Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells without any treatment were used as the untreated control (CTL). All experiments were performed in triplicates. (a) Detection of autophagic populations. Cells were treated with rapamycin and then stained with acridin orange (AO) for flow cytometric detection of the acidic vesicular organelles (AVO) in autophagic cells. (b) Determination of percentages of autophagic populations after AO staining. Significant difference between CTL and a treatment time was indicated by * P <0.05 whereas significant difference between a treatment time and another treatment time was indicated by # P <0.05. (c) Western blotting to show the protein levels of LC3 I and LC3 II forms in both cell lines. Expression of β-actin was used as a loading control in Western blotting.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Staining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells treated with 200 nM rapamycin for 24 h were used as the treated control (CTL) SK-N-BE2 cells (red bars) and IMR-32 cells (blue bars). (a) Treatments (24 h): treated CTL cells, CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, 10 µM cisplatin, and 10 µM cyclophosphamide. As presented in the bar diagrams, combination of 50 nM LC3 shRNA plasmid and 25 µM GST in SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid and 25 µM GST in IMR-32 cells showed the best synergistic efficacy, as determined by the combination index (CI) value, for the highest decrease in residual cell viability. (b) Western blotting to show the effect of GST treatment on the expression of EGFR, a receptor tyrosine kinase, in both cell lines. Cells were treated with different doses (5, 10, 25 and 50 µM) of GST for 24 h. All experiments were performed in triplicates. Significant difference between CTL and a treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, shRNA, Plasmid Preparation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Combination index (CI) values for the concentrations of LC3 shRNA plasmid and GST in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells treated with 200 nM rapamycin for 24 h were used as the treated control (CTL). Then, single and combination therapies were performed for another 24 h. Treatment groups: treated CTL, CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, and untreated CTL. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. (a) Staining of cells with AO followed by fluorescence microscopy for detection of AVO in autophagic cells. (b) Flow cytometric analysis of the AO stained cells from all treatment groups for detection and determination of AVO in autophagic cells. (c) Presentation of amounts of autophagic cells in bar diagrams. Significant difference between treated CTL and another treatment or untreated CTL was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, shRNA, Plasmid Preparation, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: (a) Time-dependent changes in expression of LC3. Cells were first treated with 200 nM rapamycin for 24 h and then transfected with LC3 shRNA plasmid for 0, 6, 12, and 24 h or treated with 10 µM 3MA for 24 h. (b) Changes in expression of key molecules involved in autophagy. Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. Representative Western blots show changes in expression of LC3 I and LC3 II, Beclin 1, TLR-4, Myd88, p62, mTOR, and β-actin.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, 3MA (10 µM) + GST, and WM (5 µM) + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 μM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 μM GST alone or in combination in IMR-32 cells. (a) In situ Wright staining was performed to show morphological features of apoptosis in both cell lines. (b) Annexin V-FITC/PI binding assay followed by flow cytometry of the cells to show accumulation of apoptotic population (quadrant A4). (c) Determination of amounts of apoptosis based on flow cytometry. All experiments were performed in triplicates. Significant difference between CTL shRNA plasmid and another treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation, In Situ, Wright Stain, Binding Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. Representative Western blots show levels of expression of Bax, Bcl-2, cytochrome c, COX-4, active caspase-3, PARP fragment, and β-actin. Expression of COX-4 (an internal control in mitochondria) was used for monitoring mitochondrial release of cytochrome c into the cytosol.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Both SK-N-BE2 and IMR32 cells were harvested, counted, and suspended in an equal volume of highly-concentrated Matrigel and then cell suspension (100 µl) was injected in nude mice for development of xenografts during 3 weeks. Xenograft bearing animals were pre-treated with rapamycin (2 mg/kg body weight/day) for 7 days and then randomly assigned to four therapeutic treatment groups: CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. Animals were daily injected with CTL shRNA plasmid (50 µg/mouse), LC3 shRNA plasmid (50 µg/mouse), or/and GST (2 mg/kg body weight) for 15 days. The data are representative of at least 3 independent experiments using 4 mice in each therapeutic treatment group. (a) Mice with SK-N-BE2 and IMR-32 xenografts. (b) Tumors after surgical removal from the animals. (c) Estimation of tumor volumes in both neuroblastoma xenografts. Significant difference between CTL shRNA plasmid and another treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05. (d) H&E staining to show hisopathological alterations in tumor sections after the therapeutic treatments. (e) Representative Western blots to show changes in expression of LC3 II, Beclin 1, Bax, Bcl-2, active caspase-3, PARP fragment, andβ-actin.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Suspension, Injection, shRNA, Plasmid Preparation, Staining, Western Blot, Expressing

Journal: Frontiers in immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome.

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: FIGURE 5 | CVL inhibited the NLRP3 inflammasome by enhancing autophagy. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 µM). The levels of LC3, p62, and ATG5 in the cell lysates were measured by Western blot. (B) J774A.1 macrophages were incubated with CVL (20 µM, 12 h) or rapamycin (100 nM, 4 h). Accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy. (C) LPS-primed J774A.1 macrophages were incubated for 0.5 h with 3-MA (5 mM) and CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. (D) LPS-primed wild-type (scramble) and LC3-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 µM) followed by incubation with CC (100 µg/ml) or MSU (100 µg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. The expression levels of LC3 in the wild-type and LC3-knockdown J774A.1 macrophages were measured by Western blot. (E,F) LPS-primed wild-type and LC3-knockdown J774A.1 macrophages were incubated for 0.5 h with CVL (20 µM) followed by incubation with MSU (100 µg/ml) for an additional 24 h. Mitochondrial integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green (E), and mitochondrial ROS was measured by staining with MitoSOX (F). The MFI of Mitotracker Deep Red are expressed as the means ± SD for three separate experiments. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Article Snippet: For generating protein knockdown J774A.1 macrophages, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (cat. No.: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (cat. No.: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Knockdown, Clone Assay, Expressing, Staining

Journal: Frontiers in immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome.

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: FIGURE 6 | CVL inhibited the NLRP3 inflammasome by enhancing the Sirt1/autophagy axis. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 µM). The Sirt1 levels in the cell lysates were measured by Western blot. (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with EX527 (10 µM) and CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The IL-1β levels in the culture medium were measured by ELISA. (C) LPS-primed wild-type (scramble) and Sirt1-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 µM) followed by incubation with CC (100 µg/ml) for an additional 24 h. The IL-1β levels in the culture medium were measured by ELISA. The expression levels of Sirt1 in the wild-type and Sirt1-knockdown J774A.1 macrophages were measured by Western blot. (D,E) J774A.1 macrophages were incubated for 0.5 h with EX527 (10 µM) followed by incubation with CVL (20 µM) for an additional 12 h. The levels of LC3 and p62 in the cell lysates were measured by Western blot (D), and the accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy (E). The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: For generating protein knockdown J774A.1 macrophages, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (cat. No.: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (cat. No.: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Clone Assay, Expressing, Confocal Microscopy