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  • 99
    Thermo Fisher lipofectamine rnaimax transfection reagent
    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post <t>transfection.</t> C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p
    Lipofectamine Rnaimax Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26019 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pre mir rna hairpins
    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post <t>transfection.</t> C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p
    Pre Mir Rna Hairpins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ãŽâ¼m pre mir rna hairpins
    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post <t>transfection.</t> C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p
    ãŽâ¼m Pre Mir Rna Hairpins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher rnaimax
    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post <t>transfection.</t> C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p
    Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher p53 sirna
    The role of <t>SRSF1</t> on translation in a representative ER(+) breast cancer cell line (T47D). (A) Immunofluorescence confocal microscopy of T47D cells. Images focused on dividing cells surrounded by nondividing cells. In T47D cells, SRSF1 is predominantly intranuclear. In dividing cells, however, SRSF1 localizes diffusely in the cytoplasm. SRSF1 colocalizes with RACK1 (a component of the IRES translation initiation complex, upper panel). Note the mitotic spindle (middle panel) and the diffusely localized CDK1 and SRSF1 in the dividing cells (lower panel). Scale bars: 10 μm. (B) Cell viability assay of T47D cells. <t>siRNA-mediated</t> downregulation of SRSF1 is associated with growth arrest in T47D cells (data presented as mean ± standard deviation, n = 3). (C) Cell cycle analysis by flow cytometry of T47D cells. siRNA mediated downregulation of SRSF1 in T47D cells is associated with an S-phase arrest (furthest left, data presented as mean ± standard deviation, n = 2). Note the nearly 0% cells in G2/M phase with siRNA2. A representative flow cytometry histogram is shown with each siRNA on the right. (D) Puromycin incorporation assay (left panel). Ponceau S staining (right panel) serves as control to confirm equal protein loading. Downregulation of SRSF1 is associated with a global downregulation in new protein synthesis especially when cells are synchronized to G2/M phase. (E) UV CLIP assay of the T47D CDK1 IRES reporter cells. The assay identifies the endogenous (empty arrow) and the stably transfected (black arrow) CDK1 5ʹUTR in the SRSF1 immunoprecipitate. RNA extracted from the non-immunoprecipitated supernatant serves as control.
    P53 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher culture medium
    The role of <t>SRSF1</t> on translation in a representative ER(+) breast cancer cell line (T47D). (A) Immunofluorescence confocal microscopy of T47D cells. Images focused on dividing cells surrounded by nondividing cells. In T47D cells, SRSF1 is predominantly intranuclear. In dividing cells, however, SRSF1 localizes diffusely in the cytoplasm. SRSF1 colocalizes with RACK1 (a component of the IRES translation initiation complex, upper panel). Note the mitotic spindle (middle panel) and the diffusely localized CDK1 and SRSF1 in the dividing cells (lower panel). Scale bars: 10 μm. (B) Cell viability assay of T47D cells. <t>siRNA-mediated</t> downregulation of SRSF1 is associated with growth arrest in T47D cells (data presented as mean ± standard deviation, n = 3). (C) Cell cycle analysis by flow cytometry of T47D cells. siRNA mediated downregulation of SRSF1 in T47D cells is associated with an S-phase arrest (furthest left, data presented as mean ± standard deviation, n = 2). Note the nearly 0% cells in G2/M phase with siRNA2. A representative flow cytometry histogram is shown with each siRNA on the right. (D) Puromycin incorporation assay (left panel). Ponceau S staining (right panel) serves as control to confirm equal protein loading. Downregulation of SRSF1 is associated with a global downregulation in new protein synthesis especially when cells are synchronized to G2/M phase. (E) UV CLIP assay of the T47D CDK1 IRES reporter cells. The assay identifies the endogenous (empty arrow) and the stably transfected (black arrow) CDK1 5ʹUTR in the SRSF1 immunoprecipitate. RNA extracted from the non-immunoprecipitated supernatant serves as control.
    Culture Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore pft α
    Suppression of IR-induced mGluR1 expression by STAT3 inhibitor in C17.2 cells. C17.2 cells were treated with STAT3 inhibitor, S3I-201 at 10 μM and p53 inhibitor, <t>Pft-α</t> at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. Level of mGluR1 mRNA was analyzed by real-time PCR (A) and expression of mGluR1 was analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p
    Pft α, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher optimem
    Suppression of IR-induced mGluR1 expression by STAT3 inhibitor in C17.2 cells. C17.2 cells were treated with STAT3 inhibitor, S3I-201 at 10 μM and p53 inhibitor, <t>Pft-α</t> at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. Level of mGluR1 mRNA was analyzed by real-time PCR (A) and expression of mGluR1 was analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p
    Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirna inhibitors
    <t>MiRNA-125b</t> regulates expression of Hh signaling and profibrotic genes in LX2. ( A ) QRT-PCR analysis of miRNA-125b expression in LX2 or transfected LX2 with miRNA-125b mimic (25 nM) or scrambled-miRNA mimic ((-) CON, 25 nM) as a negative control for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (** p
    Mirna Inhibitors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biocoat 384 well collagen coated plate
    <t>MiRNA-125b</t> regulates expression of Hh signaling and profibrotic genes in LX2. ( A ) QRT-PCR analysis of miRNA-125b expression in LX2 or transfected LX2 with miRNA-125b mimic (25 nM) or scrambled-miRNA mimic ((-) CON, 25 nM) as a negative control for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (** p
    384 Well Collagen Coated Plate, supplied by Biocoat, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Qiagen beclin
    Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following <t>RNAi</t> reduction of autophagy by targeting Atg5 or <t>beclin</t> for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant
    Beclin, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher opti mem medium
    Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following <t>RNAi</t> reduction of autophagy by targeting Atg5 or <t>beclin</t> for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant
    Opti Mem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery luciferase
    Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following <t>RNAi</t> reduction of autophagy by targeting Atg5 or <t>beclin</t> for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant
    Luciferase, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher silencer pre designed sirna
    Dampening of autophagy activation in <t>Wdfy3</t> -deficient neuronal progenitor cells is accompanied by decreased mitophagy. ( A ) Wdfy3 immunofluorescent labeling in striatal neuronal progenitor cells (NPCs) shows ubiquitous expression (scrambled). A significant down regulation of Wdfy3 was observed in <t>siRNA-transfected</t> cells. Quantification of Wdfy3 protein levels was based on fluorescent signal intensity normalized by cell area. Levels of Wdfy3 mRNA expression were assessed by qPCR normalized to Gapdh mRNA. ( B ) The effect of rapamycin (20 nM) addition was evaluated by quantifying LC3 immunostaining normalized by cell area, as well as the number of LC3 puncta per cell. Statistical analyses were performed by two-tailed Mann-Whitney test. ( C) Total mitochondrial mass was visualized in scrambled and siRNA striatal cells by staining with antibodies to ATP5B (green) whereas polarized mitochondria were stained with MitoTracker (red). Co-localization of functional and total mitochondria is shown in yellow (overlay). Insets show details of the mitochondrial morphology and co-localization of ATP5B and MitoTracker in the two genotypes. Red channel intensity has been increased for siRNA-transfected cells, due to the low MitoTracker fluorescence signal emitted by non-polarized mitochondria. Scale bars = 10 µm. Panels ( D–H ). Mitochondrial content ( D ) was quantified as the cell area occupied by mitochondria (in %). Mitochondrial footprint ( E ) was expressed as functional mitochondria (area) normalized by total mitochondria area. ( F ) Average area per mitochondrion in scrambled- and si-RNA striatal cells. Mitochondrial morphology ( G ) was reported as circularity index, with mitochondria having a more tubular shape being closer to a value of 1. All measurements shown in panels D,G were performed using a macro for ImageJ (see Methods). ( H , I ) Assessment of the mitochondrial network integrity in scrambled- and siRNA striatal neurons was performed with the MiNA macro for ImageJ and reported as number of networks per cell ( H ) and mean branch length ( I ). Statistical analysis for panels D–I was performed with a 2-tailed Mann-Whitney test. ( J ) Surface plots representing levels and mitochondrial distribution in scrambled (left) and siRNA (right) striatal cells were obtained with the ImageJ feature “surface plot”. All the box plots shown in panels F–I were obtained analyzing confocal images of mitochondria stained with MitoTracker.
    Silencer Pre Designed Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher negative control sirna
    mRNA levels for IGFBP5 and IGFBP2 are biomarkers for bladder tumor cells sensitivity to <t>IGF1R</t> kinase inhibitor, AEW541. a Heat map of the IGF receptors, ligands, and binding proteins mRNA levels in regards to sensitivity to AEW41 in 13 bladder tumor derived cell lines. Data were extracted from CCLE database; b Effect of a blocking antibody against IGF1R and of IGF1R <t>siRNA</t> on the viability of RT112 cells. IGF1R knockdown 72 h after transfection with a control or anti-IGF1R siRNA was assessed by western blotting (inset). The effect of the siRNA on cell viability was assessed in MTT assays. The effect of the anti-IGF1R blocking antibody was assessed after 72 h, in MTT assays; c Spearman’s coefficients for the correlations between the sensitivity to AEW541 and mRNA levels for IGF receptors, ligands, and binding proteins, in 13 cell bladder tumor derived cell lines. Data were extracted from CCLE database
    Negative Control Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post transfection. C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p

    Journal: PLoS ONE

    Article Title: The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

    doi: 10.1371/journal.pone.0128159

    Figure Lengend Snippet: siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post transfection. C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p

    Article Snippet: 5×103 MHCC97-H c-Met shRNA cells were plated in 96-well plates and reverse transfected (cells were added to 10 nM siRNA and 0.2 μl RNAiMAX pre-added to wells) with individual siRNA using lipid-mediated transfection with Lipofectamine RNAiMAX (Life Technologies Corporation, Grand Island, NY).

    Techniques: Microarray, Stable Transfection, Transfection, shRNA, Activity Assay, Selection, Expressing, XTT Assay, Chromatin Immunoprecipitation

    The role of SRSF1 on translation in a representative ER(+) breast cancer cell line (T47D). (A) Immunofluorescence confocal microscopy of T47D cells. Images focused on dividing cells surrounded by nondividing cells. In T47D cells, SRSF1 is predominantly intranuclear. In dividing cells, however, SRSF1 localizes diffusely in the cytoplasm. SRSF1 colocalizes with RACK1 (a component of the IRES translation initiation complex, upper panel). Note the mitotic spindle (middle panel) and the diffusely localized CDK1 and SRSF1 in the dividing cells (lower panel). Scale bars: 10 μm. (B) Cell viability assay of T47D cells. siRNA-mediated downregulation of SRSF1 is associated with growth arrest in T47D cells (data presented as mean ± standard deviation, n = 3). (C) Cell cycle analysis by flow cytometry of T47D cells. siRNA mediated downregulation of SRSF1 in T47D cells is associated with an S-phase arrest (furthest left, data presented as mean ± standard deviation, n = 2). Note the nearly 0% cells in G2/M phase with siRNA2. A representative flow cytometry histogram is shown with each siRNA on the right. (D) Puromycin incorporation assay (left panel). Ponceau S staining (right panel) serves as control to confirm equal protein loading. Downregulation of SRSF1 is associated with a global downregulation in new protein synthesis especially when cells are synchronized to G2/M phase. (E) UV CLIP assay of the T47D CDK1 IRES reporter cells. The assay identifies the endogenous (empty arrow) and the stably transfected (black arrow) CDK1 5ʹUTR in the SRSF1 immunoprecipitate. RNA extracted from the non-immunoprecipitated supernatant serves as control.

    Journal: Translational Oncology

    Article Title: Hallmarks and Determinants of Oncogenic Translation Revealed by Ribosome Profiling in Models of Breast Cancer

    doi: 10.1016/j.tranon.2019.12.002

    Figure Lengend Snippet: The role of SRSF1 on translation in a representative ER(+) breast cancer cell line (T47D). (A) Immunofluorescence confocal microscopy of T47D cells. Images focused on dividing cells surrounded by nondividing cells. In T47D cells, SRSF1 is predominantly intranuclear. In dividing cells, however, SRSF1 localizes diffusely in the cytoplasm. SRSF1 colocalizes with RACK1 (a component of the IRES translation initiation complex, upper panel). Note the mitotic spindle (middle panel) and the diffusely localized CDK1 and SRSF1 in the dividing cells (lower panel). Scale bars: 10 μm. (B) Cell viability assay of T47D cells. siRNA-mediated downregulation of SRSF1 is associated with growth arrest in T47D cells (data presented as mean ± standard deviation, n = 3). (C) Cell cycle analysis by flow cytometry of T47D cells. siRNA mediated downregulation of SRSF1 in T47D cells is associated with an S-phase arrest (furthest left, data presented as mean ± standard deviation, n = 2). Note the nearly 0% cells in G2/M phase with siRNA2. A representative flow cytometry histogram is shown with each siRNA on the right. (D) Puromycin incorporation assay (left panel). Ponceau S staining (right panel) serves as control to confirm equal protein loading. Downregulation of SRSF1 is associated with a global downregulation in new protein synthesis especially when cells are synchronized to G2/M phase. (E) UV CLIP assay of the T47D CDK1 IRES reporter cells. The assay identifies the endogenous (empty arrow) and the stably transfected (black arrow) CDK1 5ʹUTR in the SRSF1 immunoprecipitate. RNA extracted from the non-immunoprecipitated supernatant serves as control.

    Article Snippet: SRSF1 siRNAs used were siRNA1: s12725 and siRNA2: s12727 (both SilencerSelect ThermoFisher Scientific, cat# 4392420); control siRNA was SilencerSelect Negative Control No. 1 (ThermoFisher Scientific, cat# 4390843).

    Techniques: Immunofluorescence, Confocal Microscopy, Viability Assay, Standard Deviation, Cell Cycle Assay, Flow Cytometry, Cytometry, Staining, Cross-linking Immunoprecipitation, Stable Transfection, Transfection, Immunoprecipitation

    During cell division, cytoplasmic SRSF1 operates as an ITAF for the CDK1 IRES. (A) Luciferase assay (left panel) and immunoblot for SRSF1, CDK1, and p21/CDKN1A (right panel) of T47D CDK1 IRES reporter cells. T47D cells were stably transfected with the CDK1 IRES bicistronic reporter vector (upper panel). Left panel. SRSF1 expression was downregulated with siRNAs and cells were asynchronous or synchronized with nocodozale (16 h) as indicated. siRNA mediated downregulation of SRSF1 was not associated with an apparent effect on the CDK1 IRES activity in an asynchronous T47D cell population. G2/M synchronization with nozodazole captures the cell cycle dependent upregulation of CDK1 IRES mediated translation. This upregulation is blocked by siRNA mediated downregulation of the SRSF1. IRES activity (ratio of Firefly:Renilla luciferase) is normalized to control siRNA-no nocodazole (asynchronous) condition. Data presented as mean ± standard deviation, n = 4; paired Student's t-test. Right panel. CDK1 protein levels change coordinately with SRSF1 especially when cells are synchronized to the G2/M phase. Two replicates of the same experiment are shown. Note the downregulation of tubulin with SRSF1 downregulation in the synchronized cells consistent with the fact that tubulin constitutes the main component of the mitotic spindle. The downregulation of SRSF1 does not impact p21 levels in T47D cells. (B) Luciferase assay of T47D CDK1 IRES reporter cells, asynchronous or synchronized to the G2/M phase with nocodazole and concurrently treated with rapamycin (50 or 100 nM) or DMSO control (upper panel). Lower panel. G2/M synchronization with nozodazole captures the cell cycle dependent upregulation of CDK1 IRES mediated translation. Treatment with rapamycin (an mTOR inhibitor, i.e. inhibitor of cap-dependent translation), does not block this upregulation. Note also the upregulation of CDK1 IRES activity induced by rapamycin in the asynchronous population (data presented as mean ± standard deviation, n = 2; paired Student's t-test). (C) Model for the translational activity of SRSF1 in T47D cells: during the G0/G1 phase, SRSF1 is predominantly intranuclear and operates as a splicing factor. During the S, G2, and M phase, the disassembly of the nuclear membrane releases SRSF1 to the cytoplasm where it operates as an IRES trans -acting factor and upregulates the IRES mediated translation of CDK1 (and probably of other transcripts).

    Journal: Translational Oncology

    Article Title: Hallmarks and Determinants of Oncogenic Translation Revealed by Ribosome Profiling in Models of Breast Cancer

    doi: 10.1016/j.tranon.2019.12.002

    Figure Lengend Snippet: During cell division, cytoplasmic SRSF1 operates as an ITAF for the CDK1 IRES. (A) Luciferase assay (left panel) and immunoblot for SRSF1, CDK1, and p21/CDKN1A (right panel) of T47D CDK1 IRES reporter cells. T47D cells were stably transfected with the CDK1 IRES bicistronic reporter vector (upper panel). Left panel. SRSF1 expression was downregulated with siRNAs and cells were asynchronous or synchronized with nocodozale (16 h) as indicated. siRNA mediated downregulation of SRSF1 was not associated with an apparent effect on the CDK1 IRES activity in an asynchronous T47D cell population. G2/M synchronization with nozodazole captures the cell cycle dependent upregulation of CDK1 IRES mediated translation. This upregulation is blocked by siRNA mediated downregulation of the SRSF1. IRES activity (ratio of Firefly:Renilla luciferase) is normalized to control siRNA-no nocodazole (asynchronous) condition. Data presented as mean ± standard deviation, n = 4; paired Student's t-test. Right panel. CDK1 protein levels change coordinately with SRSF1 especially when cells are synchronized to the G2/M phase. Two replicates of the same experiment are shown. Note the downregulation of tubulin with SRSF1 downregulation in the synchronized cells consistent with the fact that tubulin constitutes the main component of the mitotic spindle. The downregulation of SRSF1 does not impact p21 levels in T47D cells. (B) Luciferase assay of T47D CDK1 IRES reporter cells, asynchronous or synchronized to the G2/M phase with nocodazole and concurrently treated with rapamycin (50 or 100 nM) or DMSO control (upper panel). Lower panel. G2/M synchronization with nozodazole captures the cell cycle dependent upregulation of CDK1 IRES mediated translation. Treatment with rapamycin (an mTOR inhibitor, i.e. inhibitor of cap-dependent translation), does not block this upregulation. Note also the upregulation of CDK1 IRES activity induced by rapamycin in the asynchronous population (data presented as mean ± standard deviation, n = 2; paired Student's t-test). (C) Model for the translational activity of SRSF1 in T47D cells: during the G0/G1 phase, SRSF1 is predominantly intranuclear and operates as a splicing factor. During the S, G2, and M phase, the disassembly of the nuclear membrane releases SRSF1 to the cytoplasm where it operates as an IRES trans -acting factor and upregulates the IRES mediated translation of CDK1 (and probably of other transcripts).

    Article Snippet: SRSF1 siRNAs used were siRNA1: s12725 and siRNA2: s12727 (both SilencerSelect ThermoFisher Scientific, cat# 4392420); control siRNA was SilencerSelect Negative Control No. 1 (ThermoFisher Scientific, cat# 4390843).

    Techniques: Luciferase, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Activity Assay, Standard Deviation, Blocking Assay

    Cytoplasmic SRSF1 operates as an ITAF for MYC IRES. (A) Luciferase assay of SUM159PT MYC IRES reporter cells. SRSF1 expression was downregulated with siRNAs and cells were grown under full-serum or serum-deprived (for 24 h) conditions as indicated. siRNA mediated downregulation of SRSF1 is associated with a decrease in the MYC IRES activity and leads to smaller increments of the MYC IRES activity under conditions of stress. IRES activity (ratio of Firefly:Renilla luciferase) is normalized to control siRNA full-serum condition (data presented as mean ± standard deviation, n = 5; paired Student's t-test). (B) MYC band intensity in an immunoblot ( Supplemental Figure 9 ) was divided by the respective tubulin band intensity to provide the relative MYC protein levels. The histogram illustrates the ratio of the relative MYC protein levels between serum-deprived (stress) and full-serum conditions (mean ± standard deviation, n = 2; paired Student's t-test). SUM159PT cells in which SRSF1 is downregulated cannot maintain MYC protein expression under conditions of stress. (C) UV CLIP assay of SUM159PT MYC IRES reporter cells for SRSF1 under full-serum and serum-deprived (for 24 h) conditions. SRSF1 directly associates with the MYC 5ʹUTR and this association is more pronounced under serum deprived conditions. Nonimmunoprecipitated supernatant and the vector used for the stable transfection serve as control. (D) Model for the translational activity of SRSF1 in SUM159PT cells: under normal growth conditions, SRSF1 is predominantly intranuclear and operates as a splicing factor leaving its splicing “fingerprint” on the transcriptome. Under conditions of stress, SRSF1 translocates to the cytoplasm where it operates as an IRES trans -acting factor and upregulates the IRES mediated translation of MYC mRNA (and probably of other transcripts).

    Journal: Translational Oncology

    Article Title: Hallmarks and Determinants of Oncogenic Translation Revealed by Ribosome Profiling in Models of Breast Cancer

    doi: 10.1016/j.tranon.2019.12.002

    Figure Lengend Snippet: Cytoplasmic SRSF1 operates as an ITAF for MYC IRES. (A) Luciferase assay of SUM159PT MYC IRES reporter cells. SRSF1 expression was downregulated with siRNAs and cells were grown under full-serum or serum-deprived (for 24 h) conditions as indicated. siRNA mediated downregulation of SRSF1 is associated with a decrease in the MYC IRES activity and leads to smaller increments of the MYC IRES activity under conditions of stress. IRES activity (ratio of Firefly:Renilla luciferase) is normalized to control siRNA full-serum condition (data presented as mean ± standard deviation, n = 5; paired Student's t-test). (B) MYC band intensity in an immunoblot ( Supplemental Figure 9 ) was divided by the respective tubulin band intensity to provide the relative MYC protein levels. The histogram illustrates the ratio of the relative MYC protein levels between serum-deprived (stress) and full-serum conditions (mean ± standard deviation, n = 2; paired Student's t-test). SUM159PT cells in which SRSF1 is downregulated cannot maintain MYC protein expression under conditions of stress. (C) UV CLIP assay of SUM159PT MYC IRES reporter cells for SRSF1 under full-serum and serum-deprived (for 24 h) conditions. SRSF1 directly associates with the MYC 5ʹUTR and this association is more pronounced under serum deprived conditions. Nonimmunoprecipitated supernatant and the vector used for the stable transfection serve as control. (D) Model for the translational activity of SRSF1 in SUM159PT cells: under normal growth conditions, SRSF1 is predominantly intranuclear and operates as a splicing factor leaving its splicing “fingerprint” on the transcriptome. Under conditions of stress, SRSF1 translocates to the cytoplasm where it operates as an IRES trans -acting factor and upregulates the IRES mediated translation of MYC mRNA (and probably of other transcripts).

    Article Snippet: SRSF1 siRNAs used were siRNA1: s12725 and siRNA2: s12727 (both SilencerSelect ThermoFisher Scientific, cat# 4392420); control siRNA was SilencerSelect Negative Control No. 1 (ThermoFisher Scientific, cat# 4390843).

    Techniques: Luciferase, Expressing, Activity Assay, Standard Deviation, Cross-linking Immunoprecipitation, Plasmid Preparation, Stable Transfection

    Suppression of IR-induced mGluR1 expression by STAT3 inhibitor in C17.2 cells. C17.2 cells were treated with STAT3 inhibitor, S3I-201 at 10 μM and p53 inhibitor, Pft-α at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. Level of mGluR1 mRNA was analyzed by real-time PCR (A) and expression of mGluR1 was analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p

    Journal: PLoS ONE

    Article Title: Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells

    doi: 10.1371/journal.pone.0147538

    Figure Lengend Snippet: Suppression of IR-induced mGluR1 expression by STAT3 inhibitor in C17.2 cells. C17.2 cells were treated with STAT3 inhibitor, S3I-201 at 10 μM and p53 inhibitor, Pft-α at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. Level of mGluR1 mRNA was analyzed by real-time PCR (A) and expression of mGluR1 was analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p

    Article Snippet: For p53 inhibition, PFT-α (Sigma-Aldrich) was added to the culture media at the final concentration of 20 or 40 μM 2 hr prior to the irradiation or p53-siRNA (Trp53 Silencer Select Pre-designed siRNA, ID: s75472; Invitrogen, CA, USA) was treated with Neuro-2a cells using Lipofectamine RNAiMAX (Invitorgen) using the manufacturer’s protocol at 24 hr prior to the irradiation.

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot

    Effect of STAT3 and p53 inhibitors on IR-induced neuronal properties in C17.2 cells. C17.2 cells were treated with the inhibitors of JNK (SP600125) at10 μM, STAT1 (Fludarabine) at 10 μM, STAT3 (S3I-201) at 10 μM, Rac (NSC23766) at 20 μM, B-Raf (GDC-0879) at 20 μM and p53 (Pft-α) at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. To assess rate of neurite bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software. IR-induced neurite outgrowth is not affected by the inhibitors of JNK, STAT1, Rac and B-Raf but was blocked by the STAT3 inhibitor, S3I-201 and the p53 inhibitor, Pft-α (A). Expression of NSC marker, nestin, and neuronal marker, β-III tubulin was analyzed by Western blot (B, upper panel), and then quantified by Image J software (B, down panel). **p

    Journal: PLoS ONE

    Article Title: Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells

    doi: 10.1371/journal.pone.0147538

    Figure Lengend Snippet: Effect of STAT3 and p53 inhibitors on IR-induced neuronal properties in C17.2 cells. C17.2 cells were treated with the inhibitors of JNK (SP600125) at10 μM, STAT1 (Fludarabine) at 10 μM, STAT3 (S3I-201) at 10 μM, Rac (NSC23766) at 20 μM, B-Raf (GDC-0879) at 20 μM and p53 (Pft-α) at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. To assess rate of neurite bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software. IR-induced neurite outgrowth is not affected by the inhibitors of JNK, STAT1, Rac and B-Raf but was blocked by the STAT3 inhibitor, S3I-201 and the p53 inhibitor, Pft-α (A). Expression of NSC marker, nestin, and neuronal marker, β-III tubulin was analyzed by Western blot (B, upper panel), and then quantified by Image J software (B, down panel). **p

    Article Snippet: For p53 inhibition, PFT-α (Sigma-Aldrich) was added to the culture media at the final concentration of 20 or 40 μM 2 hr prior to the irradiation or p53-siRNA (Trp53 Silencer Select Pre-designed siRNA, ID: s75472; Invitrogen, CA, USA) was treated with Neuro-2a cells using Lipofectamine RNAiMAX (Invitorgen) using the manufacturer’s protocol at 24 hr prior to the irradiation.

    Techniques: Incubation, Software, Expressing, Marker, Western Blot

    MiRNA-125b regulates expression of Hh signaling and profibrotic genes in LX2. ( A ) QRT-PCR analysis of miRNA-125b expression in LX2 or transfected LX2 with miRNA-125b mimic (25 nM) or scrambled-miRNA mimic ((-) CON, 25 nM) as a negative control for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (** p

    Journal: Scientific Reports

    Article Title: MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells

    doi: 10.1038/srep14135

    Figure Lengend Snippet: MiRNA-125b regulates expression of Hh signaling and profibrotic genes in LX2. ( A ) QRT-PCR analysis of miRNA-125b expression in LX2 or transfected LX2 with miRNA-125b mimic (25 nM) or scrambled-miRNA mimic ((-) CON, 25 nM) as a negative control for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (** p

    Article Snippet: Before treating cells with miRNA inhibitors, the diluted miRNA-125b inhibitor or negative control inhibitor in Opti-MEM® I reduced serum medium (Gibco) was incubated with the diluted Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM® I for 20 minutes at RT to make the transfection complexes.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Negative Control

    MiRNA-125b is expressed in CP-MSCs. ( A ) QRT-PCR analysis of the expression of miRNA-125b, miRNA-324-5 p and miRNA-326 in human normal liver (Normal), LX2 (human HSC line) and human CP-MSCs cultured during 72 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Journal: Scientific Reports

    Article Title: MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells

    doi: 10.1038/srep14135

    Figure Lengend Snippet: MiRNA-125b is expressed in CP-MSCs. ( A ) QRT-PCR analysis of the expression of miRNA-125b, miRNA-324-5 p and miRNA-326 in human normal liver (Normal), LX2 (human HSC line) and human CP-MSCs cultured during 72 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Article Snippet: Before treating cells with miRNA inhibitors, the diluted miRNA-125b inhibitor or negative control inhibitor in Opti-MEM® I reduced serum medium (Gibco) was incubated with the diluted Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM® I for 20 minutes at RT to make the transfection complexes.

    Techniques: Quantitative RT-PCR, Expressing, Cell Culture

    MiRNA-125b-downregulated CP-MSCs fail to downregulate expression of Hh signaling and profibrotic genes in LX2. QRT-PCR analysis of the expression of Hh signals, including shh , smo , gli2 , and gli3 , and profibrotic genes, including vimentin and mmp9 , in LX2 mono-culture (alone), LX2 treated with 1 μM GDC-0449 (Smo antagonist), and co-cultured LX2 with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Journal: Scientific Reports

    Article Title: MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells

    doi: 10.1038/srep14135

    Figure Lengend Snippet: MiRNA-125b-downregulated CP-MSCs fail to downregulate expression of Hh signaling and profibrotic genes in LX2. QRT-PCR analysis of the expression of Hh signals, including shh , smo , gli2 , and gli3 , and profibrotic genes, including vimentin and mmp9 , in LX2 mono-culture (alone), LX2 treated with 1 μM GDC-0449 (Smo antagonist), and co-cultured LX2 with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Article Snippet: Before treating cells with miRNA inhibitors, the diluted miRNA-125b inhibitor or negative control inhibitor in Opti-MEM® I reduced serum medium (Gibco) was incubated with the diluted Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM® I for 20 minutes at RT to make the transfection complexes.

    Techniques: Expressing, Quantitative RT-PCR, Cell Culture

    Hh signaling and profibrotic genes were highly expressed in activated primary HSCs co-cultured with CP-MSCs containing miRNA-125b inhibitor. QRT-PCR analysis of the expression of Hh signals, including shh , smo , gli2 , and gli3 , and profibrotic genes, including vimentin and mmp9 , in mono-cultured primary HSCs isolated from rats with CCl 4 -induced fibrosis (alone), and co-cultured primary rat HSCs with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Journal: Scientific Reports

    Article Title: MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells

    doi: 10.1038/srep14135

    Figure Lengend Snippet: Hh signaling and profibrotic genes were highly expressed in activated primary HSCs co-cultured with CP-MSCs containing miRNA-125b inhibitor. QRT-PCR analysis of the expression of Hh signals, including shh , smo , gli2 , and gli3 , and profibrotic genes, including vimentin and mmp9 , in mono-cultured primary HSCs isolated from rats with CCl 4 -induced fibrosis (alone), and co-cultured primary rat HSCs with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (* p

    Article Snippet: Before treating cells with miRNA inhibitors, the diluted miRNA-125b inhibitor or negative control inhibitor in Opti-MEM® I reduced serum medium (Gibco) was incubated with the diluted Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM® I for 20 minutes at RT to make the transfection complexes.

    Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Isolation

    Maternal diabetes in vivo and high glucose in vitro down-regulate miR-17. A. miR-17-5p/3p levels in E8.75 embryos determined by the miRNA profiling ( n = 3 litters). B. miR-17-5p/3p levels in E8.75 embryos assessed by RT-qPCR. ND, non-diabetic; DM, diabetic mellitus. C. miR-17-5p levels in cells treated with normal glucose (5 mM) or high glucose (16.7, 25, 33.3 mM) for 24 h. D. miR-17-5p levels in cells under normal (5 mM) or high (25 mM) glucose conditions for 12, 24, and 48 h. E. miR-17-3p levels in cells treated with different glucose concentrations for 24 h. F. miR-17-3p levels in cells under normal (5 mM) or high (25 mM) glucose conditions for 12, 24, and 48 h. G. miR-17-5p levels in cells under normal glucose (5 mM) conditions with or without high mannitol (11.7, 20, and 28.3 mM) for 24 h. H. miR-17-5p levels under normal glucose (5 mM) conditions with or without 20 mM of mannitol for 12, 24, and 48 h. I. miR-17-3p levels in cells under normal glucose (5 mM) conditions with or without high mannitol (11.7, 20, and 28.3 mM) for 24 h. J. miR-17-3p levels in cells under normal glucose (5 mM) conditions with or without 20 mM of mannitol for 12, 24, and 48 h. G : glucose, M , mannitol. Experiments were repeated 3 times ( n = 3). Values are means ± SEM from 3 separate experiments. *indicates significant difference ( P

    Journal: Toxicological Sciences

    Article Title: MiR-17 Downregulation by High Glucose Stabilizes Thioredoxin-Interacting Protein and Removes Thioredoxin Inhibition on ASK1 Leading to Apoptosis

    doi: 10.1093/toxsci/kfv313

    Figure Lengend Snippet: Maternal diabetes in vivo and high glucose in vitro down-regulate miR-17. A. miR-17-5p/3p levels in E8.75 embryos determined by the miRNA profiling ( n = 3 litters). B. miR-17-5p/3p levels in E8.75 embryos assessed by RT-qPCR. ND, non-diabetic; DM, diabetic mellitus. C. miR-17-5p levels in cells treated with normal glucose (5 mM) or high glucose (16.7, 25, 33.3 mM) for 24 h. D. miR-17-5p levels in cells under normal (5 mM) or high (25 mM) glucose conditions for 12, 24, and 48 h. E. miR-17-3p levels in cells treated with different glucose concentrations for 24 h. F. miR-17-3p levels in cells under normal (5 mM) or high (25 mM) glucose conditions for 12, 24, and 48 h. G. miR-17-5p levels in cells under normal glucose (5 mM) conditions with or without high mannitol (11.7, 20, and 28.3 mM) for 24 h. H. miR-17-5p levels under normal glucose (5 mM) conditions with or without 20 mM of mannitol for 12, 24, and 48 h. I. miR-17-3p levels in cells under normal glucose (5 mM) conditions with or without high mannitol (11.7, 20, and 28.3 mM) for 24 h. J. miR-17-3p levels in cells under normal glucose (5 mM) conditions with or without 20 mM of mannitol for 12, 24, and 48 h. G : glucose, M , mannitol. Experiments were repeated 3 times ( n = 3). Values are means ± SEM from 3 separate experiments. *indicates significant difference ( P

    Article Snippet: The mirVana miRNA mimic, the miRNA inhibitor for miR-17 and the negative control (NC) were purchased from Ambion (Life Technologies).

    Techniques: In Vivo, In Vitro, Quantitative RT-PCR

    Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following RNAi reduction of autophagy by targeting Atg5 or beclin for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant

    Journal:

    Article Title: Metabolic Activity Determines Efficacy of Macroautophagic Clearance of Pathological Oligomeric ?-Synuclein

    doi: 10.2353/ajpath.2009.080928

    Figure Lengend Snippet: Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following RNAi reduction of autophagy by targeting Atg5 or beclin for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant

    Article Snippet: For RNA interference (RNAi) experiments, a cell suspension containing 50,000 cells was added to a pre-made mixture of 5 μl of RNAiMax (Invitrogen) plus 20 nmol/L of RNAi targeting Atg5 or beclin (sequences from Qiagen), plated onto a 6 well plate, and incubated for 48 hours before harvesting.

    Techniques: Inhibition

    Dampening of autophagy activation in Wdfy3 -deficient neuronal progenitor cells is accompanied by decreased mitophagy. ( A ) Wdfy3 immunofluorescent labeling in striatal neuronal progenitor cells (NPCs) shows ubiquitous expression (scrambled). A significant down regulation of Wdfy3 was observed in siRNA-transfected cells. Quantification of Wdfy3 protein levels was based on fluorescent signal intensity normalized by cell area. Levels of Wdfy3 mRNA expression were assessed by qPCR normalized to Gapdh mRNA. ( B ) The effect of rapamycin (20 nM) addition was evaluated by quantifying LC3 immunostaining normalized by cell area, as well as the number of LC3 puncta per cell. Statistical analyses were performed by two-tailed Mann-Whitney test. ( C) Total mitochondrial mass was visualized in scrambled and siRNA striatal cells by staining with antibodies to ATP5B (green) whereas polarized mitochondria were stained with MitoTracker (red). Co-localization of functional and total mitochondria is shown in yellow (overlay). Insets show details of the mitochondrial morphology and co-localization of ATP5B and MitoTracker in the two genotypes. Red channel intensity has been increased for siRNA-transfected cells, due to the low MitoTracker fluorescence signal emitted by non-polarized mitochondria. Scale bars = 10 µm. Panels ( D–H ). Mitochondrial content ( D ) was quantified as the cell area occupied by mitochondria (in %). Mitochondrial footprint ( E ) was expressed as functional mitochondria (area) normalized by total mitochondria area. ( F ) Average area per mitochondrion in scrambled- and si-RNA striatal cells. Mitochondrial morphology ( G ) was reported as circularity index, with mitochondria having a more tubular shape being closer to a value of 1. All measurements shown in panels D,G were performed using a macro for ImageJ (see Methods). ( H , I ) Assessment of the mitochondrial network integrity in scrambled- and siRNA striatal neurons was performed with the MiNA macro for ImageJ and reported as number of networks per cell ( H ) and mean branch length ( I ). Statistical analysis for panels D–I was performed with a 2-tailed Mann-Whitney test. ( J ) Surface plots representing levels and mitochondrial distribution in scrambled (left) and siRNA (right) striatal cells were obtained with the ImageJ feature “surface plot”. All the box plots shown in panels F–I were obtained analyzing confocal images of mitochondria stained with MitoTracker.

    Journal: Scientific Reports

    Article Title: Beyond autophagy: a novel role for autism-linked Wdfy3 in brain mitophagy

    doi: 10.1038/s41598-018-29421-7

    Figure Lengend Snippet: Dampening of autophagy activation in Wdfy3 -deficient neuronal progenitor cells is accompanied by decreased mitophagy. ( A ) Wdfy3 immunofluorescent labeling in striatal neuronal progenitor cells (NPCs) shows ubiquitous expression (scrambled). A significant down regulation of Wdfy3 was observed in siRNA-transfected cells. Quantification of Wdfy3 protein levels was based on fluorescent signal intensity normalized by cell area. Levels of Wdfy3 mRNA expression were assessed by qPCR normalized to Gapdh mRNA. ( B ) The effect of rapamycin (20 nM) addition was evaluated by quantifying LC3 immunostaining normalized by cell area, as well as the number of LC3 puncta per cell. Statistical analyses were performed by two-tailed Mann-Whitney test. ( C) Total mitochondrial mass was visualized in scrambled and siRNA striatal cells by staining with antibodies to ATP5B (green) whereas polarized mitochondria were stained with MitoTracker (red). Co-localization of functional and total mitochondria is shown in yellow (overlay). Insets show details of the mitochondrial morphology and co-localization of ATP5B and MitoTracker in the two genotypes. Red channel intensity has been increased for siRNA-transfected cells, due to the low MitoTracker fluorescence signal emitted by non-polarized mitochondria. Scale bars = 10 µm. Panels ( D–H ). Mitochondrial content ( D ) was quantified as the cell area occupied by mitochondria (in %). Mitochondrial footprint ( E ) was expressed as functional mitochondria (area) normalized by total mitochondria area. ( F ) Average area per mitochondrion in scrambled- and si-RNA striatal cells. Mitochondrial morphology ( G ) was reported as circularity index, with mitochondria having a more tubular shape being closer to a value of 1. All measurements shown in panels D,G were performed using a macro for ImageJ (see Methods). ( H , I ) Assessment of the mitochondrial network integrity in scrambled- and siRNA striatal neurons was performed with the MiNA macro for ImageJ and reported as number of networks per cell ( H ) and mean branch length ( I ). Statistical analysis for panels D–I was performed with a 2-tailed Mann-Whitney test. ( J ) Surface plots representing levels and mitochondrial distribution in scrambled (left) and siRNA (right) striatal cells were obtained with the ImageJ feature “surface plot”. All the box plots shown in panels F–I were obtained analyzing confocal images of mitochondria stained with MitoTracker.

    Article Snippet: For silencing experiments, siRNA-lipid complexes were formed using 1 ml Opti-MEM I Medium (Gibco) without serum, 43 μl of Lipofectamine RNAiMAX (Thermo Fisher Scientific) and 145 pmol scrambled siRNA or Wdfy3 siRNA (Thermo Fisher Scientific, #AM4611 and AM16708, ID: 95113, respectively).

    Techniques: Activation Assay, Labeling, Expressing, Transfection, Real-time Polymerase Chain Reaction, Immunostaining, Two Tailed Test, MANN-WHITNEY, Staining, Functional Assay, Fluorescence

    mRNA levels for IGFBP5 and IGFBP2 are biomarkers for bladder tumor cells sensitivity to IGF1R kinase inhibitor, AEW541. a Heat map of the IGF receptors, ligands, and binding proteins mRNA levels in regards to sensitivity to AEW41 in 13 bladder tumor derived cell lines. Data were extracted from CCLE database; b Effect of a blocking antibody against IGF1R and of IGF1R siRNA on the viability of RT112 cells. IGF1R knockdown 72 h after transfection with a control or anti-IGF1R siRNA was assessed by western blotting (inset). The effect of the siRNA on cell viability was assessed in MTT assays. The effect of the anti-IGF1R blocking antibody was assessed after 72 h, in MTT assays; c Spearman’s coefficients for the correlations between the sensitivity to AEW541 and mRNA levels for IGF receptors, ligands, and binding proteins, in 13 cell bladder tumor derived cell lines. Data were extracted from CCLE database

    Journal: BMC Cancer

    Article Title: IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

    doi: 10.1186/s12885-017-3618-5

    Figure Lengend Snippet: mRNA levels for IGFBP5 and IGFBP2 are biomarkers for bladder tumor cells sensitivity to IGF1R kinase inhibitor, AEW541. a Heat map of the IGF receptors, ligands, and binding proteins mRNA levels in regards to sensitivity to AEW41 in 13 bladder tumor derived cell lines. Data were extracted from CCLE database; b Effect of a blocking antibody against IGF1R and of IGF1R siRNA on the viability of RT112 cells. IGF1R knockdown 72 h after transfection with a control or anti-IGF1R siRNA was assessed by western blotting (inset). The effect of the siRNA on cell viability was assessed in MTT assays. The effect of the anti-IGF1R blocking antibody was assessed after 72 h, in MTT assays; c Spearman’s coefficients for the correlations between the sensitivity to AEW541 and mRNA levels for IGF receptors, ligands, and binding proteins, in 13 cell bladder tumor derived cell lines. Data were extracted from CCLE database

    Article Snippet: A negative control siRNA and a pre-validated siRNA specific for IGF1R were purchased from Ambion.

    Techniques: Binding Assay, Derivative Assay, Blocking Assay, Transfection, Western Blot, MTT Assay