect of Photofrin-mediated photocytotoxicity on a panel of human pancreatic cancer cells

doi: 10.1016/j.pdpdt.2012.11.001

Figure Lengend Snippet: Western blot analysis. PDT-induced apoptosis was confirmed by detecting the cleaved caspase-3, caspase-8, caspase-9, and PARP fragments (arrows) and by the disappearance of the uncleaved precursors of these proteins in pancreatic cancer cells.

Article Snippet: The moderately well to poorly differentiated human pancreatic cancer cell lines (BxPc-3, HPAF-II, Mia PaCa-2, MPanc-96, PANC-1 and PL-45) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 medium (HyClone Laboratories, Inc., Logan, UT) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc.) and antibiotics.

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: MET inhibitor, capmatinib overcomes osimertinib resistance via suppression of MET/Akt/snail signaling in non-small cell lung cancer and decreased generation of cancer-associated fibroblasts

doi: 10.18632/aging.202547

Figure Lengend Snippet: Osimertinib-resistant cells promoted CAF transformation. ( A ) Expression profile analysis of TCGA lung cancer cohorts showed that an elevated MET mRNA level in both lung adenocarcinoma and squamous cell lung cancer tissues as compared to the normal lung tissue. *P<0.05. ( B ) Western blots analysis showed that osimertinib resistant (OR) NSCLC samples contained a higher EGFR, Akt and MET activities (p-, phosphorylated form) and higher expression of EMT markers, vimentin and snail, than osimertinib sensitive (OS) counterparts. ( C ) ELISA assay of medium collected from OR and OS cells showed that OR cells produced a significantly higher amount of TGFβ1 as compared to OS cells. ( D ) Immunofluorescence examination of fibroblasts after co-cultured with OR or OS cells. Fibroblasts co-cultured with OR cells showed a markedly stronger fluorescence intensity of both CAF makers, α-SMA (green) and vimentin (red), comparing to fibroblasts co-cultured with OS cells (magnification 200X).

Article Snippet: The amount of secreted TGFβ1 into the culture medium by CAFs was determined using an ELISA kit (Catalog# OKCD06012, Aviva Systems Biology Corporation, USA); secreted IL-6 by cancer cells into the culture medium was measured using a human IL-6 ELISA kit (Catalog # OKBB00189, Aviva Systems Biology Corporation, USA).

Techniques: Transformation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Produced, Immunofluorescence, Cell Culture, Fluorescence

Journal: Aging (Albany NY)

Article Title: MET inhibitor, capmatinib overcomes osimertinib resistance via suppression of MET/Akt/snail signaling in non-small cell lung cancer and decreased generation of cancer-associated fibroblasts

doi: 10.18632/aging.202547

Figure Lengend Snippet: Combination treatment of capmatinib and osimertinib overcame osimertinib resistance and improved survival in PDX mouse model. ( A ) Tumor burden over time curve shows that the combination treatment (Cap+Osi) group with the lowest tumor burden followed by capmatinib only (cap) group while no difference between the control and osimertinib (osi) only groups. ( B ) Survival ratio versus time curve indicates that the best survival rate in the following order, combination treatment (cap+osi), capmatinib (cap) only, and lowest in both control and osimertinib (osi) only groups. ( C ) Immunohistochemical analysis of tumor samples. The lowest staining intensity of Snail MET and α-SMA was found in the combination group followed by the capmatinib only samples while the immunostaining of all three markers were highest in the control and osimertinib only tumor sections. ( D ) Comparative tumor formation assay shows that the lowest number of tumor spheres were formed in the samples which received the combination treatment followed by capmatinib only. Tumor samples from control and osimertinib only groups showed similar tumor forming ability. ( E ) ELISA assay of secreted TGFβ1 secretion by the tumor samples with different treatment regimens. The lowest amount of TGFβ1 secreted into the medium was observed in the tumor samples received combination treatment. **P<0.01, ***P<0.001, NS, no significance.

Article Snippet: The amount of secreted TGFβ1 into the culture medium by CAFs was determined using an ELISA kit (Catalog# OKCD06012, Aviva Systems Biology Corporation, USA); secreted IL-6 by cancer cells into the culture medium was measured using a human IL-6 ELISA kit (Catalog # OKBB00189, Aviva Systems Biology Corporation, USA).

Techniques: Control, Immunohistochemical staining, Staining, Immunostaining, Tube Formation Assay, Enzyme-linked Immunosorbent Assay