mrx ip gfp lc3 rfplc3δg vector  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence and late clinical metastasis in a Beclin-1-dependent manner

doi: 10.1101/2022.05.05.490726

Figure Lengend Snippet: A) Growth of cells seeded at 10 4 cells per well in cell-repellent surface 12-well plate and cultured for 10 days in regular growth medium supplemented with none (none) and 50% conditioned medium (CM) from senescent mouse embryo fibroblasts cultures. The stars indicate the statistical significance of the differences with cells grown in regular growth medium. Labels indicate mean cell number. B) Representative pictures of cells grown for 7 days in cell-repellent surface plates in regular growth medium supplemented with none (3D + none) or Conditioned Medium (3D + CM). The scale bar indicates 0.25 mm. C) RT-qPCR analysis of mRNA species levels according to the indicated cell-culture conditions. Stars indicate statistical significance of differences with the KC-2D condition (n=4 independent experiments). D) CE of cells harvested from 3D or 2D cultures that were dissociated by the same method. The relatively high frequency of cell clones in the clonogenic assays gem d0→ none and low FCS→ gem d3→ none resulted from the spontaneous reversion of cells in SSQ after gemcitabine selection when their density exceeded 10% of the seeded cells. E ) Schematic representation of the fusion protein encoded by the reporter gene EGFP-LC3B-mRFP-LC3BΔG encoding the autophagy sensitive EGFP-LC3B and the autophagy resistant mRFP-LC3ΔG fluorescent proteins. F) GFP to mRFP fluorescence ratio in EGFP-LC3B-mRFP-LC3BDG transduced cells cultured in 2D with none, bafilomycin A1 (baf1) or Torin1 for 1 day or cultured in 3D with none (3D+none) or CM (3D+CM) for 7 days. Stars indicate the significance of the difference with untreated 2D-cultured (control) cells. G ) Data from a representative flow cytometry experiment showing the distribution of the ratio of GFP to mRFP fluorescence in transduced cells cultured as indicated. H) CE of cells pretreated at medium cell density in 2D-cultures with none or Torin1 for 1 day (pretreatment) before cell harvesting for CE assay. Labels above bars indicate the percentage of seeded cells having formed a cell clone after SSQi supplementation. Stars indicate statistical significance of CE differences with 2D control in panel D and with non-pretreated cells in panel H in clonogenic assays with SSQi supplementation.

Article Snippet: Cell transduced with MRX-IP-GFP-LC3-RFP-LC3ΔG vector were seeded at 4 x 10 5 cells per 6 cm diameter dish (day 0), treated at day 1 as indicated and dissociated and harvested in cold culture medium at day 2 by strong pipetting after Tryp-EDTA-Pyr treatment, pelleted by centrifugation, resuspended in 500 µL of cold PBS 1X and eventually fixed by adding 4.5 mL of 4 % paraformaldehyde in saline (sc-281692, Santa Cruz Biotechnology, France) and incubating at room temperature for 5 minutes.

Techniques: Cell Culture, Quantitative RT-PCR, Clone Assay, Selection, Fluorescence, Flow Cytometry, Cell Harvesting

Journal: bioRxiv

Article Title: Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence and late clinical metastasis in a Beclin-1-dependent manner

doi: 10.1101/2022.05.05.490726

Figure Lengend Snippet: A) and B) CE of KC-DT66066 cell populations transduced with shRNA targeting GFP (KCsh gfp ) or Beclin-1 (KCsh becn1 ) and pretreated with none (A ) or Torin1 ( B ) as depicted in . Labels above bars indicate the percentage of seeded cells having formed a cell clone after SSQi supplementation. * indicate statistical significance (p) of CE differences after SSQi supplementation between KCsh gfp and KCsh becn1 cells. C ) Western Blot analysis of KCsh gfp and KCsh Becn1 cell populations seeded at medium cell density at d0 and treated from d1 to d2 as indicated (baf1 and tor1 were for bafilomycin A1 and Torin1 respectively). This is one representative western blot of 3 analyzed. The star indicates a non-specific band and the arrow the SQSTM1 band. D ) left : GFP/mRFP fluorescence ratio showing no difference in autophagic flux in KC shgfp and KC shbecn1 cell populations transduced with the EGFP-LC3B-mRFP-LC3BΔG reporter gene. Cells were passaged at medium cell density at d0, treated from d1 to d2 with the indicated drugs (none, bafilomycin A1 (baf1), and/or Torin1) and analyzed as in . Dotted lines connect data points derived from a same experiment. Labels indicate the number of independent reporter gene transduction experiments. Right : A representative flow cytometry experiment shown.

Article Snippet: Cell transduced with MRX-IP-GFP-LC3-RFP-LC3ΔG vector were seeded at 4 x 10 5 cells per 6 cm diameter dish (day 0), treated at day 1 as indicated and dissociated and harvested in cold culture medium at day 2 by strong pipetting after Tryp-EDTA-Pyr treatment, pelleted by centrifugation, resuspended in 500 µL of cold PBS 1X and eventually fixed by adding 4.5 mL of 4 % paraformaldehyde in saline (sc-281692, Santa Cruz Biotechnology, France) and incubating at room temperature for 5 minutes.

Techniques: Transduction, shRNA, Western Blot, Fluorescence, Derivative 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: 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