Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: EPEC induces internalization of Crb polarity proteins Crb3 and Pals1, but not Patj in intestinal epithelial cells. SKCO-15 cells were plated on Transwells and infected on the apical side, or not (UI), with EPEC for 2h. Immunolocalization of endogenous Crb3/Pals1/Patj and E-cad was performed. (A and B) Representative confocal images and quantification of the fluorescence intensity are shown. Arrows indicate the loss of membrane-associated Crb3 and Pals1 induced by EPEC. Hoechst was used to mark the nuclei (blue) in all the images. Scale bar, 10 μm. Data represent the mean ± SEM (n=3); ***P < 0.001 values were calculated using ANOVA Tukey’s Multiple Comparison Test.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Fluorescence

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: EPEC and C. rodentium redistribute Crb complex proteins from the plasma membrane to the cytoplasm of murine colonocytes. Mice were infected with EPEC or C. rodentium strain by oral gavage, sacrificed on day 3 or day 10 post-infection respectively; intestinal tissues were processed for immunofluorescence. (A–D) Representative confocal images of Crb3/Pals1/Patj and quantification of the fluorescence intensity of the colonic tissues are shown. Scale bar, 40 μm. Data represent the mean ± SEM (n=3); ***P < 0.001 values were calculated using ANOVA Tukey’s Multiple Comparison Test.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Immunofluorescence, Fluorescence

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: Deletion of espF protects against Crb3 mislocalization while deletion of either map or espF attenuates the internalization of Pals1. SKCO-15 cells were plated on Transwells and infected apically with wild-type EPEC, ΔespF, Δmap, or complemented strains (ΔespF/pespF and Δmap/pmap) for 2h. Cells were immunostained for Crb3/Pals1/Patj and E-cad was used to label the lateral membrane. (A–F) Representative confocal images and quantification of the fluorescence intensity of membrane, cytoplasm, and total protein are shown. Arrows show the absence of membrane-associated Crb3 and Pals1 in infected monolayers. Scale bar, 10 μm. Data represent the mean ± SEM (n=3); ***P < 0.001 values were calculated using ANOVA Tukey’s Multiple Comparison Test.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Fluorescence

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: Ectopic expression of EspF, but not Map, displaces Crb3 from the membrane. A) espF-HA or map-HA was cloned into the doxycycline-inducible pRetroX-Tight-Pur vector and transfected into SKCO-15 cells. Tet-On SKCO-15 cells were plated in absence of doxycycline (−dox) and transgene expression was induced with doxycycline (+dox) for 3 days. B) SKCO-15 cells were transiently transfected or not (control), with GFP-vector or GFP-EspF. Ectopic expression of EspF or Map was evaluated by immunodetection of HA (Green) or GFP (A and B, respectively). The impact of EspF or Map on localization of Crb3 and Patj was evaluated. Scale bar, 10 μm.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Transfection, Immunodetection

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: Depletion of espF or treatment with dynasore inhibits EPEC-induced cytoplasmic internalization of Crb3. (A–B) SKCO-15 cells were infected with wild-type EPEC, ΔespF or complemented strain (ΔespF/pespF) for 4h and processed for immunodetection of Crb3 and Rab5. (A and B) Representative confocal images and quantification of the area of the co-localization vesicles Crb3/Rab5 are shown. Data represent the mean ± SEM (n=3); ***P < 0.001 values were calculated using ANOVA Tukey’s Multiple Comparison Test. C) SKCO-15 cells were plated in Transwells and treated with DMSO or Dyn, then cells were infected or not (UI) with EPEC for 2h. Samples were processed for immunodetection of Crb3. Scale bar, 10 μm.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Immunodetection

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: The interaction of EspF with SNX9 is crucial for Crb3 endocytosis. SKCO-15 cells were infected with wild-type EPEC, ΔespF, or ΔespF complemented to express specific site-directed EspF mutations (ΔespF/pSer47A, ΔespF/pSer47/50A and ΔespF/pespFD3) for 2h. (A and B) Representative images of Crb3 localization and quantification of the fluorescence intensity are shown. Only infection with ΔespF and ΔespF/pespF-D3, which cannot bind SNX9, preserved Crb3 localization. Scale bar, 10 μm. Data represent the mean ± SEM (n=3); ***P < 0.001 values were calculated using ANOVA Tukey’s Multiple Comparison Test.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Fluorescence

Journal: Cellular microbiology

Article Title: EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

doi: 10.1111/cmi.12757

Figure Lengend Snippet: Model depicting the effect of EPEC on apical-basal polarity. Polarized epithelial cells consist of the apical membrane facing the lumen and the basolateral domain contacting the underlying basement membrane. The apical polarity complex Crb (Crb3/Pals1/Patj) localizes at the TJ (Control). i) During EPEC infection, the injected EPEC effectors, including EspF and Map, induce the endocytosis of polarity proteins Crb3 and Pals1 and the basolateral protein Na+/K+ ATPase. ii) Increased endocytosis of Crb3, Pals1 and likely other polarity proteins leads to a loss in the apical-basal polarity, as demonstrated by the redistribution of Na+/K+ ATPase to the apical membrane.

Article Snippet: Antibodies and Reagents Antibodies used include (species-antigen): rat-Crb3 (ab180835, Abcam), rabbit-Patj (ab102113, Abcam), rabbit-Pals1 (07-708, Millipore), rabbit-ZO-1 (61-7300, Zymed), rabbit-EspF (Hecht Lab), rabbit-cleaved caspase-3 (9661S, Cell Signaling), rabbit-actin (A2066, Sigma), rabbit-HA (C29F4, Cell Signaling), rabbit-GFP ( {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 , Life Technologies), mouse-Rab5 (sc-46692, Santa Cruz), mouse-E-cad (610181, BD), mouse-occludin (33-1500, ThermoFisher Scientific), mouse-Na + /K + ATPase (610992, BD), Phalloidin Alexa Fluor 568 (Life Technologies).

Techniques: Infection, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: IL‐38 was increased in mice after MI. IL‐38 mRNA and protein expression levels were assessed by real‐time PCR and Western blot, respectively. Relative mRNA expression of IL‐38 in the border zone (A) and remote zone (B) of infarcted hearts in C57BL/6 at different time points post‐MI. Representative image of western blot for IL‐38 protein expression levels in the border zone (C) and remote zone (D) of infarcted hearts in C57BL/6 at different time points post‐MI. E, IL‐38 expression was reflected by immunohistochemical staining with IL‐38 antibody in infarcted heart at different time points post‐MI. (sham n = 6, 24 h n = 8, 48 h n = 8, 7 d n = 8).* P < .05 vs sham; ** P < .01 vs sham

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: Cardiomyocytes are responsible for producing IL‐38 in infarcted heart. A, Representative images of infarcted heart sections stained with antibodies against cardiomyocyte marker α‐actinin (green), macrophage marker CD68 (green), and the cytokine IL‐38(red), and a nuclear stain (blue). Scar bar: 50 μm (each time point n = 8). B, IL‐38 mRNA expression levels in cultured cardiomyocytes with H 2 O 2 stimulation for 6h and 12 h. C, IL‐38 protein expression levels in cultured cardiomyocytes with H 2 O 2 stimulation for 6 and 12 h (control n = 6, 6 h n = 8, 12 h n = 8). D, Real‐time analysis of Bcl‐2 and Bax mRNA levels after incubation with rIL‐38 (50 ng/mL) for the indicated times. The results are expressed as the Bax/Bcl‐2 ratio (control n = 6, 6 h n = 8, 12 h n = 8). * P < .05 vs sham; ** P < .01 vs sham

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: Staining, Marker, Expressing, Cell Culture, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: IL‐38 improves left ventricular function post‐MI. A, Survival analysis in PBS‐treated and IL‐38‐treated mice after MI within 28 days (sham n= 15, MI+PBS n=40 and MI+IL‐38 n = 40). B, Representative M‐mode echocardiography images of the left ventricle on day 28 post‐MI in different groups. C‐F, Ejection fraction (EF), ventricular end‐diastolic diameter (LVEDD), left ventricular end‐systolic diameter (LVESD) and fractional shortening (FS) on day 7 and day 28 post‐MI (sham n = 6, MI+PBS n = 8 and MI+IL‐38 n = 8). * P < .05 vs sham; ** P < .01 vs sham; # P < .05 vs MI+PBS; ## P < .01 vs MI+PBS

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: IL‐38 inhibited cardiomyocyte apoptosis and cardiac fibrosis in vivo. A, Representative images of terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL)‐stained heart sections from different experimental groups 1 and 28 d post‐MI. TUNEL (green) and 4, 6‐diamidino‐2‐phenylindole (blue) staining of nuclei in apoptotic cardiomyocytes (red) in the peri‐infarct zone. Scar bar: 100 μm. B, Quantitative analysis of the percentages of TUNEL‐positive nuclei (each group n = 6). C, Real‐time PCR determined mRNA expression levels of Bax and Bcl‐2 in the in infarcted heart on day 1 after MI. The results were also expressed as ratio of Bax/Bcl‐2 (n = 4). D, Representative Masson’s trichrome staining images of collagen deposition (blue) in both infarct and remote areas on day7 post‐MI. E, The extent of fibrosis, as assessed by the fibrotic area/left ventricle and interstitial fibrosis, was compared among the different groups (each group n = 6). ** P < .01 vs sham; # P < .05 vs MI+PBS; ## P < .01 vs MI+PBS

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: In Vivo, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: IL‐38 inhibits inflammatory response in the infarcted heart. A, Representative images of haematoxylin and eosin (HE) staining, infiltration of myeloperoxidase (MPO + ) neutrophils and mouse CD68 + macrophages in the border area of infarcted hearts. Images for HE staining and neutrophils are from day 3 after MI, and images for macrophages are from day7 post‐MI. B, Infiltration of neutrophils and macrophages were compared between the different groups at set time points (each group n = 6). C–F, Analysis of mRNA levels of IL‐6, IL‐1β, TNF‐α and IL‐17A on day 3 and 7 after MI. Data are depicted as fold changes vs sham and shown as the mean ± SEM of 3‐6 independent experiments. ** P < .01 vs sham; # P < 0.05 . vs MI+PBS; ## P < .01 vs MI+PBS

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: The role of IL‐38 on DCs. Bone marrow‐derived DCs were cultured in the absence of stimulus (imDCs) or in the presence of 100 ng/mL LPS (mDCs) or/and 100 ng/mL LPS plus 50 ng/mL IL‐38. DCs were stained with specific antibodies against major histocompatibility complex class II (MHC‐II), CD40 and CD86 and analyzed by flow cytometry. A, Comparison of the CD45 + CD11b + CD11c + cells in infarcted heart. B, Comparison of the CD11c + MHC‐II + , CD11c + CD40 + and CD11c + CD86 + cells in different cultured DCs. C, TNF‐α, IL‐23 and IFN‐γ expression levels were assessed by ELISA in cultured supernatants of different DCs. D, IDO and IL‐10 mRNA expressions in cultured DCs.( each group n = 4 and repeat at least three times).* P < .05, ** P < .01 vs sham

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: Derivative Assay, Cell Culture, Staining, Flow Cytometry, Comparison, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

doi: 10.1111/jcmm.14741

Figure Lengend Snippet: IL‐38 plus TNI treated DCs augment the percentage of regulatory T cells in vitro. We used small doses of LPS (10 ng/mL) and TNI (1 μg/mL) to induce the tolerance of DCs. A‐B, IL‐10 and IDO mRNA expression levels in cultured DCs. C‐E, IL‐23, TNF‐α, and IFN‐γ expression levels were assessed by ELISA in cultured supernatants of different DCs. F, Splenic CD4 + T cells (1 × 10 6 cells/mL) were cultured for 3 d in the presence of medium alone or combined with imDCs, mDCs, or tDCs (2 × 10 5 cells/mL). The cells were stained with anti‐CD4, anti‐CD25, and anti‐Foxp3 and analyzed by flow cytometry. G‐J, Analysis of the expression levels of IFN‐γ, IL‐17A, IL‐22 and IL‐10 in culture supernatants of CD4 + T cells using ELISA in different groups. (each group n=4 and repeat at least three times). * P < .05,** P < .01

Article Snippet: The following primary antibodies were used: anti‐rat GAPDH (Immunoway, USA) and anti‐mouse IL‐38 (R&D Systems, USA).

Techniques: In Vitro, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Biology Open

Article Title: Immunofluorescent detection of protein CoAlation in mammalian cells under oxidative stress

doi: 10.1242/bio.061685

Figure Lengend Snippet: IF detection of anti-CoA mAb immunoreactive signal in HEK293 (A) and HEK293/Pank1β (B) cells. Cells were untreated or treated with 0.5 mM H 2 O 2 before being fixed with formalin, permeabilised, and processed for immunofluorescence confocal microscopy using anti-CoA mAb (1F10) and FITC-anti-mouse secondary antibodies (green). DAPI (blue) was used to visualise the nuclei. Scale bars: 10 µm.

Article Snippet: They were then incubated for 2 h at RT or overnight at 4°C with 1F10 anti-CoA mAbs (1 μg/ml) and either rabbit anti-VDAC antibody (Cell Signalling Technology #4866, 1:4000 dilutions), or rabbit anti-PARP antibody (Cell Signalling Technology #9532, 1:6000 dilutions), or rabbit polyclonal anti-S6K1 C-terminal antibodies (1:4000 dilutions) have been previously described , all diluted in TBST.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Biology Open

Article Title: Immunofluorescent detection of protein CoAlation in mammalian cells under oxidative stress

doi: 10.1242/bio.061685

Figure Lengend Snippet: IF detection of anti-CoA mAb immunoreactive signal in MCF7 (A) and MDA-MB-231 (B) cells. Cells were untreated or treated with 0.5 mM H 2 O 2 before being fixed with formalin, permeabilised, and processed for immunofluorescence confocal microscopy using anti-CoA mAb (1F10) and FITC-anti-mouse secondary antibodies (green). Nuclei were visualised by DAPI staining (blue). Scale bars: 10 µm.

Article Snippet: They were then incubated for 2 h at RT or overnight at 4°C with 1F10 anti-CoA mAbs (1 μg/ml) and either rabbit anti-VDAC antibody (Cell Signalling Technology #4866, 1:4000 dilutions), or rabbit anti-PARP antibody (Cell Signalling Technology #9532, 1:6000 dilutions), or rabbit polyclonal anti-S6K1 C-terminal antibodies (1:4000 dilutions) have been previously described , all diluted in TBST.

Techniques: Immunofluorescence, Confocal Microscopy, Staining