Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The association of p62 with cytosolic Salmonella increases shortly after initiation of intracellular replication. (A) GFP-p62- or GFP-expressing HeLa cells were infected with wild-type (WT) or ∆ sifA Salmonella . Cells at 1 hpi and 5 hpi were stained with LAMP-1 (red) and DAPI (cyan). “Zoom” represents a magnified picture of the boxed area. Scale bar, 10 µm. (B to D) Quantification of the percentage of p62 + WT or p62 + ∆ sifA bacteria (B), the percentage of p62 + WT or p62 + ∆ sifA bacteria that are not associated with LAMP-1 (C), and the percentage of LAMP-1 − WT or LAMP-1 − ∆ sifA bacteria (D) in GFP-p62-expressing cells at 1, 5, and 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Staining, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Dynamic association of p62 and/or LC3 correlates with Salmonella replication in the cytosol of HeLa cells. (A) GFP-LC3-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 min), cells were imaged at 30-s intervals with a spinning-disc confocal microscope. A series of live-cell imaging data is shown, and the elapsed time (minutes) is shown in the corner of each picture. Arrowheads 1, 2, 3, and 4 indicate replicating bacteria, whereas arrowheads 5 and 6 indicate nonreplicating bacteria. Scale bar, 10 µm. (B) GFP-p62-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 h), the cells were imaged at 20-s intervals with a spinning-disc confocal microscope. Boxed areas indicate where p62 associates with cytosolic Salmonella . Arrows 1 to 5 indicate bright bacteria that are not replicating; arrowheads 6 and 7 in column 1 (0 h) indicate light-red bacteria that start to replicate. Scale bar, 5 µm.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Microscopy, Live Cell Imaging, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: p62 and autophagy favors cytosolic replication of Salmonella . (A) Western blot analyses of HeLa cells treated with control siRNA (si-NT) or siRNA targeting p62 (si-p62), LC3 (si-LC3), Atg5 (si-Atg5), and p62 and LC3 (si-p62+LC3). These cells were infected with WT Salmonella . Calnexin was used as a loading control. (B) Microscopic analyses of infected cells containing 1 to 5, 6 to 10, 11 to 20, or greater than 20 bacteria per cell at 2 hpi (top) and 6 hpi (bottom). The number of bacteria in at least 50 infected cells was enumerated, and the percentage of cells containing the indicated number of bacteria was presented (mean ± SD, n = 3). (C) Replication of Salmonella was compared in HeLa cells used in panel A. The fold replication was calculated as fold increases of the number of bacteria at late time points compared to the number of bacteria at 2 hpi (mean ± SD, n = 3). (D) HeLa cells were treated with control siRNA (si-NT) or siRNA targeting TBK1 (TANK-binding kinase-1) (si-TBK1), TBK1 and LC3 (si-TBK1+LC3), and TBK1 and ATG5 (si-TBK1+ATG5) and infected with Salmonella for 2 h (top) or 6 h (bottom). The percentage of cells containing the indicated number of bacteria was quantified as in panel B (mean ± SD; n = 3). (E) GFP-LC3-expressing HeLa cells were treated with si-NT, si-TBK1, si-TBK1+LC3, or si-TBK1+Atg5. The percentage of LC3 + bacteria or LAMP-1 − bacteria at 5 hpi was quantified (mean ± SD, n = 3). (F) Western blot analyses of HeLa cells used in panel D.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Western Blot, Control, Infection, Bacteria, Binding Assay, Expressing

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The effector SopB is required for Salmonella to associate with autophagosomes. (A) GFP-LC3-expressing cells were infected with the indicated strains. The ∆ sopB /pACDE mutant expresses the wild-type SopB in a ∆ sopB background. Unlike other strains that are individually used to infect cells, ∆ invA and ∆ sipB bacteria were used in a coinfection model. The percentage of LC3 + bacteria at 5 hpi was quantified for each strain (mean ± SD, n = 3). (B) Western blot analysis of HeLa cells used in panel A. (C) HeLa cells treated with si-NT or si-LC3 were infected with the indicated strains. The fold replication of these strains at 6 hpi was determined (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Mutagenesis, Bacteria, Western Blot

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Cells containing hyperreplicating bacteria and showing decreased autophagy are positive for caspase-1 and caspase-3/7 activation and undergo cell death. (A) GFP-p62-expressing HeLa cells were infected with Salmonella for 8 h and stained with DAPI (blue). A cell containing hyperreplicating bacteria (more than 50 bacteria per cell, i.e., HRBC cells) and showing abnormal nuclear morphology is shown. Scale bar, 10 µm. (B) At least 100 HRBC cells were divided into two groups. The first group exhibited a low level of p62 signals colocalized with Salmonella (<1% of intracellular bacteria), and the second group exhibited a relatively high level of p62 signals (>2% of intracellular bacteria). The number of cells showing abnormal nuclear morphology (such as chromatin condensation and DNA fragmentation) was further quantified and expressed as the percentage of cells with abnormal nuclei in each group of cells (mean ± SD, n = 3). (C) A GFP-p62-expressing HRBC cell shows a relatively enhanced autophagy and normal nuclear morphology. Scale bar, 10 µm. (D) Cells stably expressing monomeric red fluorescent protein (mRFP)-LC3 were infected with WT Salmonella for 7 h and stained with DAPI (blue) and active caspase-1 probe FAM-YVAD-FMK (green) or caspase-3/7 probe FAM-DEVD-FMK (similar to caspase-1 staining [data not shown]). (E) Quantification of the percentage of casapase-1 (left)- or caspase-3/7 (right)-positive cells in uninfected cells, HRBC cells showing decreased autophagy, and HRBC cells showing normal autophagy at 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Activation Assay, Expressing, Infection, Staining, Stable Transfection

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: A proposed model for p62-dependent autophagy favoring Salmonella replication in the cytosol of HeLa cells. In this proposed model, Salmonella likely acquires nutrients supplied by autophagy for its replication. At least three populations of bacteria are present in infected HeLa cells. The first population of bacteria remains in intact Salmonella -containing vacuoles (SCVs). These bacteria replicate slowly, as they do not associate with autophagosomes and are difficult to acquire nutrients from the cytosol. The second population of bacteria is partially exposed to the cytosol through the gaps between damaged SCVs . These bacteria are ineffectively coated with ubiquitin and associated with autophagosomes to a low level. They acquire a limited amount of nutrients for replication and replicate faster than the first population of bacteria. The third population of bacteria is completely exposed to the cytosol. These bacteria are effectively coated with ubiquitin and associated with autophagosomes to a high level. They acquire sufficient nutrients from the cytosol and replicate the fastest. Notably, some cells containing the third population of bacteria will undergo cell death and detach from the epithelial layer, resulting in the release of hyperreplicating bacteria that are capable of initiating secondary infections in neighboring cells. The whereabouts of the first and second populations of bacteria at 8 hpi were not determined (ND).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Infection, Ubiquitin Proteomics

Journal:

Article Title: Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

doi: 10.1002/ijc.25059

Figure Lengend Snippet: Effect of betulinic acid on constitutively active STAT3. A, The structure betulinic acid. B, Betulinic acid suppresses p-STAT3 levels. U266 cells (2 × 106/mL) were treated with the indicated concentrations of betulinic acid for 4 h, then whole-cell extracts were prepared, and 30 μg of protein was resolved on 7.5% SDS-PAGE gel, electro transferred onto nitrocellulose membranes, and probed for p-STAT3 C, Betulinic acid suppresses p-STAT3 levels in a time-dependent manner. U266 cells (2 × 106/mL) were treated with 50 μM betulinic acid for the indicated durations and analyzed for p-STAT3 levels. D, Betulinic acid causes inhibition of STAT3 translocation to the nucleus. U266 cells (1 × 105/mL) were incubated with or without 50 μM betulinic acid for 4 h and then analyzed for the intracellular distribution of STAT3 by immuno-cytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/mL) for 5 min. E, Betulinic acid inhibits STAT3 DNA binding. U266 cells (2 × 106/mL) were treated with the indicated concentrations of betulinic acid for 4 h and analyzed for STAT3 DNA binding by EMSA. F, Cells were treated with 50 μM betulinic acid for the indicated durations and analyzed for nuclear STAT3 levels by EMSA. CV, cell viability

Article Snippet: We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA).

Techniques: SDS Page, Inhibition, Translocation Assay, Incubation, Immunocytochemistry, Binding Assay

Journal:

Article Title: Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

doi: 10.1002/ijc.25059

Figure Lengend Snippet: Effect of betulinic acid on c-Src, p-JAK1 and JAK2 activity. U266 cells (2 × 106/mL) were treated with betulinic acid (50 μM) for indicated time points, whole-cell extracts were prepared, and 30 μg aliquots of those extracts were resolved on 10% SDS-PAGE, electro transferred onto nitrocellulose membranes, and probed for either p-Src or p-JAK1 antibody. The same blots were stripped and reprobed with Src or JAK1 antibody to verify equal protein loading. For JAK2 kinase activity U266 cells (4 × 106/mL) were treated betulinic acid, whole-cell extracts were prepared, and kinase assay was performed.

Article Snippet: We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA).

Techniques: Activity Assay, SDS Page, Kinase Assay

Journal:

Article Title: Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

doi: 10.1002/ijc.25059

Figure Lengend Snippet: A, Pervanadate reverses the p-STAT3 inhibitory effect of betulinic acid. U266 cells (2 × 106/mL) were first treated with the indicated concentration of pervanadate for 30 min followed by 50 μM betulinic acid for 4 h, and whole-cell extracts were subjected to Western blot analysis for p-STAT3 and STAT3. B, Betulinic acid induces the levels of SHP-1 in U266 cells. U266 cells (2 × 106/mL) were treated with betulinic acid (50μM) for indicated time points. After treatment, whole-cell extracts were prepared and 30 μg portions of those extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. C, Effect of SHP-1 knockdown on betulinic acid -induced expression of SHP-1. SCC4 cells (1 × 105/ml) were transfected with either SHP-1-specific or scrambled siRNA (50 nM). After 48 h, cells were treated with 50 μM betulinic acid for 4 h, and whole-cell extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. Transfection with SHP-1 siRNA reverses betulinic acid -induced suppression of STAT3 activation. The same whole-cell extracts were subjected to p-STAT3 and STAT3. D,Effect of SHP-1 knockdown on betulinic acid -induced cell death. SCC4 cells were transfected with either SHP-1-specific or scrambled siRNA (50 nM). After 48 h, cells were treated with 25 μM betulinic acid for 24 h, live and dead assay was performed and 20 random fields were counted.

Article Snippet: We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA).

Techniques: Concentration Assay, Western Blot, Knockdown, Expressing, Transfection, Activation Assay

Journal:

Article Title: Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

doi: 10.1002/ijc.25059

Figure Lengend Snippet: A, Betulinic acid downregulates the expression of antiapoptotic proteins. U266 cells (2 × 106/mL) were treated with 25 μM betulinic acid for indicated time intervals, whole-cell extracts were prepared, and 30 μg portions of those extracts were resolved on 10% SDS-PAGE, and probed against bcl-2, bcl-xL, survivin, and cyclin D1 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. B, U266 cells were treated with 25 μM betulinic acid for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis against the caspase-3 and PARP antibody. The same blot was stripped and reprobed with β-actin antibody to show equal protein loading. C, Betulinic acid causes accumulation of cells in the sub-G1 phase. U266 cells (2 × 106/mL) were synchronized by incubation overnight in the absence of serum and then treated with 25 μM betulinic acid for the indicated times, after which the cells were washed, fixed, stained with propidium iodine, and analyzed for DNA content by flow cytometry.

Article Snippet: We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, SDS Page, Western Blot, Incubation, Staining, Flow Cytometry

Journal:

Article Title: Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

doi: 10.1002/ijc.25059

Figure Lengend Snippet: A, Cytotoxic effects of betulinic acid on MDA-MB-231, MCF-7, U266, MM.1S and Du-145, PC-3 cells. Cells were plated in triplicate, treated with indicated concentrations of betulinic acid for 72 h, and then subjected to MTT assay to analyze the viability of cells. B, Over expression of constitutive STAT3 rescues A293 cells from betulinic acid -induced cytotoxicity. First, A293 cells were transfected with constitutive STAT3 plasmid. After 24 h of transfection, the cells were treated with indicated concentration of betulinic acid for 24 h, and then the cytotoxicity was determined by Live/Dead assay and 20 random fields were counted. C, Betulinic acid potentiates the apoptotic effect of bortezomib and thalidomide. U266 cells (5000 cells/well) were treated with indicated amounts of betulinic acid for 4h followed by indicated amounts of thalidomide or bortezomib for 24 h at 37°C. Cell viability was assessed by MTT uptake method. For live and dead assay, U266 cells (1 × 106/mL) were treated with 25 μM betulinic acid and 10 μg/mL thalidomide or 20 nM bortezomib alone or in combination for 24 h at 37°C. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope and 20 random fields were counted.

Article Snippet: We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA).

Techniques: MTT Assay, Over Expression, Transfection, Plasmid Preparation, Concentration Assay, Live Dead Assay, Staining, Fluorescence, Microscopy