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: Nucleic acids research

Article Title: DIS3L2 ribonuclease degrades terminal-uridylated RNA to ensure oocyte maturation and female fertility.

doi: 10.1093/nar/gkad061

Figure Lengend Snippet: Figure 2. DIS3L2 genetic depletion stabilizes uridylated-poly(A) RNA. (A) Schematic of the floxed allele of the Dis3l2 gene. Two loxP sites are inserted surrounding exon 2 using CRISPR/Cas9. (B) Dot plot showing the litter size of control and Dis3l2cKO female mice of 6-month-old harem breeders. The number of females is labeled below each group. Note that none of the Dis3l2cKO female mice had a litter. (C) Ex vivo culture of the GV oocytes at 0, 3 and 16 h. The number of oocytes is labeled below each group and is combined from three independent assays. (D) Live imaging of GV oocytes after 3 h ex vivo culture. Inset shows an individual oocyte magnified 2×. The white dashed circle labels the intact nuclear envelope in Dis3l2cKO oocytes which breaks down in control oocytes. Scale bar, 100 m. (E) qPCR validation of Dis3l2 transcripts in growing oocytes (70 m diameter obtained from P20 females) and fully grown GV oocytes. *0.04, ****4.06e-07. (F) Bar graph showing the percentage of surrounded nucleolus (SN) and non-surrounded nucleolus (NSN) oocytes in control and Dis3l2cKO oocytes. (G) Density plot showing the distribution of transcripts of different poly(A) lengths in control and Dis3l2cKO oocytes. Arrows indicate the left shift of the red line. (H) The number and percentage of uridylated-poly(A) RNA and poly(A) RNA in Dis3l2cKO oocytes. (I) Distribution of the poly(A) length of uridylated-poly(A) RNA and poly(A) RNA in Dis3l2cKO oocytes. Arrows indicate the left shift of the red line. (J) Boxplot showing the length of the MBA region of uridylated-poly(A) RNA and poly(A) RNA (nt) in Dis3l2cKO oocytes. The red dashed lines label the median values in poly(A) RNA (1 nt) and uridylated-poly(A) RNA (24 nt). The outliers having MBA length >100 nt (<5% of all transcripts) are not included. (K) Boxplot showing the base composition in the MBA region in uridylated-poly(A) RNA and poly(A) RNA in Dis3l2cKO oocytes. (L) The number and percentage of uridylated-poly(A) genes and poly(A) genes in Dis3l2cKO oocytes. (M) The ratio between uridylated-poly(A) RNA and poly(A) RNA of the 1542 genes that have both uridylated-poly(A) RNA and poly(A) RNA in control and Dis3l2cKO oocytes. ****6.60E-62, one-way ANOVA test. (N) In vitro synthesized mVenus mRNA was microinjected into either control or Dis3l2cKO oocytes, purified, adaptor ligated, amplified and sequenced. The blue background indicates the mVenus coding region up to the stop codon (UAA, underlined). Placement of the restriction enzyme site (XbaI, TCTAGA) ensures the end of the DNA template after which RNA polymerase disengages during in vitro RNA synthesis. (O) PCR validation of individual genes from PAIso-seq results. As an example, the H1f1 transcript is amplified for Sanger sequencing. The mapped coding region, MBA region, poly(A) region, adaptor and vector regions are indicated.

Article Snippet: Oocytes were imaged using an LSM 780 confocal microscope (Carl Zeiss). mRNA synthesis for microinjection mRNA was in vitro synthesized using a mMESSAGE mMACHINE® T7 ULTRA Transcription Kit (Thermo Fisher Scientific AM1345). mVenus DNA template was from pcDNA4-TO-mVenus-MAP-Puro (Addgene #44118).

Techniques: CRISPR, Control, Labeling, Ex Vivo, Imaging, Biomarker Discovery, In Vitro, Synthesized, Purification, Amplification, Sequencing, Plasmid Preparation

Journal: Journal of Inflammation (London, England)

Article Title: Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei -infected macrophages

doi: 10.1186/s12950-024-00395-w

Figure Lengend Snippet: Role of autophagy in CA-mediated IL-1β inhibition in S. sonnei -infected macrophages. ( A-C ) J774A.1 macrophages were incubated with 40 µM CA for 4–20 h or 1 µM rapamycin for 4 h. The expression levels of LC3 ( A ), ATG5 ( B ) and p62 ( C ) in the cell lysates were analysed by Western blotting. ( D ) J774A.1 macrophages were primed for 4 h with LPS in the presence or absence of 1 mM 3-MA, followed by incubation with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. ( E ) Wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 20 h. The expression levels of LC3 in the cell lysates were analysed by Western blotting. ( F ) LPS-primed wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. The Western blotting images are representative of separate experiments. The ELISA data are expressed as the means ± SD of three separate experiments. * p < 0.05 and *** p < 0.001 as indicated

Article Snippet: Mouse LC3 CRISPR/Cas9 knockout plasmids (sc-426,563 and sc-417,828-HDR) and antibodies against IL-18 (SC-6177), ASC (SC-22,514-R), cathepsin B (SC-365,558) and actin (SC-47,778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Infection, Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown