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

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: List of antibodies and isotypes used for cell‐ and cell derived large EV flow cytometric analysis. The antibodies were titrated against their matching isotype controls prior use and applied according to the manufacturers’ instructions.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Antibody validation on cultured THP‐1 cells for EV detection and general used large EV gating strategy . (a) In vitro human THP‐1 monocyte cells were treated with PMA alone or PMA & LPS (each n ≥ 3), increasing CD14 expression. (b) Expression of CD14 on surface of AnnV + large EVs (lEVs) derived from non‐stimulated THP‐1 cells and from PMA alone or PMA & LPS stimulated THP‐1 cells. AnnV is used as a general large EV marker. Values given as mean with SEM. Three column statistical analysis was done by Kruskal–Wallis test including Dunn's post hoc test for multiple comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (c) Thresholds testing for cell culture and serum derived large EV detection and gating. Prior use of flow cytometry gates sensitivity of used flow cytometer was set taking advantage of 1000, 500 and not detectable 200 nm beads. (d) Gating strategy for the detection of CD14‐APC on AnnV + large EVs controlled by matching REA isotype control.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Biomarker Discovery, Cell Culture, In Vitro, Expressing, Derivative Assay, Marker, Flow Cytometry, Control

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Semi quantification of human serum derived AnnV + CD14 + large EVs . (a) Workflow SOP for isolation of large EVs (lEVs) from 1 mL of human serum collected from polytrauma patients (ISS > 15, at day 0, at day 1 and day 7 post trauma). (b) AnnV + CD14 + large EVs counts in small EVs in polytrauma (ISS > 15) with internal organ damage (polytrauma OD) versus polytrauma (ISS ≥ 15) without internal organ damage (polytrauma w/o OD) upon admission (day 0). (c) AnnV + CD14 + large EVs counts in polytrauma (ISS > 15) polytrauma OD versus polytrauma w/o OD (ISS > 15) 24 h post trauma (day 1). (d) Day 1, corresponding AUROC, sensitivity, specificity, and cut‐off value, respectively. (e) AnnV + CD14 + large EVs counts in small EVs in polytrauma (ISS ≥ 15) polytrauma OD versus polytrauma w/o OD (ISS ≥ 15) 7 days post trauma (day 7). Values given as median with 95% CI. Three column statistical analysis was done by Kruskal–Wallis test including Dunn's post hoc test for multiple comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001; not significant, n as indicated). Horizontal dotted line in panels B, C and E indicates calculated associated cut‐off (summarized in Table ).

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay, Isolation

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: AnnV + CD14 + large EVs kinetics at day 1 and day 7 in polytrauma (ISS > 15) and correlation with the Injury Severity Score (ISS) . (a) depicted are pairs of AnnV + CD14 + large EVs (lEVs) measured at day 1 and day 7 of enrolled polytrauma (ISS > 15). Values given as median. Statistical significance was assessed by two tailed paired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (b) Spearman ( r sp ) correlation between percentages of AnnV + CD14 + large EVs and ISS ( n = 24). p and r sp values as indicated.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Two Tailed Test

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Summarized data FACS of AnnV + CD14 + large EVS in polytrauma including associated SEM, percentage change and statistics at day 1.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques:

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: List of antibodies used for human derived large EV surface marker analysis. The antibody was added according to manufacturer's recommendations and titrated against the matching isotype control prior use.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay, Marker, Control

Journal: iScience

Article Title: Clonal hematopoiesis is associated with cardiovascular events in patients with stable coronary artery disease

doi: 10.1016/j.isci.2024.109472

Figure Lengend Snippet:

Article Snippet: CD14-phycoerythrin, clone REA599 , Miltenyi Biotec, Bergisch Gladbach, Germany , Cat#130-110-519; RRID: AB_2655052.

Techniques: Enzyme-linked Immunosorbent Assay, Software, Flow Cytometry