Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Autophagy is induced in MT cells. ( A ) Mutant UQCRB-expressing cell lines were examined by Western blotting using anti-UQCRB and anti-Myc antibodies. ( B ) HEK293 and MT cells treated with either 10% serum-containing medium or serum-free medium for 24 h and then analyzed by Western blot analysis using antibodies against LC3B and p62. shows treatment of either 10% serum-containing medium or serum-free medium for 4 h. ( C ) Confocal microscopy images of HEK293 and MT cells treated with serum-free medium for 24 h after immunostaining with anti-LC3 antibody. Scale bar, 50 μm. ( D ) Western blot analysis results of LC3B and p62 levels in cells after treatment with 5 μM chloroquine (CQ) treatment for 0–6 h. ( E ) HEK293 and MT cells were treated with either deionized water (DIW) or CQ for 6 h. Cell extracts were subjected to Western blotting using LC3B and p62 antibodies. The densitometric value of LC3-II was quantitated based on Western blot data and normalized to LC3-I levels. ( F ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 for 24 h. Scale bar, 20 μm. ( G ) Numbers of mRFP-LC3 puncta were counted in HEK293 and MT cells ( n = 8). The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. Data are presented as the mean ± SD; statistical significance was assessed using an unpaired t -test. ns, p > 0.05.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Confocal Microscopy, Immunostaining, Transfection, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: TRPML1-induced lysosomal Ca 2+ release and TFEB activation are increased in MT cells. HEK293 and MT cells were transfected with GCaMP3-TRPML1 encoding a lysosome-specific Ca 2+ probe, 40 µM ML-SA1, for 120 s. ( A ) Confocal microscopy images of GCaMP green fluorescence showing HEK293 and ( B ) MT cells. ML-SA1 treatment for 120 s. ( C ) Confocal microscopy images of HEK293 and MT cells transfected with EGFP-TFEB and treated with the DMSO control or A1938 for 6 h. ( D ) The number of cells with a nuclear (Nuc) or cytoplasmic (Cyt) TFEB localization ratio is represented in the graph. Scale bar, 20 μm. Statistical significance was assessed using Student’s t -test. ** p < 0.01.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activation Assay, Transfection, Confocal Microscopy, Fluorescence, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Lysosome activity in MT cells with or without A1938 treatment. ( A ) Confocal microscopy images and ( B ) numbers of acidic lysosome red puncta were counted in HEK293 and MT cells ( n = 15). The cells were treated with DMSO as a control or 30 µM A1938 for 24 h and then stained with LTR. Scale bar, 20 μm. ( C ) Confocal microscopy images of MT cells treated with DMSO as a control or 30 µM A1938 for 24 h, then the live cells were stained with 2 μg/mL acridine orange for 25 min and fixed. Scale bar, 20 μm. ( D ) Images of HEK293 and MT cell extracts treated with the DMSO control or 30 µM A1938 for 24 h in a serum-free medium, then subjected to Western blot analysis using antibodies against LAMP1. ( E ) Intensities of the LAMP1 immunoblot bands were normalized to β-actin expression. Western blot analysis out of three independent experiments. Statistical significance was assessed using Student’s t -test. ns, p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activity Assay, Confocal Microscopy, Control, Staining, Western Blot, Expressing

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: A1938 regulates autophagy flux by suppression of mROS generation in MT cells. ( A ) Cells treated with A1938 for 24 h in a serum-free medium. The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. ( B ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 and treated with DMSO, A1938, or Mito-Tempo (mTP) for 24 h. Scale bar, 20 μm. Quantification of data indicating red puncta (autolysosome) versus yellow puncta (autophagosome) is shown in ( C ). Values are the means ± SEM; statistical significance was assessed using a paired t -test. n = 10 cells, ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Control, Confocal Microscopy, Transfection

Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration