Journal: The Journal of Neuroscience

Article Title: Mediation by Protein Kinases C and A of G o -Linked Slow Responses of Enteric Neurons to 5-HT

doi: 10.1523/JNEUROSCI.17-03-01011.1997

Figure Lengend Snippet: Inhibitors of PKC antagonize slow responses to 5-HT. A, Slow responses to 5-HT of an AH/type 2 neuron are antagonized by the intracellular injection of the PKC pseudosubstrate PKC[19–31] (top trace,left) and by the superfusion of the membrane permeable PKC pseudosubstrate myr-PKC[19–27] (myr-FARKGALRQ;bottom trace, left). Slow responses are also inhibited by the superfusion of chelerythrine (3.0 μm; top trace, right) and by the selective PKC α and β antagonist Gö 6976 (10 μm; bottom trace,right). B,C, The abilities of PKC[19–31] chelerythrine and K252a to reduce the amplitude of the 5-HT-induced slow depolarization of AH/type 2 cells are compared.

Article Snippet: These included the phosphatidylcholine-phospholipase C (PC-PLC) inhibitor D609 (Kamiya Biomedical, Thousand Oaks, CA); the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine (DDA, Biomol Research Laboratory, Plymouth Meeting, PA); and the PKC inhibitors chelerythrine (LC Laboratories), staurosporine (Research Biochemical International), K252a (Kamiya), Gö 6976, an indolcabazole that selectively inhibits the PKC isozymes, α, β1, β2, and γ, but not δ, ε, or ζ ( Martiny-Baron et al., 1993 ) (Calbiochem, La Jolla, CA); and a myristolated pseudosubstrate sequence from PKC α and β, myr-FARKGALRQ (Biomol).

Techniques: Injection

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Phospholipase D and diacylglycerol lipase contribute arachidonic acid for PGE2 biosynthesis. (a) RAW 264.7 macrophages labeled with [3H]arachidonic acid were treated with 10 μM VU0155056 1 hour prior to inoculation with F. tularensis LVS. Twenty hours after inoculation, radioactivity in the supernatant and cell fraction was measured by liquid scintillation and expressed as percent release (n = 3). (b) BMDMs or RAW 264.7 macrophages were pretreated with inhibitors and then inoculated with F. tularensis LVS at an MOI of 200:1. Three hours after inoculation, 5 μM arachidonic acid was added to culture medium. PGE2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation (n = 4). Asterisks denote a statistical difference compared to the LVS-infected group (a) or the corresponding AA-treated group (b) (P ≤ 0.05). For clarity, the data from some experiments were presented in multiple figures. However, these experiments were run simultaneously and as such utilize the same controls (uninfected, infected, and COX-2 inhibitor treated). This includes Fig. 2c and ​anddd and panels a and b of this figure. Graphs are means of pooled experiments ± SEMs.

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques: Labeling, Radioactivity, Enzyme-linked Immunosorbent Assay, Infection

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Inhibitors used in this study a

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques:

Journal: Infection and Immunity

Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

doi: 10.1128/IAI.02060-14

Figure Lengend Snippet: Inhibition of macrophage cPLA2 (pyrrophenone), PAP (propranolol), DAGL (RHC80267), or PLD (VU0155056) reduced F. tularensis-induced prostaglandin E2 biosynthesis by infected macrophages. (a) F. tularensis LVS was inoculated in broth medium and left untreated or incubated with inhibitors/vehicle and allowed to grow over 12 hours. Bacterial growth was measured as a function of absorbance at 600 nm. Curves represent average growth from 3 independent experiments. (b) BMDMs or RAW 264.7 macrophages were pretreated with inhibitors and then inoculated with F. tularensis LVS at an MOI of 200:1; PGE2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation. Data represent means ± SEMs. n = 9 (BMDMs); n = 17 (RAW cells). (c) LIVE/DEAD Fixable Yellow stain analysis was performed to verify that inhibitors were not causing cell death or increased cell death during F. tularensis infection. BMDMs or RAW 264.7 macrophages were incubated with vehicle or inhibitor for 20 hours at 37°C (n = 6). Asterisks denote a statistical difference compared to the untreated infected group (P ≤ 0.05). Graphs are means of pooled experiments ± SEMs.

Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA 2 ), CAY10590 (for sPLA 2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA 2 ), U73122 (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

Techniques: Inhibition, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: Human colonocytes in normal tissues express CD14 constitutively in the absence of TLR4, whereas cancerous tissues display enhanced levels of CD14 and TLR4. (a) Representative images of human surgical specimens of colonic adenocarcinoma from three clinical cases examined for the expression of LPS receptors. Fluorescent images indicate immunoreactivity to CD14, TLR4, or MD2 (green) superimposed with nuclear staining (blue). Apical expression of CD14 and the absence of TLR4 and MD2 were found on epithelial cells of morphologically normal tissues. Increased CD14 and TLR4 levels were observed in tumorous regions with no change in MD2 patterns. The levels of proliferating cell nuclear antigen (PCNA) in colonic tissues are in brown. Scale bar: 50 μm. (b and c) Immunoreactivity score of CD14 and TLR4 expression in normal epithelium and tumorous regions from thirty specimens. *P<0.05 versus normal

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Expressing, Staining

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: Baseline levels of CD14/TLR4/MD2 in mouse colonocytes and modification of expression levels in tumorous tissues. (a) Transcript levels of CD14, TLR4, and MD2 in mouse colonocyte cell preparations, showing the absence of CD68 transcripts (a marker of monocytes/macrophages). Mononuclear leukocytes (ML) and monocytic RAW264.7 (RAW) cells were used as positive controls for CD68. (b) Representative images of immunofluorescent staining of CD14, TLR4, or MD2 (red) in mouse colonic tissues, superimposed with nuclear staining (blue) for visual orientation. CD14 and TLR4 were localized to the apical membrane of the surface epithelium in untreated mouse colonic tissues, whereas MD2 was found on the crypt epithelium. Following three cycles of AOM/DSS, enhanced levels of CD14, TLR4, and MD2 were seen in tumorous tissues compared with adjacent normal tissues or untreated tissues. Scale bar: 50 μm. n=10/group

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Modification, Expressing, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: CD14-dependent epithelial apoptosis is augmented in colonic tissues of TLR4-mut mice after induction of CO or LPS challenge. WT and TLR4-mut mouse colons were subjected to non-ischemic loop ligation for induction of CO or were mounted on organ baths for LPS challenge from the mucosal side. (a) Augmented colonic mucosal apoptosis was detected in TLR4-mut but not WT mice after induction of CO. Sham-operated (sham) colons were without ligation. (b) Increased gut-associated and Gram-negative bacterial counts were confirmed after induction of CO in both WT and TLR4-mut mice. Each data point represents the bacterial count from one animal, and the median values are shown as bars. (c) Elevated mucosa-associated LPS levels were observed after CO in both mouse strains. (d) Colonic tissues obtained from untreated mice were challenged from the mucosal side with LPS or PBS, and increased mucosal apoptosis was seen in TLR4-mut mice compared with WT mice. (e) Apoptotic TUNEL-positive cells (arrowheads) were detected on the colonic epithelium after LPS challenge in TLR4-mut, but not WT, mice. Scale bar: 50 μm. (f) Intracolonic administration of neutralizing anti-CD14 antibody, but not its isotype IgG2b, decreased LPS-induced cell apoptosis. (g) Binding of anti-CD14 to epithelial surfaces was confirmed by immunofluorescent staining (green). Scale bar: 50 μm. n=6–8/group. *P<0.05 versus WT; #P<0.05 versus sham or PBS

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Ligation, TUNEL Assay, Binding Assay, Staining

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: LPS/CD14-mediated epithelial apoptosis is prevented by TLR4 expression in primary mouse colonic organoids. (a) Representative images of colonic organoid cultures derived from colonic crypts of WT and TLR4-mut mice. Original magnification: × 200. (b) Transcript levels of LPS receptors (CD14, TLR4, and MD2) and Lgr5 in WT colonic organoids. (c) Apoptotic levels by LPS challenge in WT colonic organoids with gene silencing of TLR4 and/or CD14. Organoids were either LPS challenged or untreated as controls (CON). (d) Apoptotic levels in TLR4-mut colonic organoids with gene silencing of CD14. n=6/group. *P<0.05 versus respective CON

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Expressing, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: Apical exposure to LPS induces apoptosis in Caco-2 cells (CD14+TLR4−), but not in HT29 (CD14+TLR4+) or T84 (CD14-TLR4+) cells. The expression pattern of LPS receptors and death response to LPS were examined in three well-established human colorectal adenocarcinoma cell lines. (A) Levels of CD14, TLR4, and MD2 transcripts in control (CON) and LPS-challenged Caco-2, T84, and HT29 cells. Promonocytic THP-1 cells were used as positive controls. (B) Representative immunostaining of CD14 and TLR4 proteins in control (A and C) or LPS-exposed (B) cells. Confocal microscopic imaging in C indicates apical staining of CD14 or TLR4 (red) superimposed with nuclei (blue) and phalloidin-labeled F-actin (green). Scale bar: 5 μm. (C and D) Caco-2 cells were challenged with various doses of LPS for 24 h and displayed increased DNA fragmentation and caspase-3 activation in a dose-dependent manner. *P<0.05 and **P<0.005 versus 0 μg/ml LPS. (E) LPS-induced apoptosis was observed in Caco-2 cells, but not in T84 or HT29 cells. *P<0.05 versus CON. (F) Western blots showing increased caspase-3 cleavage after LPS challenge in Caco-2 cells, but not in T84 or HT29 cells. Experiments were repeated at least twice. Scale bar: 5 μm. n=4–6/group

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Expressing, Immunostaining, Imaging, Staining, Labeling, Activation Assay, Western Blot

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: CD14-mediated lipid signaling is involved in LPS-induced apoptosis in Caco-2 cells. (a) Quantification of TUNEL-positive cells in control (CON) and LPS-challenged groups. The increase in the percentage of TUNEL-positive cells following LPS challenge could be inhibited with neutralizing anti-CD14 antibody, but not with anti-TLR4 or isotype antibodies. (b) LPS-induced apoptosis was inhibited by anti-CD14 but not anti-TLR4. (c) siRNA-mediated gene silencing of CD14 and PKCζ prevented LPS-induced cell apoptosis. Western blotting confirmed the knockdown efficiency of CD14 and PKCζ, and indicated that MD2 levels were not affected by siRNA treatment. (d) Hyperphosphorylation of PKCζ, caused by LPS exposure, was reduced in CD14 knockdowns. Representative blots of total (t)- and phospho (p)-PKCζ after CD14 knockdown. The p-PKCζ to t-PKCζ ratio was quantified by densitometric analysis. (e) Pretreatment with D609 (a PC-PLC inhibitor), imipramine (imip; a SMase inhibitor), Gö6983 (a PKC inhibitor), or PKCζ pseudosubstrate (ps; a specific inhibitor of PKCζ) suppressed LPS-induced cell apoptosis. No effects were observed when vehicle (veh) or necrostatin-1 (nec1; a necroptosis inhibitor) were used. Experiments were repeated at least twice. *P<0.05 versus respective CON; #P<0.05 versus LPS or LPS+veh. n=4–6/group

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: TUNEL Assay, Western Blot

Journal: Cell Death and Differentiation

Article Title: LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

doi: 10.1038/cdd.2014.240

Figure Lengend Snippet: Expression of functional TLR4 inhibits LPS/CD14-induced apoptosis in Caco-2 cells. Caco-2 cells were transfected with mock plasmids or pMyc-huTLR4 expressing WT or mutant (Asp299Gly, Thr399Ile, or Pro714His) TLR4 before LPS challenge. (a) Apical expression of TLR4 proteins in Caco-2 cells was verified after transfection. Representative en face views of TLR4 staining (red) are shown in the upper panels, and superimposed confocal images of TLR4 (red), F-actin (green), and nuclei (blue) are shown in the lower panels. Scale bar: 10 μm. (b) Representative blots of TLR4, CD14, and MD2 in transfected cells. Expression of TLR4 did not affect CD14 or MD2 levels in control (CON) or LPS-challenged cells. (c) LPS-induced apoptosis was abolished in cells transfected with pMyc-huTLR4 harboring the WT TLR4 insert, but not with mock plasmids. Site-directed mutagenesis of TLR4 proteins eliminated the anti-apoptotic effects in Caco-2 cells. (d) Functional TLR4 signaling, defined as LPS-induced JNK and IκBα phosphorylation, was observed in Caco-2 cells transfected with WT TLR4, but not in cells harboring mutated TLR4. (e and f) Spheroid cultures of Caco-2 cells showed increase of apoptosis following LPS challenge, which was ablated by knockdown of CD14 or overexpression of TLR4. *P<0.05 versus respective CON. (g) HT29 cells underwent single or double knockdown of CD14, TLR4, or PKCζ, as verified by PCR. (h) LPS challenge induced elevated apoptosis in HT29 cells with TLR4 knockdown, whereas no change in apoptosis was observed in CD14 or PKCζ knockdowns or in double knockdowns. All experiments were repeated twice. *P<0.05 versus respective CON. n=4/group

Article Snippet: These included 120 μ M Z-DEVD-FMK (z-DEVD, a caspase-3 inhibitor; Merck), 10 μ g/ml PMB (a LPS antagonist; Sigma), 50 μ M D609 (a PC-PLC inhibitor; Sigma), 5 μ M imipramine (a SMase inhibitor; Sigma), 60 nM Gö6983 (a broad spectrum PKC inhibitor; Merck), 20 μ M inhibitory PKC ζ pseudosubstrate (Merck), 100 μ M necrostatin-1 (a necroptosis inhibitor; Sigma), 10 μ g/ml neutralizing mouse anti-human CD14 (R&D systems), 20 μ g/ml neutralizing mouse anti-human TLR4 (eBioscience, San Diego, CA, USA), or their respective IgG1and IgG2a isotype controls.

Techniques: Expressing, Functional Assay, Transfection, Mutagenesis, Staining, Over Expression