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  • 99
    Thermo Fisher taqman microrna reverse transcription kit
    Carv induces the expression of miR-199a-3p and miR-214 under sI/R in HL-1 and H9c2 cells. A – C : HL-1 cells were treated with either vehicle (DMSO) or 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using <t>TaqMan</t> <t>microRNA</t> assay. Carv elicits upregulation of miR-199a-3p and miR-214 in simulated I/R, but not under normoxic (basal) conditions. D – F : H9c2 cells were treated with 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using TaqMan microRNA assay. Carv activates the expression of miR-199a-3p and miR-214 in sI/R, but not under basal conditions. * P
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sirna
    BMP3 suppresses BMP signaling via Acvr2b. A, Transcript levels of BMPR-II , Acvr2a , and Acvr2b were determined in W20-17 cells using quantitative real-time PCR. B–D, W20-17 cells were <t>transfected</t> with <t>siRNA</t> specific for Acvr2b or a scrambled siRNA.
    Sirna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sirna
    Cellular uptake. Cellular uptake of <t>DP-CLPs–PTX–survivin</t> <t>siRNA</t> (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirna
    Model of RNA pathogenic mechanism in HD. Several RNA dependent mechanisms contribute to HD pathogenesis. Dicer activity on hairpin-like structures in the mutant HTT gene or in double stranded sense and antisense transcripts induces the formation of sCAG or <t>CAG/CTG</t> <t>siRNA</t> that are incorporated into the RISC complex and trigger abnormal gene silencing. In addition mutant HTT mRNA may induce gene expression deregulation through sequestration of RNA binding proteins that have affinity for CAG repeats, including the transcriptional regulator MBLN. miRNA deregulation produced at least by cellular stress and REST transcriptional malfunction may also contribute to gene expression deregulation in HD.
    Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 16150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Santa Cruz Biotechnology sirna
    DRG2 localizes on EEA1- and Rab5-containing endosomes in a PI3K-dependent manner and interacts with EEA1 and Rab5 on endosomes. (A, B) DRG2 colocalizes with Rab5 and EEA1 on endosomes in a PI3K-depenednt manner. MCF7 cells expressing (A) mRFP-DRG2 and EGFP-Rab5 or (B) mRFP-DRG2 and EGFP-EEA1 were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002. (C) Activation status of Rab5 affects localization of DRG2 on Rab5 endosomes. MCF7 cells expressing EGFP-DRG2 and the dn mutant Rab5(S35N) or constitutively active mutant Rab5(Q79L) fused with mCherry. (D) EEA1 inhibition blocks the localization of DRG2 on Rba5 endosomes. EEA1 was inhibited by transfecting MCF7 cells with <t>siRNA</t> against EEA1. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5. (E) Serum deprivation inhibits colocalization of DRG2 with Rab5 on endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5 were incubated with serum-free medium for 12 h. (F) Expression of dn mutant Rab11(S25N) does not affect localization of DRG2 to endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab11(S25N). Representative confocal images with magnified insets of boxed areas. Blue, DAPI staining. Cells from A–E were analyzed for Pearson’s R ( r ). Values are mean ± SD from three separate experiments, with 10 different cells per group per experiment. (G, J) DRG2 interacts with EEA1 and Rab5 on endosomes in vivo. (G, H) MCF7 cells were <t>transfected</t> with (G) EGFP-EEA1 or (H) EGFP-Rab5(wild type), EGFP-Racb5(Q79L), or EGFP-Rab5(S35N). GFP-tagged Rab5 was immunoprecipitated from cell lysates and immunoblotted for endogenous DRG2. (I, J) MCF7 cells expressing DRG2-EGFP and (I) EEA1-mCherry or (J) Rab5-mCherry were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002 and analyzed for protein–protein interaction using FRET. The FRET efficiency percentage is depicted using discrete colors representing FRET values ranging from 0 to 10%. Values are mean ± SD from two separate experiments, with at least 10 different cells per group per experiment. ** p
    Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 9855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery sirnas
    Statistics on used siRNA libraries and hits. (A) We weighted <t>siRNAs</t> based on their library quality. Each vertical compartment in the plot corresponds to a training set of siRNAs. We averaged data in the training set from the siRNAs of the specific manufacturers. Each boxplot corresponds to a test set of single siRNAs from different manufacturers (except “Dharm. siRNA mean” which is the average of 4 <t>Dharmacon</t> unpooled siRNAs). Y-axis refers to Pearson correlation coefficients R between the training and test sets. A star corresponds to significant differences in the correlation coefficients (Mann–Whitney-U-test p
    Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 11504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher sirnas
    Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting <t>siRNA</t> (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with <t>siRNAs</t> targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.
    Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 47833 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenePharma Company sirna
    Knockdown of <t>MALAT1</t> inhibits ox-LDL-induced β-catenin translocation. HUVECs were transfected with <t>MALAT1-siRNA</t> or scramble control (scr) for 24 h. a The protein expression of β-catenin in nucleus (n = 3, * P
    Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 94/100, based on 4882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen allstars negative control sirna
    The effect of miR-26a and let-7a on the cell viability of SKMEL-28 and WM1552C melanoma cells. Cell viability was determined using the MTT assay after 48 h transfection with transfection reagent alone (control), <t>AllStars</t> negative control <t>siRNA</t> (100 nM), miR-26a mimics, and let-7a mimics at a final concentration of 50 or 100 nM. Each experiment was repeated six times and the results are presented as mean±S.D. Asterisks indicate a significant difference ( P
    Allstars Negative Control Sirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 5667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher silencer select negative control no 1 sirna
    Small interfering RNA suppression of STAT6 expression in vitro. A549 lung epithelial cells were transfected with a range of <t>siRNA</t> concentrations (0.1 – 100 nM) and total RNA and protein extracted 72 hours later. The percent STAT6 mRNA remaining at each of the concentrations tested was used to calculate the 50% inhibitory concentration of 372u (a) and 372 m (b); 0.27nM and 0.35nM, respectively. Western blot analysis was then used to confirm STAT6 protein ablation, which was observed at all concentrations of 372u tested (c) and at concentrations ≥ 5 nM for 372 m (d). SC = Silencer Select Negative Control No. 1 siRNA, vehicle = transfection reagent only. Values presented are mean ± S.E.M (n = 3).
    Silencer Select Negative Control No 1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen sirna
    Inhibition of cytoadherence but not adhesive strength on HDMEC transfected with small interference RNA of α 5 <t>integrin.</t> (A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative <t>siRNA</t> and siRNA for α 5 integrin ‘C’ and ‘D, and CD36. Blots were probed with a polyclonal anti-α 5 integrin antibody (top) and a monoclonal anti-α-tubulin antibody (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of α 5 integrin (n = 3). (C) Adhesion of IRBC to α 5 integrin knockdown endothelial monolayers (n = 3). (D) Force of detachment for IRBC on α 5 integrin knockdown endothelial monolayers (n = 2). (E) Force of detachment for IRBC on endothelial monolayers pre-incubated with the anti-α 5 mAb JBS5 (n = 2). Results for (D) and (E) are shown as mean ± SD.
    Sirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 15050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher negative control sirna
    The effects of depleting the expression of <t>MAGE-A9</t> on cell proliferation, migration and invasiveness of lung carcinoma cells ( A ) MAGE-A9 protein expression in four NSCLC cell lines. β-Actin was used as a loading control. ( B ) Western blots were used to select the most effective silencing <t>siRNA</t> for targeting human MAGE-A9. ( C ) and ( D ) The proliferation ability of the two experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24 h, 48 h, 72 h and 96 h. The cells were then incubated for an additional 3 h. The absorbance was determined at a wavelength of 450 nm. ( E ) Migration and ( F ) invasion ability were presented as the total number of cells that migrated to the bottom chamber without or with the Transwell precoated with Matrigel, as calculated in at least six random fields (total magnification ×200) per filter. The lower “F” is photo of six random fields represent invasion ability. It is corresponding to upper “F”. (* P
    Negative Control Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher silencer select pre designed sirna
    <t>RanBP2</t> depletion does not inhibit GFP expression in SVGA-GFP transgenic cells. Stable SVGA-GFP cells were transfected overnight either with 100 nM RanBP2 <t>siRNA,</t> 200 nM RanBP3 siRNA (negative control), 100 nM scrambled siRNA (control), or 100 nM GFP siRNA as a positive control. The cells were then followed up for 5 days. On day 3 images were captured and the GFP positive cells were analyzed by flow cytometry.
    Silencer Select Pre Designed Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen control sirna
    Immunofluorescence staining and protein blot analysis of CDCP1 in HS5 stromal cells before and after stimulation. Panel A : HS5 cells were treated with and without P3D9 stimulating antibody against CDCP1 for 30 minutes (right and left panels, respectively). They were then fixed and stained for CDCP1 (green), Actin (red) and Nuclei (DAPI, blue). Scale bars, 20 µm. Panel B : Control and stimulated HS5 cells were fixed and stained for VASP (green), CDCP1 (red), phosphotyrosine (pY, blue; detected by 4G10 antibody) and nuclei (DAPI, gray). Orthogonal images of the control and stimulated cells are shown. Pink fluorescence indicates the co-localization of CDCP1 and pY, suggesting that CDCP1 was tyrosine-phosphorylated. Original objective, X60. Scale bars, 20 µm. Panel C : Western blot analysis of detergent extracts of HS27a and HS5 cells before (−) and after (+) P3D9 stimulation. 4G10 antibody against pY was used. The bands of phosphorylated CDCP1, pSFK and <t>PKC-δ</t> were indicated according to the published studies [25] , [46] . Identification of the PKC-δ band was confirmed by knock-down experiments of HS5 cells using <t>siRNA</t> for PKC-δ ( Figure S3 in File S1 ). Panel D : Integrated pixel intensity of the bands of phosphorylated CDCP1 and PKC-δ in the blot of Panel C was quantitated by using Odyssey application software. Blue and red bars indicate HS5 cells before and after stimulation, respectively.
    Control Sirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 4715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher silencer negative control no 1 sirna
    Immunofluorescence staining and protein blot analysis of CDCP1 in HS5 stromal cells before and after stimulation. Panel A : HS5 cells were treated with and without P3D9 stimulating antibody against CDCP1 for 30 minutes (right and left panels, respectively). They were then fixed and stained for CDCP1 (green), Actin (red) and Nuclei (DAPI, blue). Scale bars, 20 µm. Panel B : Control and stimulated HS5 cells were fixed and stained for VASP (green), CDCP1 (red), phosphotyrosine (pY, blue; detected by 4G10 antibody) and nuclei (DAPI, gray). Orthogonal images of the control and stimulated cells are shown. Pink fluorescence indicates the co-localization of CDCP1 and pY, suggesting that CDCP1 was tyrosine-phosphorylated. Original objective, X60. Scale bars, 20 µm. Panel C : Western blot analysis of detergent extracts of HS27a and HS5 cells before (−) and after (+) P3D9 stimulation. 4G10 antibody against pY was used. The bands of phosphorylated CDCP1, pSFK and <t>PKC-δ</t> were indicated according to the published studies [25] , [46] . Identification of the PKC-δ band was confirmed by knock-down experiments of HS5 cells using <t>siRNA</t> for PKC-δ ( Figure S3 in File S1 ). Panel D : Integrated pixel intensity of the bands of phosphorylated CDCP1 and PKC-δ in the blot of Panel C was quantitated by using Odyssey application software. Blue and red bars indicate HS5 cells before and after stimulation, respectively.
    Silencer Negative Control No 1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sirna  (Amaxa)
    92
    Amaxa sirna
    HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Src and Akt. A , <t>MonoMac6</t> cells were cultured with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV) or treated with HIV-1 Tat (100 pg/ml). Culture lysates were examined by immunoblot for expression of PTEN and actin. B , MDM were incubated with or without HIV-1 Tat and examined for expression of Src, phospho-Src, FAK and phospho-FAK by immunoblot. C , MDM were incubated with PP2 (20 µM) or vehicle followed by incubation with HIV-1 Tat. Expression of total Akt and phospho-Akt were examined by immunoblot. D , MonoMac6 cells were transfected with control <t>siRNA</t> or siRNA directed against Src and examined for expression of Src and actin. E , MonoMac6 cells transfected with control siRNA or siRNA directed against Src were transfected with LC3-eGFP and incubated with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV)-infected MonoMac6 cells overnight. Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. Data are representative of 3 independent experiments presented as means ± SEM; ***p≤0.001, ∧p≥0.05.
    Sirna, supplied by Amaxa, used in various techniques. Bioz Stars score: 92/100, based on 3705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Carv induces the expression of miR-199a-3p and miR-214 under sI/R in HL-1 and H9c2 cells. A – C : HL-1 cells were treated with either vehicle (DMSO) or 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using TaqMan microRNA assay. Carv elicits upregulation of miR-199a-3p and miR-214 in simulated I/R, but not under normoxic (basal) conditions. D – F : H9c2 cells were treated with 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using TaqMan microRNA assay. Carv activates the expression of miR-199a-3p and miR-214 in sI/R, but not under basal conditions. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Carvedilol-responsive microRNAs, miR-199a-3p and -214 protect cardiomyocytes from simulated ischemia-reperfusion injury

    doi: 10.1152/ajpheart.00807.2015

    Figure Lengend Snippet: Carv induces the expression of miR-199a-3p and miR-214 under sI/R in HL-1 and H9c2 cells. A – C : HL-1 cells were treated with either vehicle (DMSO) or 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using TaqMan microRNA assay. Carv elicits upregulation of miR-199a-3p and miR-214 in simulated I/R, but not under normoxic (basal) conditions. D – F : H9c2 cells were treated with 1 μM Carv for 4 h and subjected to either normoxia (basal) or sI/R. The expression of mature miR-199a-3p and miR-214 was detected using TaqMan microRNA assay. Carv activates the expression of miR-199a-3p and miR-214 in sI/R, but not under basal conditions. * P

    Article Snippet: For detection of mature miR-199a-3p or miR-214, the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) was used to synthesize cDNA for TaqMan microRNA assays.

    Techniques: Expressing, TaqMan microRNA Assay

    PRIMA-1 upregulates expression of microRNA-34a. ( a ) TaqMan microRNA real-time PCR analysis was used to evaluate the expression of microRNA-34a, b and c. Cells were treated with the PRIMA-1 (100 μM), and cells were harvested at 6, 24 and 48 hr

    Journal: International journal of cancer. Journal international du cancer

    Article Title: MicroRNA-34a is an important component of PRIMA-1-induced apoptotic network in human lung cancer cells

    doi: 10.1002/ijc.25049

    Figure Lengend Snippet: PRIMA-1 upregulates expression of microRNA-34a. ( a ) TaqMan microRNA real-time PCR analysis was used to evaluate the expression of microRNA-34a, b and c. Cells were treated with the PRIMA-1 (100 μM), and cells were harvested at 6, 24 and 48 hr

    Article Snippet: Quantitation of micro-RNAs was carried out using TaqMan microRNA assays (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Quantitative RT-PCR validation for miR-222, miR148a and miR-21 in independent CLL patients . A) MiRNAs expression in a novel cohort of not responder (NR) and complete responder (CR) patients, before fludarabine treatment, quantified by using TaqMan reverse transcription qPCR. Each expression data is normalized on endogenous U6 RNA levels by 2 -ΔCt method. Each sample has been analyzed in triplicate. Data are displayed using vertical scatter plot (GraphPad v.5), bars represent means ± SEM. Two-tailed t-test was used to determine the p-values. B) Prediction of response to treatment in newly diagnosed CLL patients in accordance to a 3 miRNAs-based score. A threshold useful to predict response to therapy was established based on miRNA relative expression. Each patient with a final score > 1 was classified as refractory.

    Journal: Molecular Cancer

    Article Title: MicroRNAs involvement in fludarabine refractory chronic lymphocytic leukemia

    doi: 10.1186/1476-4598-9-123

    Figure Lengend Snippet: Quantitative RT-PCR validation for miR-222, miR148a and miR-21 in independent CLL patients . A) MiRNAs expression in a novel cohort of not responder (NR) and complete responder (CR) patients, before fludarabine treatment, quantified by using TaqMan reverse transcription qPCR. Each expression data is normalized on endogenous U6 RNA levels by 2 -ΔCt method. Each sample has been analyzed in triplicate. Data are displayed using vertical scatter plot (GraphPad v.5), bars represent means ± SEM. Two-tailed t-test was used to determine the p-values. B) Prediction of response to treatment in newly diagnosed CLL patients in accordance to a 3 miRNAs-based score. A threshold useful to predict response to therapy was established based on miRNA relative expression. Each patient with a final score > 1 was classified as refractory.

    Article Snippet: Mature miRNAs expression was evaluated by Taqman MiRNA assays (Applied Biosystem) specific for miR-21, miR-222, miR-148a and RNU6B as reference gene according to the manufacturer's protocol.

    Techniques: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    Expression pattern of miR-216a. ( a and b ) The dynamic expression profile of miR-216a during osteogenesis in hAMSCs was detected by miRNA microarray and validated by TaqMan qRT-PCR. ( c ) Analysis of miR-216a conservation in mammals. ( d and e ) The tissue

    Journal: Cell Death and Differentiation

    Article Title: miR-216a rescues dexamethasone suppression of osteogenesis, promotes osteoblast differentiation and enhances bone formation, by regulating c-Cbl-mediated PI3K/AKT pathway

    doi: 10.1038/cdd.2015.99

    Figure Lengend Snippet: Expression pattern of miR-216a. ( a and b ) The dynamic expression profile of miR-216a during osteogenesis in hAMSCs was detected by miRNA microarray and validated by TaqMan qRT-PCR. ( c ) Analysis of miR-216a conservation in mammals. ( d and e ) The tissue

    Article Snippet: For the qRT-PCR analysis of miR-216a, the TaqMan miRNA reverse transcription kit and TaqMan miRNA assay kit (Applied Biosystems) were used.

    Techniques: Expressing, Microarray, Quantitative RT-PCR

    Comparison of the sensitivity of three different miRNA qPCR assays. Total RNAs were reverse transcribed into cDNA using S-Poly(T) RT primer, oligo(dT) RT primer and Stem-loop RT primer, respectively. Hsa-miR-21 (A), hsa-miR-16 (B) and has-miR-210 (C) were amplified and detected with SYBR Green I followed by melting curve analysis. Hsa-miR-140-5p was reverse transcribed into cDNA using the S-Poly(T) RT primer and the RT primer from TaqMan® microRNA assays kit (Applied Biosystems). The cDNAs were then amplified and detected with either a universal Taqman probe (S-Poly(T) method) or a specific Taqman probe (stem-loop method). The threshold was set at 0.2 on PRISM 7300 Real-time PCR system (Applied Biosystems). All PCR reactions were run in triplicate.

    Journal: PLoS ONE

    Article Title: A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

    doi: 10.1371/journal.pone.0048536

    Figure Lengend Snippet: Comparison of the sensitivity of three different miRNA qPCR assays. Total RNAs were reverse transcribed into cDNA using S-Poly(T) RT primer, oligo(dT) RT primer and Stem-loop RT primer, respectively. Hsa-miR-21 (A), hsa-miR-16 (B) and has-miR-210 (C) were amplified and detected with SYBR Green I followed by melting curve analysis. Hsa-miR-140-5p was reverse transcribed into cDNA using the S-Poly(T) RT primer and the RT primer from TaqMan® microRNA assays kit (Applied Biosystems). The cDNAs were then amplified and detected with either a universal Taqman probe (S-Poly(T) method) or a specific Taqman probe (stem-loop method). The threshold was set at 0.2 on PRISM 7300 Real-time PCR system (Applied Biosystems). All PCR reactions were run in triplicate.

    Article Snippet: For the comparison experiment, the expression of hsa-miR-140-5p was measured with the TaqMan microRNA assay kit (Applied Biosystems) according to the manufacturer’s instructions.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay, Polymerase Chain Reaction

    Depletion of NPM leads to a disorganized nucleolar structure in HeLa cells ( A ) Western-blot analysis at 48-h post-transfection of mock and NPM RNAi cells. The NPM expression level was reduced to ∼80% by RNAi. Other nucleolar proteins, such as fibrillarin and nucleolin, an NE protein, lamin A/C, and a cytoskeleton protein, β-actin, were immunoblotted. Western-blot analysis of α-tubulin served as loading controls. ( B ) Quantification of NPM, nucleolin (NCL) and fibrillarin levels in mock- and NPM-siRNA-treated cells in Western blots. Values are the means±S.D. of three independent blots. ( C ) Immunostaining for NPM (red) and DNA (blue) in mock and NPM RNAi cells. Scale bar, 10 μm. ( D ) Phase-contrast images of fixed cells showing the pattern of nucleolar organization in mock-treated cells and the distortion of this pattern in NPM-depleted cells. Scale bar, 10 μm. ( E – G ) Immunostaining for nucleolar proteins in mock and NPM RNAi cells. ( E ) Fibrillarin (red), ( F ) nucleolin (red) and ( G ) Ki-67 (red); DNA is shown in blue. Scale bars, 5 μm.

    Journal: Biochemical Journal

    Article Title: Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells

    doi: 10.1042/BJ20081411

    Figure Lengend Snippet: Depletion of NPM leads to a disorganized nucleolar structure in HeLa cells ( A ) Western-blot analysis at 48-h post-transfection of mock and NPM RNAi cells. The NPM expression level was reduced to ∼80% by RNAi. Other nucleolar proteins, such as fibrillarin and nucleolin, an NE protein, lamin A/C, and a cytoskeleton protein, β-actin, were immunoblotted. Western-blot analysis of α-tubulin served as loading controls. ( B ) Quantification of NPM, nucleolin (NCL) and fibrillarin levels in mock- and NPM-siRNA-treated cells in Western blots. Values are the means±S.D. of three independent blots. ( C ) Immunostaining for NPM (red) and DNA (blue) in mock and NPM RNAi cells. Scale bar, 10 μm. ( D ) Phase-contrast images of fixed cells showing the pattern of nucleolar organization in mock-treated cells and the distortion of this pattern in NPM-depleted cells. Scale bar, 10 μm. ( E – G ) Immunostaining for nucleolar proteins in mock and NPM RNAi cells. ( E ) Fibrillarin (red), ( F ) nucleolin (red) and ( G ) Ki-67 (red); DNA is shown in blue. Scale bars, 5 μm.

    Article Snippet: The siRNA sequences for fibrillarin and nucleolin have been published previously [ , ]. siRNA transfection was performed according to manufacturer's protocol (Invitrogen).

    Techniques: Western Blot, Transfection, Expressing, Immunostaining

    BMP3 suppresses BMP signaling via Acvr2b. A, Transcript levels of BMPR-II , Acvr2a , and Acvr2b were determined in W20-17 cells using quantitative real-time PCR. B–D, W20-17 cells were transfected with siRNA specific for Acvr2b or a scrambled siRNA.

    Journal: Molecular Endocrinology

    Article Title: BMP3 Suppresses Osteoblast Differentiation of Bone Marrow Stromal Cells via Interaction with Acvr2b

    doi: 10.1210/me.2011-1168

    Figure Lengend Snippet: BMP3 suppresses BMP signaling via Acvr2b. A, Transcript levels of BMPR-II , Acvr2a , and Acvr2b were determined in W20-17 cells using quantitative real-time PCR. B–D, W20-17 cells were transfected with siRNA specific for Acvr2b or a scrambled siRNA.

    Article Snippet: Cells were transfected with siRNA using N-TER Nanoparticle siRNA Transfection System according to the manufacturer's instructions (Sigma-Aldrich).

    Techniques: Real-time Polymerase Chain Reaction, Transfection

    Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: Fluorescence, Microscopy, Incubation

    Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: Agarose Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Microscopy, In Vitro, Transmission Electron Microscopy

    (A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: (A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques:

    (A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: (A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: In Vivo, Fluorescence, Imaging, Mouse Assay, In Situ, Magnetic Resonance Imaging, Injection

    Silencing of PML facilitates transrepression of BAX and PUMA by SMAR1. ( A ) Comparative expression levels of SMAR1, Ac-p53 and p53 in HCT116 p53 +/+ cells transfected with scrambled siRNA (left half) versus PML siRNA (right panel) on UV (100 J/m 2 ) treatment. ( B ) Confocal staining of SMAR1 and PML in HCT116 p53 +/+ cells transfected with PML siRNA and treated with UV. Images are representative of > 50 fields ( n > 50) from two independent experiments. ( C ) ChIP assay in HCT116 p53 +/+ cells exposed to low dose (5 J/m 2 ) and high dose (100 J/m 2 ) UV irradiation. ( D ) ChIP showing SMAR1 occupancy on BAX and PUMA promoter after low dose (5 J/m 2 , 48 h) and at high dose (100 J/m 2 , 24 h) in PML knockdown HCT116 p53 +/+ cells.

    Journal: The EMBO Journal

    Article Title: Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element

    doi: 10.1038/emboj.2009.395

    Figure Lengend Snippet: Silencing of PML facilitates transrepression of BAX and PUMA by SMAR1. ( A ) Comparative expression levels of SMAR1, Ac-p53 and p53 in HCT116 p53 +/+ cells transfected with scrambled siRNA (left half) versus PML siRNA (right panel) on UV (100 J/m 2 ) treatment. ( B ) Confocal staining of SMAR1 and PML in HCT116 p53 +/+ cells transfected with PML siRNA and treated with UV. Images are representative of > 50 fields ( n > 50) from two independent experiments. ( C ) ChIP assay in HCT116 p53 +/+ cells exposed to low dose (5 J/m 2 ) and high dose (100 J/m 2 ) UV irradiation. ( D ) ChIP showing SMAR1 occupancy on BAX and PUMA promoter after low dose (5 J/m 2 , 48 h) and at high dose (100 J/m 2 , 24 h) in PML knockdown HCT116 p53 +/+ cells.

    Article Snippet: PML siRNAs and control siRNA (Ambion) were used at 300 nM concentration for 24 h. UV treatment was given using UVP cross-linker (Amersham) at two doses: 5 and 100 J/m2 .

    Techniques: Expressing, Transfection, Staining, Chromatin Immunoprecipitation, Irradiation

    Model of RNA pathogenic mechanism in HD. Several RNA dependent mechanisms contribute to HD pathogenesis. Dicer activity on hairpin-like structures in the mutant HTT gene or in double stranded sense and antisense transcripts induces the formation of sCAG or CAG/CTG siRNA that are incorporated into the RISC complex and trigger abnormal gene silencing. In addition mutant HTT mRNA may induce gene expression deregulation through sequestration of RNA binding proteins that have affinity for CAG repeats, including the transcriptional regulator MBLN. miRNA deregulation produced at least by cellular stress and REST transcriptional malfunction may also contribute to gene expression deregulation in HD.

    Journal: PLoS Genetics

    Article Title: A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity

    doi: 10.1371/journal.pgen.1002481

    Figure Lengend Snippet: Model of RNA pathogenic mechanism in HD. Several RNA dependent mechanisms contribute to HD pathogenesis. Dicer activity on hairpin-like structures in the mutant HTT gene or in double stranded sense and antisense transcripts induces the formation of sCAG or CAG/CTG siRNA that are incorporated into the RISC complex and trigger abnormal gene silencing. In addition mutant HTT mRNA may induce gene expression deregulation through sequestration of RNA binding proteins that have affinity for CAG repeats, including the transcriptional regulator MBLN. miRNA deregulation produced at least by cellular stress and REST transcriptional malfunction may also contribute to gene expression deregulation in HD.

    Article Snippet: Transfections All the transfection experiments were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instruction and at a 60% cell confluence. (CAG)7 ( 5′CAGCAGCAGCAGCAGCAGCAG-3′ ) and control, scrambled siRNA ( 5′-GCGACGUUCCUGAAACCAC-3′ ) were purchased from Dharmacon and were administered at a final concentration of 50 nM, unless otherwise indicated.

    Techniques: Activity Assay, Mutagenesis, CTG Assay, Expressing, RNA Binding Assay, Produced

    Knockdown of STAT3 decreases binding of ROCK2 to Irf4 promoter. Human peripheral blood CD4 + T cells were transfected with control or STAT3 siRNA, before stimulated under Th17-skewing conditions for 48 hours ( a–d ) or treated with a JAK inhibitor, Baricitinib while being stimulated under Th17-skewing conditions for 48 hours ( e ). Chromatin was purified and proceeds to ChIP-qPCR analysis with anti-ROCK2 ( a , c , e ) or anti-STAT3 antibodies ( b , d , e ). The average of three or four different experiments is shown. *p

    Journal: Scientific Reports

    Article Title: ROCK2, but not ROCK1 interacts with phosphorylated STAT3 and co-occupies TH17/TFH gene promoters in TH17-activated human T cells

    doi: 10.1038/s41598-018-35109-9

    Figure Lengend Snippet: Knockdown of STAT3 decreases binding of ROCK2 to Irf4 promoter. Human peripheral blood CD4 + T cells were transfected with control or STAT3 siRNA, before stimulated under Th17-skewing conditions for 48 hours ( a–d ) or treated with a JAK inhibitor, Baricitinib while being stimulated under Th17-skewing conditions for 48 hours ( e ). Chromatin was purified and proceeds to ChIP-qPCR analysis with anti-ROCK2 ( a , c , e ) or anti-STAT3 antibodies ( b , d , e ). The average of three or four different experiments is shown. *p

    Article Snippet: RNA interference Mixture of four ON-TARGETplus SMARTpool siRNAs specific to STAT3 (L-003544-00) and control siRNA (D-001810-10-20) were purchased from Dharmacon (Thermo Fisher Scientific Inc., Waltham, MA).

    Techniques: Binding Assay, Transfection, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    DRG2 localizes on EEA1- and Rab5-containing endosomes in a PI3K-dependent manner and interacts with EEA1 and Rab5 on endosomes. (A, B) DRG2 colocalizes with Rab5 and EEA1 on endosomes in a PI3K-depenednt manner. MCF7 cells expressing (A) mRFP-DRG2 and EGFP-Rab5 or (B) mRFP-DRG2 and EGFP-EEA1 were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002. (C) Activation status of Rab5 affects localization of DRG2 on Rab5 endosomes. MCF7 cells expressing EGFP-DRG2 and the dn mutant Rab5(S35N) or constitutively active mutant Rab5(Q79L) fused with mCherry. (D) EEA1 inhibition blocks the localization of DRG2 on Rba5 endosomes. EEA1 was inhibited by transfecting MCF7 cells with siRNA against EEA1. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5. (E) Serum deprivation inhibits colocalization of DRG2 with Rab5 on endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5 were incubated with serum-free medium for 12 h. (F) Expression of dn mutant Rab11(S25N) does not affect localization of DRG2 to endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab11(S25N). Representative confocal images with magnified insets of boxed areas. Blue, DAPI staining. Cells from A–E were analyzed for Pearson’s R ( r ). Values are mean ± SD from three separate experiments, with 10 different cells per group per experiment. (G, J) DRG2 interacts with EEA1 and Rab5 on endosomes in vivo. (G, H) MCF7 cells were transfected with (G) EGFP-EEA1 or (H) EGFP-Rab5(wild type), EGFP-Racb5(Q79L), or EGFP-Rab5(S35N). GFP-tagged Rab5 was immunoprecipitated from cell lysates and immunoblotted for endogenous DRG2. (I, J) MCF7 cells expressing DRG2-EGFP and (I) EEA1-mCherry or (J) Rab5-mCherry were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002 and analyzed for protein–protein interaction using FRET. The FRET efficiency percentage is depicted using discrete colors representing FRET values ranging from 0 to 10%. Values are mean ± SD from two separate experiments, with at least 10 different cells per group per experiment. ** p

    Journal: Molecular Biology of the Cell

    Article Title: Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    doi: 10.1091/mbc.E15-08-0558

    Figure Lengend Snippet: DRG2 localizes on EEA1- and Rab5-containing endosomes in a PI3K-dependent manner and interacts with EEA1 and Rab5 on endosomes. (A, B) DRG2 colocalizes with Rab5 and EEA1 on endosomes in a PI3K-depenednt manner. MCF7 cells expressing (A) mRFP-DRG2 and EGFP-Rab5 or (B) mRFP-DRG2 and EGFP-EEA1 were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002. (C) Activation status of Rab5 affects localization of DRG2 on Rab5 endosomes. MCF7 cells expressing EGFP-DRG2 and the dn mutant Rab5(S35N) or constitutively active mutant Rab5(Q79L) fused with mCherry. (D) EEA1 inhibition blocks the localization of DRG2 on Rba5 endosomes. EEA1 was inhibited by transfecting MCF7 cells with siRNA against EEA1. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5. (E) Serum deprivation inhibits colocalization of DRG2 with Rab5 on endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab5 were incubated with serum-free medium for 12 h. (F) Expression of dn mutant Rab11(S25N) does not affect localization of DRG2 to endosomes. MCF7 cells expressing mRFP-DRG2 and EGFP-Rab11(S25N). Representative confocal images with magnified insets of boxed areas. Blue, DAPI staining. Cells from A–E were analyzed for Pearson’s R ( r ). Values are mean ± SD from three separate experiments, with 10 different cells per group per experiment. (G, J) DRG2 interacts with EEA1 and Rab5 on endosomes in vivo. (G, H) MCF7 cells were transfected with (G) EGFP-EEA1 or (H) EGFP-Rab5(wild type), EGFP-Racb5(Q79L), or EGFP-Rab5(S35N). GFP-tagged Rab5 was immunoprecipitated from cell lysates and immunoblotted for endogenous DRG2. (I, J) MCF7 cells expressing DRG2-EGFP and (I) EEA1-mCherry or (J) Rab5-mCherry were incubated for 30 min in the absence or presence of 100 nM PI3K inhibitor LY294002 and analyzed for protein–protein interaction using FRET. The FRET efficiency percentage is depicted using discrete colors representing FRET values ranging from 0 to 10%. Values are mean ± SD from two separate experiments, with at least 10 different cells per group per experiment. ** p

    Article Snippet: To transiently inhibit EEA1 expression, cells were transfected with siRNA against EEA1 (3′-AAGTTTCAGATTCTTTACAAA-5′; ) or scrambled siRNA as a control (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Incubation, Activation Assay, Mutagenesis, Inhibition, Staining, In Vivo, Transfection, Immunoprecipitation

    TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p

    Journal: Nature Communications

    Article Title: Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization

    doi: 10.1038/s41467-018-07470-w

    Figure Lengend Snippet: TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p

    Article Snippet: For silencing experiments, cells were transfected with siRNAs targeting Trp53 and Tgfbr2 as well as control siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations.

    Techniques: RNA Sequencing Assay, Irradiation, Generated, Migration, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay

    Statistics on used siRNA libraries and hits. (A) We weighted siRNAs based on their library quality. Each vertical compartment in the plot corresponds to a training set of siRNAs. We averaged data in the training set from the siRNAs of the specific manufacturers. Each boxplot corresponds to a test set of single siRNAs from different manufacturers (except “Dharm. siRNA mean” which is the average of 4 Dharmacon unpooled siRNAs). Y-axis refers to Pearson correlation coefficients R between the training and test sets. A star corresponds to significant differences in the correlation coefficients (Mann–Whitney-U-test p

    Journal: BMC Genomics

    Article Title: Simultaneous analysis of large-scale RNAi screens for pathogen entry

    doi: 10.1186/1471-2164-15-1162

    Figure Lengend Snippet: Statistics on used siRNA libraries and hits. (A) We weighted siRNAs based on their library quality. Each vertical compartment in the plot corresponds to a training set of siRNAs. We averaged data in the training set from the siRNAs of the specific manufacturers. Each boxplot corresponds to a test set of single siRNAs from different manufacturers (except “Dharm. siRNA mean” which is the average of 4 Dharmacon unpooled siRNAs). Y-axis refers to Pearson correlation coefficients R between the training and test sets. A star corresponds to significant differences in the correlation coefficients (Mann–Whitney-U-test p

    Article Snippet: In addition, the data showed that the averaged single siRNAs of Dharmacon performed at most as good as the single pooled siRNA consisting of the same siRNAs.

    Techniques: MANN-WHITNEY

    Using more siRNAs adds power and yields reproducible results. (A) The three boxplots show Pearson correlation coefficients R between screens performed using the same siRNA set. The numbers 1 to 3 correspond to the total number of replicate screens that we averaged and compared to another distinct set of replicate screens, averaged over the same number. We resampled the replicate screens up to 500 times. The scatter plot shows an example for the correlation of infection indices from the duplicate of Adenovirus Dharmacon pooled screen. (B) The set of six boxplots shows the Pearson correlation coefficients of the averaged readouts from 1 to 6 siRNA sets. The scatter plots depict the correlation of infection indices for Adenovirus , the first between two different single siRNAs and the second between each an average over six siRNAs.

    Journal: BMC Genomics

    Article Title: Simultaneous analysis of large-scale RNAi screens for pathogen entry

    doi: 10.1186/1471-2164-15-1162

    Figure Lengend Snippet: Using more siRNAs adds power and yields reproducible results. (A) The three boxplots show Pearson correlation coefficients R between screens performed using the same siRNA set. The numbers 1 to 3 correspond to the total number of replicate screens that we averaged and compared to another distinct set of replicate screens, averaged over the same number. We resampled the replicate screens up to 500 times. The scatter plot shows an example for the correlation of infection indices from the duplicate of Adenovirus Dharmacon pooled screen. (B) The set of six boxplots shows the Pearson correlation coefficients of the averaged readouts from 1 to 6 siRNA sets. The scatter plots depict the correlation of infection indices for Adenovirus , the first between two different single siRNAs and the second between each an average over six siRNAs.

    Article Snippet: In addition, the data showed that the averaged single siRNAs of Dharmacon performed at most as good as the single pooled siRNA consisting of the same siRNAs.

    Techniques: Infection

    Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Journal: Oncogene

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    doi: 10.1038/onc.2014.97

    Figure Lengend Snippet: Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Article Snippet: siRNA sequences used for study All siRNAs used for study were ordered from Thermo Fisher Scientific Biosciences Inc. Lafayette, CO 80026, USA.

    Techniques: Expressing, Western Blot, Transfection, Fluorescence, Staining

    UBQLN1 loss induces cell migration and invasion (a) Migration assay in A549 and H358 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hrs post siRNA transfections a pipette tip was used to scratch the dish to make a “wound”. Cells were examined after wound has been formed and successively for 24hr and 48hr post wound formation and photographed. (b) Invasion assay in A549 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hours after siRNA transfection cells were seeded into Boyden chambers without (left) or with (right) matrigel. The lower chamber contained media with serum, whereas the upper chamber containing the cells was without serum. 48 hrs later cells on the underside of the membrane were fixed and stained. (c) Quantification of relative number of cells migrated or invaded through matrigel (*P

    Journal: Oncogene

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    doi: 10.1038/onc.2014.97

    Figure Lengend Snippet: UBQLN1 loss induces cell migration and invasion (a) Migration assay in A549 and H358 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hrs post siRNA transfections a pipette tip was used to scratch the dish to make a “wound”. Cells were examined after wound has been formed and successively for 24hr and 48hr post wound formation and photographed. (b) Invasion assay in A549 cells. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU1-2). 24 hours after siRNA transfection cells were seeded into Boyden chambers without (left) or with (right) matrigel. The lower chamber contained media with serum, whereas the upper chamber containing the cells was without serum. 48 hrs later cells on the underside of the membrane were fixed and stained. (c) Quantification of relative number of cells migrated or invaded through matrigel (*P

    Article Snippet: siRNA sequences used for study All siRNAs used for study were ordered from Thermo Fisher Scientific Biosciences Inc. Lafayette, CO 80026, USA.

    Techniques: Migration, Transfection, Transferring, Invasion Assay, Staining

    Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.

    Journal: Oncogene

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    doi: 10.1038/onc.2014.97

    Figure Lengend Snippet: Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.

    Article Snippet: siRNA sequences used for study All siRNAs used for study were ordered from Thermo Fisher Scientific Biosciences Inc. Lafayette, CO 80026, USA.

    Techniques: Transfection, Expressing, Fluorescence, Staining

    Loss of UBQLN1 does not induce ER stress or alter ERAD (a) Western blot analysis of proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2) and two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. (b) Western Blot analysis of proteins involved in ER stress response. A549 cells were treated with vehicle or with indicated concentrations of Thapsigargin (TH). (c) Western Blot analysis of proteins involved in ER stress response. A549 cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2). 48 hrs post siRNA transfections cells were treated with vehicle or with 20nM of Thapsigargin (TH) for 30 min and harvested 24 hrs later. (d) UBQLN1 loss does not alter ERAD. 293T cells stably expressing TCRyfp, a well-known ERAD substrate, were untreated (NT), treated with MG132, or transfected with the indicated siRNA. Cells were subjected to flow cytometry 24 hrs after MG132 treatment or 72 hrs post siRNA transfection to determine the intensity of yfp signal. MG132 treatment is used as a positive control, as ERAD is known to rely on the proteasome for destruction of substrates.

    Journal: Oncogene

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    doi: 10.1038/onc.2014.97

    Figure Lengend Snippet: Loss of UBQLN1 does not induce ER stress or alter ERAD (a) Western blot analysis of proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2) and two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. (b) Western Blot analysis of proteins involved in ER stress response. A549 cells were treated with vehicle or with indicated concentrations of Thapsigargin (TH). (c) Western Blot analysis of proteins involved in ER stress response. A549 cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting UBQLN1 (siU1 and siU2). 48 hrs post siRNA transfections cells were treated with vehicle or with 20nM of Thapsigargin (TH) for 30 min and harvested 24 hrs later. (d) UBQLN1 loss does not alter ERAD. 293T cells stably expressing TCRyfp, a well-known ERAD substrate, were untreated (NT), treated with MG132, or transfected with the indicated siRNA. Cells were subjected to flow cytometry 24 hrs after MG132 treatment or 72 hrs post siRNA transfection to determine the intensity of yfp signal. MG132 treatment is used as a positive control, as ERAD is known to rely on the proteasome for destruction of substrates.

    Article Snippet: siRNA sequences used for study All siRNAs used for study were ordered from Thermo Fisher Scientific Biosciences Inc. Lafayette, CO 80026, USA.

    Techniques: Western Blot, Transfection, Expressing, Stable Transfection, Flow Cytometry, Cytometry, Positive Control

    Knockdown of MALAT1 inhibits ox-LDL-induced β-catenin translocation. HUVECs were transfected with MALAT1-siRNA or scramble control (scr) for 24 h. a The protein expression of β-catenin in nucleus (n = 3, * P

    Journal: Lipids in Health and Disease

    Article Title: LncRNA MALAT1 modulates ox-LDL induced EndMT through the Wnt/β-catenin signaling pathway

    doi: 10.1186/s12944-019-1006-7

    Figure Lengend Snippet: Knockdown of MALAT1 inhibits ox-LDL-induced β-catenin translocation. HUVECs were transfected with MALAT1-siRNA or scramble control (scr) for 24 h. a The protein expression of β-catenin in nucleus (n = 3, * P

    Article Snippet: For MALAT1 knockdown, three MALAT1-targeting siRNAs (MALAT1-siRNA1, 5′-GGUGGUGG UAUUUAGAUAATTUUAUCUAAA UACCACCACCTT-3′; MALAT1-siRNA2, 5′-GCGUCAUUUAAAGCCUAGUTTA CUAGGCUUUAAAUGACGCTT3’; MALAT1-siRNA3, 5′-GGGCUGACAUUAAC UACAATTUUGUAGUUAAUGUCA GCCCTT-3′) and a scrambled siRNA (sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAA TT-3′) (Scramble) were designed and synthesized at GenePharma (Shanghai, China).

    Techniques: Translocation Assay, Transfection, Expressing

    The effect of miR-26a and let-7a on the cell viability of SKMEL-28 and WM1552C melanoma cells. Cell viability was determined using the MTT assay after 48 h transfection with transfection reagent alone (control), AllStars negative control siRNA (100 nM), miR-26a mimics, and let-7a mimics at a final concentration of 50 or 100 nM. Each experiment was repeated six times and the results are presented as mean±S.D. Asterisks indicate a significant difference ( P

    Journal: Cell Death Discovery

    Article Title: MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene

    doi: 10.1038/cddiscovery.2017.28

    Figure Lengend Snippet: The effect of miR-26a and let-7a on the cell viability of SKMEL-28 and WM1552C melanoma cells. Cell viability was determined using the MTT assay after 48 h transfection with transfection reagent alone (control), AllStars negative control siRNA (100 nM), miR-26a mimics, and let-7a mimics at a final concentration of 50 or 100 nM. Each experiment was repeated six times and the results are presented as mean±S.D. Asterisks indicate a significant difference ( P

    Article Snippet: The hsa-let-7a-5p (MSY0000062), miR-26a-5p (MSY0000082) and AllStars negative control siRNA were purchased from Qiagen (Germantown, MD, USA) and transfected into the cells using DharmFECT transfection reagent 2 (Thermo Scientific, Pittsburgh, PA, USA) at a final concentration of 50 or 100 nM.

    Techniques: MTT Assay, Transfection, Negative Control, Concentration Assay

    Small interfering RNA suppression of STAT6 expression in vitro. A549 lung epithelial cells were transfected with a range of siRNA concentrations (0.1 – 100 nM) and total RNA and protein extracted 72 hours later. The percent STAT6 mRNA remaining at each of the concentrations tested was used to calculate the 50% inhibitory concentration of 372u (a) and 372 m (b); 0.27nM and 0.35nM, respectively. Western blot analysis was then used to confirm STAT6 protein ablation, which was observed at all concentrations of 372u tested (c) and at concentrations ≥ 5 nM for 372 m (d). SC = Silencer Select Negative Control No. 1 siRNA, vehicle = transfection reagent only. Values presented are mean ± S.E.M (n = 3).

    Journal: PLoS ONE

    Article Title: Development of Pre-Clinical Models for Evaluating the Therapeutic Potential of Candidate siRNA Targeting STAT6

    doi: 10.1371/journal.pone.0090338

    Figure Lengend Snippet: Small interfering RNA suppression of STAT6 expression in vitro. A549 lung epithelial cells were transfected with a range of siRNA concentrations (0.1 – 100 nM) and total RNA and protein extracted 72 hours later. The percent STAT6 mRNA remaining at each of the concentrations tested was used to calculate the 50% inhibitory concentration of 372u (a) and 372 m (b); 0.27nM and 0.35nM, respectively. Western blot analysis was then used to confirm STAT6 protein ablation, which was observed at all concentrations of 372u tested (c) and at concentrations ≥ 5 nM for 372 m (d). SC = Silencer Select Negative Control No. 1 siRNA, vehicle = transfection reagent only. Values presented are mean ± S.E.M (n = 3).

    Article Snippet: Scrambled (SC) siRNA used within the A549 lung epithelial cell experiments was Silencer Select Negative Control No. 1 siRNA (Life Technologies, Paisley, UK).

    Techniques: Small Interfering RNA, Expressing, In Vitro, Transfection, Concentration Assay, Western Blot, Negative Control

    Inhibition of cytoadherence but not adhesive strength on HDMEC transfected with small interference RNA of α 5 integrin. (A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for α 5 integrin ‘C’ and ‘D, and CD36. Blots were probed with a polyclonal anti-α 5 integrin antibody (top) and a monoclonal anti-α-tubulin antibody (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of α 5 integrin (n = 3). (C) Adhesion of IRBC to α 5 integrin knockdown endothelial monolayers (n = 3). (D) Force of detachment for IRBC on α 5 integrin knockdown endothelial monolayers (n = 2). (E) Force of detachment for IRBC on endothelial monolayers pre-incubated with the anti-α 5 mAb JBS5 (n = 2). Results for (D) and (E) are shown as mean ± SD.

    Journal: PLoS Pathogens

    Article Title: CD36 Recruits ?5?1 Integrin to Promote Cytoadherence of P. falciparum-Infected Erythrocytes

    doi: 10.1371/journal.ppat.1003590

    Figure Lengend Snippet: Inhibition of cytoadherence but not adhesive strength on HDMEC transfected with small interference RNA of α 5 integrin. (A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for α 5 integrin ‘C’ and ‘D, and CD36. Blots were probed with a polyclonal anti-α 5 integrin antibody (top) and a monoclonal anti-α-tubulin antibody (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of α 5 integrin (n = 3). (C) Adhesion of IRBC to α 5 integrin knockdown endothelial monolayers (n = 3). (D) Force of detachment for IRBC on α 5 integrin knockdown endothelial monolayers (n = 2). (E) Force of detachment for IRBC on endothelial monolayers pre-incubated with the anti-α 5 mAb JBS5 (n = 2). Results for (D) and (E) are shown as mean ± SD.

    Article Snippet: GmBH, Hilden, Germany) and 20 nM siRNA for β1 integrin (Qiagen) or α5 integrin or 20 nM scrambled siRNA (All Stars Negative Control, Qiagen) was added in 100 µl of Optimem.

    Techniques: Inhibition, Transfection, Western Blot, Incubation

    Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β 1 integrin. (A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β 1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β 1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β 1 integrin (n = 3). (C) Adhesion of IRBC to β 1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β 1 integrin and CD36 knock down endothelial monolayers (n = 4).

    Journal: PLoS Pathogens

    Article Title: CD36 Recruits ?5?1 Integrin to Promote Cytoadherence of P. falciparum-Infected Erythrocytes

    doi: 10.1371/journal.ppat.1003590

    Figure Lengend Snippet: Inhibition of cytoadherence and adhesive strength on HDMEC transfected with small interference RNA of β 1 integrin. (A) Western blot analysis of HDMEC lysates 72 h after transfection with 20 nM of negative siRNA and siRNA for β 1 integrin ‘B’ and ‘D’, and CD36. Blots were probed with mAb anti-β 1 integrin TS2/16 (top) and anti-α-tubulin (bottom). Results shown are representative of 3 independent experiments. (B) Densitometric analysis showing the effectiveness of the knockdown of β 1 integrin (n = 3). (C) Adhesion of IRBC to β 1 integrin and CD36 knock down endothelial monolayers (n = 3). (D) Force of detachment for IRBC on β 1 integrin and CD36 knock down endothelial monolayers (n = 4).

    Article Snippet: GmBH, Hilden, Germany) and 20 nM siRNA for β1 integrin (Qiagen) or α5 integrin or 20 nM scrambled siRNA (All Stars Negative Control, Qiagen) was added in 100 µl of Optimem.

    Techniques: Inhibition, Transfection, Western Blot

    The effects of depleting the expression of MAGE-A9 on cell proliferation, migration and invasiveness of lung carcinoma cells ( A ) MAGE-A9 protein expression in four NSCLC cell lines. β-Actin was used as a loading control. ( B ) Western blots were used to select the most effective silencing siRNA for targeting human MAGE-A9. ( C ) and ( D ) The proliferation ability of the two experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24 h, 48 h, 72 h and 96 h. The cells were then incubated for an additional 3 h. The absorbance was determined at a wavelength of 450 nm. ( E ) Migration and ( F ) invasion ability were presented as the total number of cells that migrated to the bottom chamber without or with the Transwell precoated with Matrigel, as calculated in at least six random fields (total magnification ×200) per filter. The lower “F” is photo of six random fields represent invasion ability. It is corresponding to upper “F”. (* P

    Journal: Oncotarget

    Article Title: High expression levels of MAGE-A9 are correlated with unfavorable survival in lung adenocarcinoma

    doi: 10.18632/oncotarget.6741

    Figure Lengend Snippet: The effects of depleting the expression of MAGE-A9 on cell proliferation, migration and invasiveness of lung carcinoma cells ( A ) MAGE-A9 protein expression in four NSCLC cell lines. β-Actin was used as a loading control. ( B ) Western blots were used to select the most effective silencing siRNA for targeting human MAGE-A9. ( C ) and ( D ) The proliferation ability of the two experimental cell lines was examined using CCK-8 at 450 nm. Specifically, 5 × 10 3 cells were seeded in 100 μL of medium per well into 96-well plates (three wells per each group). Then, 10 μL of CCK8 solution was added to the culture medium in each well after 24 h, 48 h, 72 h and 96 h. The cells were then incubated for an additional 3 h. The absorbance was determined at a wavelength of 450 nm. ( E ) Migration and ( F ) invasion ability were presented as the total number of cells that migrated to the bottom chamber without or with the Transwell precoated with Matrigel, as calculated in at least six random fields (total magnification ×200) per filter. The lower “F” is photo of six random fields represent invasion ability. It is corresponding to upper “F”. (* P

    Article Snippet: SiRNA transfection Three different siRNA sequences targeting human MAGE-A9 and negative control siRNA were designed by, and obtained from, Shanghai Invitrogen Corporation.

    Techniques: Expressing, Migration, Western Blot, CCK-8 Assay, Incubation

    RanBP2 depletion does not inhibit GFP expression in SVGA-GFP transgenic cells. Stable SVGA-GFP cells were transfected overnight either with 100 nM RanBP2 siRNA, 200 nM RanBP3 siRNA (negative control), 100 nM scrambled siRNA (control), or 100 nM GFP siRNA as a positive control. The cells were then followed up for 5 days. On day 3 images were captured and the GFP positive cells were analyzed by flow cytometry.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion does not inhibit GFP expression in SVGA-GFP transgenic cells. Stable SVGA-GFP cells were transfected overnight either with 100 nM RanBP2 siRNA, 200 nM RanBP3 siRNA (negative control), 100 nM scrambled siRNA (control), or 100 nM GFP siRNA as a positive control. The cells were then followed up for 5 days. On day 3 images were captured and the GFP positive cells were analyzed by flow cytometry.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Expressing, Transgenic Assay, Transfection, Negative Control, Positive Control, Flow Cytometry, Cytometry

    RanBP2 depletion does not interfere with nuclear export of viral mRNA species. ( A ) Latently HIV infected monocytic (THP89) cells were transfected with 100 nM RanBP2 siRNA and total proteins were extracted 48 or 72 h post transfection. Western blot analysis revealed the levels of RanBP2 protein. RanBP2 level in cells that were transfected with 100 nM RanBP2 siRNA followed by bryostatin treatment were also examined the performance on viral activity. THP89 cells either transfected with scramble siRNA or left untransfected were used controls. ( B–D ) THP89 cells were first transfected either with RanBP2 siRNA, RanBP3 siRNA, scrambled siRNA or GFP siRNA as a positive control. 48 h later, the cells were reactivated with 25 ng/ml bryostatin (potent reactivator of latent HIV infection). The GFP expression was examined 48 hrs after bryostatin treatment. ( B ) The Images were captured and ( C ) GFP positive cells were analyzed by flow cytometry. ( D ) Supernatants collected in ( B ) were analyzed for p24 levels by ELISA.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion does not interfere with nuclear export of viral mRNA species. ( A ) Latently HIV infected monocytic (THP89) cells were transfected with 100 nM RanBP2 siRNA and total proteins were extracted 48 or 72 h post transfection. Western blot analysis revealed the levels of RanBP2 protein. RanBP2 level in cells that were transfected with 100 nM RanBP2 siRNA followed by bryostatin treatment were also examined the performance on viral activity. THP89 cells either transfected with scramble siRNA or left untransfected were used controls. ( B–D ) THP89 cells were first transfected either with RanBP2 siRNA, RanBP3 siRNA, scrambled siRNA or GFP siRNA as a positive control. 48 h later, the cells were reactivated with 25 ng/ml bryostatin (potent reactivator of latent HIV infection). The GFP expression was examined 48 hrs after bryostatin treatment. ( B ) The Images were captured and ( C ) GFP positive cells were analyzed by flow cytometry. ( D ) Supernatants collected in ( B ) were analyzed for p24 levels by ELISA.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Infection, Transfection, Western Blot, Activity Assay, Positive Control, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    RanBP2 depletion inhibits 2-LTR circle formation (HIV DNA) but not viral reverse transcription. SVGA and Magi cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control), scrambled siRNA or TNPO3 siRNA as a positive control. 48 h later, the cells were transduced with 200 ng/ml p24 equivalent VSV-NL4-3 (HIV). AZT, a reverse transcriptase inhibitor, was used as a positive control to inhibit the reverse transcription and added 30 min before VSV-HIV transduction. ( A ) Total DNA was extracted 24 h after VSV-HIV transduction and 2-LTR levels were examined by real-time PCR. ( B ) Total DNA from SVGA cells was extracted 12 hrs after VSV-HIV transduction and the viral reverse transcription was examined by real-time PCR.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion inhibits 2-LTR circle formation (HIV DNA) but not viral reverse transcription. SVGA and Magi cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control), scrambled siRNA or TNPO3 siRNA as a positive control. 48 h later, the cells were transduced with 200 ng/ml p24 equivalent VSV-NL4-3 (HIV). AZT, a reverse transcriptase inhibitor, was used as a positive control to inhibit the reverse transcription and added 30 min before VSV-HIV transduction. ( A ) Total DNA was extracted 24 h after VSV-HIV transduction and 2-LTR levels were examined by real-time PCR. ( B ) Total DNA from SVGA cells was extracted 12 hrs after VSV-HIV transduction and the viral reverse transcription was examined by real-time PCR.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Transfection, Negative Control, Positive Control, Transduction, Real-time Polymerase Chain Reaction

    RanBP2 depletion inhibits global ectopic gene expression. ( A ) Schematic diagram of Tat responsive SVGA-LTR-GFP reporter cells is shown. Cells were either transfected with 500 ng pCMV-Tat or pcDNA vector (control) and images were captured 48 h after transfection. ( B ) SVGA-LTR-GFP reporter cells were transfected either with series of concentrations of RanBP2 siRNA or scrambled siRNA. 48 h later, the cells were transfected with 500 ng pCMV-Tat. After 48 h the images were captured and GFP positive cells were analyzed by flow cytometry. ( C ) SVGA cells were transfected either with 100 nM RanBP2 siRNA, 200 nM RanBP3 siRNA or 100 nM scrambled siRNA. 48 h later, the cells were then transfected either with 100 ng pEGFP, 100 ng pTat-GFP or 100 ng pΔTat-GFP plasmids respectively. The GFP expression was examined 48 h later by capturing the images and analysis by flow cytometry.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion inhibits global ectopic gene expression. ( A ) Schematic diagram of Tat responsive SVGA-LTR-GFP reporter cells is shown. Cells were either transfected with 500 ng pCMV-Tat or pcDNA vector (control) and images were captured 48 h after transfection. ( B ) SVGA-LTR-GFP reporter cells were transfected either with series of concentrations of RanBP2 siRNA or scrambled siRNA. 48 h later, the cells were transfected with 500 ng pCMV-Tat. After 48 h the images were captured and GFP positive cells were analyzed by flow cytometry. ( C ) SVGA cells were transfected either with 100 nM RanBP2 siRNA, 200 nM RanBP3 siRNA or 100 nM scrambled siRNA. 48 h later, the cells were then transfected either with 100 ng pEGFP, 100 ng pTat-GFP or 100 ng pΔTat-GFP plasmids respectively. The GFP expression was examined 48 h later by capturing the images and analysis by flow cytometry.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    siRNA-mediated depletion of RanBP2 and RanBP3. SVGA cells were transfected either with RanBP2 siRNA or RanBP3 siRNA, respectively. In parallel, control SVGA cells were either transfected with 200 nM scrambled siRNA or without siRNA. Total proteins were extracted 72 h post siRNA transfection. Western blot analysis was performed to examine levels of RanBP2 and RanBP3, respectively. Actin was used as internal control. Image J was used to analyze the intensities of the bands.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: siRNA-mediated depletion of RanBP2 and RanBP3. SVGA cells were transfected either with RanBP2 siRNA or RanBP3 siRNA, respectively. In parallel, control SVGA cells were either transfected with 200 nM scrambled siRNA or without siRNA. Total proteins were extracted 72 h post siRNA transfection. Western blot analysis was performed to examine levels of RanBP2 and RanBP3, respectively. Actin was used as internal control. Image J was used to analyze the intensities of the bands.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Transfection, Western Blot

    RanBP2 depletion inhibits HIV-1 replication and lentiviral vector gene expression. ( A ) Magi cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control), TNPO3 siRNA (positive control) or scrambled siRNA (control). 48 h later, the cells were transduced with 200 ng/ml p24 equivalent VSV-NLENY1 (recombinant HIV virus containing YFP) for 16 h. The fluorescent images were taken 48 h post transduction followed by collection of the culture supernatants for p24 (ELISA). The YFP positive cells were analyzed by flow cytometry (p

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion inhibits HIV-1 replication and lentiviral vector gene expression. ( A ) Magi cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control), TNPO3 siRNA (positive control) or scrambled siRNA (control). 48 h later, the cells were transduced with 200 ng/ml p24 equivalent VSV-NLENY1 (recombinant HIV virus containing YFP) for 16 h. The fluorescent images were taken 48 h post transduction followed by collection of the culture supernatants for p24 (ELISA). The YFP positive cells were analyzed by flow cytometry (p

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Plasmid Preparation, Expressing, Transfection, Negative Control, Positive Control, Transduction, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    RanBP2 depletion impairs biological activities of ectopically expressed Tat and Rev HIV proteins. ( A ) Schematic diagram of Tat/Rev responsive SVGA-LTR-gag-GFP cells is shown. The cells were transfected with either 50 ng NL4-3 plasmid, or 50 ng NL4-3 (Rev-). Fluorescent images were captured 48 h post transfection. ( B ) SVGA-LTR-gag-GFP reporter cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control) or scrambled siRNA (control). 48 h later, the cells were then co-transfected with 50 ng pCMV-Tat and 200 ng pCMV-Rev. In parallel, Leptomycin B was used as positive control and was added 4 h after the transfection of reporter cells (pre-transfected with scrambled siRNA) with pCMV-Tat and pCMV-Rev. The images were captured and GFP positive cells were analyzed by flow cytometry 48 h post plasmid transfection.

    Journal: PLoS ONE

    Article Title: Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

    doi: 10.1371/journal.pone.0015620

    Figure Lengend Snippet: RanBP2 depletion impairs biological activities of ectopically expressed Tat and Rev HIV proteins. ( A ) Schematic diagram of Tat/Rev responsive SVGA-LTR-gag-GFP cells is shown. The cells were transfected with either 50 ng NL4-3 plasmid, or 50 ng NL4-3 (Rev-). Fluorescent images were captured 48 h post transfection. ( B ) SVGA-LTR-gag-GFP reporter cells were transfected either with RanBP2 siRNA, RanBP3 siRNA (negative control) or scrambled siRNA (control). 48 h later, the cells were then co-transfected with 50 ng pCMV-Tat and 200 ng pCMV-Rev. In parallel, Leptomycin B was used as positive control and was added 4 h after the transfection of reporter cells (pre-transfected with scrambled siRNA) with pCMV-Tat and pCMV-Rev. The images were captured and GFP positive cells were analyzed by flow cytometry 48 h post plasmid transfection.

    Article Snippet: RNA interference RanBP2 siRNA (sequence 5′- CCGUUUUGGUGAGUCAACAtt - 3′ , siRNA ID s11773, cat no. 4390824), positive control GFP siRNA (cat no. AM4626) and negative control RanBP3 siRNA (sequence 5′- GGAAGCCUGUGAGAAAAAAtt - 3′ , siRNA ID 19354, cat no. AM16708) were purchased from Ambion.

    Techniques: Transfection, Plasmid Preparation, Negative Control, Positive Control, Flow Cytometry, Cytometry

    Immunofluorescence staining and protein blot analysis of CDCP1 in HS5 stromal cells before and after stimulation. Panel A : HS5 cells were treated with and without P3D9 stimulating antibody against CDCP1 for 30 minutes (right and left panels, respectively). They were then fixed and stained for CDCP1 (green), Actin (red) and Nuclei (DAPI, blue). Scale bars, 20 µm. Panel B : Control and stimulated HS5 cells were fixed and stained for VASP (green), CDCP1 (red), phosphotyrosine (pY, blue; detected by 4G10 antibody) and nuclei (DAPI, gray). Orthogonal images of the control and stimulated cells are shown. Pink fluorescence indicates the co-localization of CDCP1 and pY, suggesting that CDCP1 was tyrosine-phosphorylated. Original objective, X60. Scale bars, 20 µm. Panel C : Western blot analysis of detergent extracts of HS27a and HS5 cells before (−) and after (+) P3D9 stimulation. 4G10 antibody against pY was used. The bands of phosphorylated CDCP1, pSFK and PKC-δ were indicated according to the published studies [25] , [46] . Identification of the PKC-δ band was confirmed by knock-down experiments of HS5 cells using siRNA for PKC-δ ( Figure S3 in File S1 ). Panel D : Integrated pixel intensity of the bands of phosphorylated CDCP1 and PKC-δ in the blot of Panel C was quantitated by using Odyssey application software. Blue and red bars indicate HS5 cells before and after stimulation, respectively.

    Journal: PLoS ONE

    Article Title: CDCP1 Identifies a CD146 Negative Subset of Marrow Fibroblasts Involved with Cytokine Production

    doi: 10.1371/journal.pone.0109304

    Figure Lengend Snippet: Immunofluorescence staining and protein blot analysis of CDCP1 in HS5 stromal cells before and after stimulation. Panel A : HS5 cells were treated with and without P3D9 stimulating antibody against CDCP1 for 30 minutes (right and left panels, respectively). They were then fixed and stained for CDCP1 (green), Actin (red) and Nuclei (DAPI, blue). Scale bars, 20 µm. Panel B : Control and stimulated HS5 cells were fixed and stained for VASP (green), CDCP1 (red), phosphotyrosine (pY, blue; detected by 4G10 antibody) and nuclei (DAPI, gray). Orthogonal images of the control and stimulated cells are shown. Pink fluorescence indicates the co-localization of CDCP1 and pY, suggesting that CDCP1 was tyrosine-phosphorylated. Original objective, X60. Scale bars, 20 µm. Panel C : Western blot analysis of detergent extracts of HS27a and HS5 cells before (−) and after (+) P3D9 stimulation. 4G10 antibody against pY was used. The bands of phosphorylated CDCP1, pSFK and PKC-δ were indicated according to the published studies [25] , [46] . Identification of the PKC-δ band was confirmed by knock-down experiments of HS5 cells using siRNA for PKC-δ ( Figure S3 in File S1 ). Panel D : Integrated pixel intensity of the bands of phosphorylated CDCP1 and PKC-δ in the blot of Panel C was quantitated by using Odyssey application software. Blue and red bars indicate HS5 cells before and after stimulation, respectively.

    Article Snippet: Transfection of HS5 cells with siRNA HS5 cells were transfected with siRNA for CDCP1 (FlexiTube siRNA Hs_CDCP1__4, SI00341446, Qiagen, Hilden Germany; Stealth RNAiTM 10620312, Invitrogen), SDC1 (Hs_SDC1__1, SI00020587, Qiagen), KDM3B (Hs_JMJD1B__7, SI04270357, Qiagen), PKC-δ (Stealth RNAiTM 10620318, Invitrogen) or control siRNA (Luciferase GL2, Qiagen) using Lipofectamine RNAiMAX (Life Technologies, Grand Island NY) according to the manufacturer's protocol.

    Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Software

    HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Src and Akt. A , MonoMac6 cells were cultured with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV) or treated with HIV-1 Tat (100 pg/ml). Culture lysates were examined by immunoblot for expression of PTEN and actin. B , MDM were incubated with or without HIV-1 Tat and examined for expression of Src, phospho-Src, FAK and phospho-FAK by immunoblot. C , MDM were incubated with PP2 (20 µM) or vehicle followed by incubation with HIV-1 Tat. Expression of total Akt and phospho-Akt were examined by immunoblot. D , MonoMac6 cells were transfected with control siRNA or siRNA directed against Src and examined for expression of Src and actin. E , MonoMac6 cells transfected with control siRNA or siRNA directed against Src were transfected with LC3-eGFP and incubated with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV)-infected MonoMac6 cells overnight. Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. Data are representative of 3 independent experiments presented as means ± SEM; ***p≤0.001, ∧p≥0.05.

    Journal: PLoS ONE

    Article Title: HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3

    doi: 10.1371/journal.pone.0011733

    Figure Lengend Snippet: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Src and Akt. A , MonoMac6 cells were cultured with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV) or treated with HIV-1 Tat (100 pg/ml). Culture lysates were examined by immunoblot for expression of PTEN and actin. B , MDM were incubated with or without HIV-1 Tat and examined for expression of Src, phospho-Src, FAK and phospho-FAK by immunoblot. C , MDM were incubated with PP2 (20 µM) or vehicle followed by incubation with HIV-1 Tat. Expression of total Akt and phospho-Akt were examined by immunoblot. D , MonoMac6 cells were transfected with control siRNA or siRNA directed against Src and examined for expression of Src and actin. E , MonoMac6 cells transfected with control siRNA or siRNA directed against Src were transfected with LC3-eGFP and incubated with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV)-infected MonoMac6 cells overnight. Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. Data are representative of 3 independent experiments presented as means ± SEM; ***p≤0.001, ∧p≥0.05.

    Article Snippet: Assessment of Autophagy MonoMac6 cells were transfected with LC3-eGFP or double transfected with LC3-eGFP and siRNA for Akt1 , Src, or STAT3 by Amaxa Nucleofection (Amaxa) per manufacturer's protocol.

    Techniques: Cell Culture, Infection, Expressing, Incubation, Transfection

    HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Akt. A , MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B , MonoMac6 cells were transfected with siRNA against Akt 1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C , MonoMac6 cells transfected with control or siRNA directed against Akt 1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. D , MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. E , MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.

    Journal: PLoS ONE

    Article Title: HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3

    doi: 10.1371/journal.pone.0011733

    Figure Lengend Snippet: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Akt. A , MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B , MonoMac6 cells were transfected with siRNA against Akt 1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C , MonoMac6 cells transfected with control or siRNA directed against Akt 1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. D , MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. E , MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.

    Article Snippet: Assessment of Autophagy MonoMac6 cells were transfected with LC3-eGFP or double transfected with LC3-eGFP and siRNA for Akt1 , Src, or STAT3 by Amaxa Nucleofection (Amaxa) per manufacturer's protocol.

    Techniques: Incubation, Expressing, Transfection, Infection

    STAT3 and Akt are required for HIV-1 and IL-10 to inhibit autophagy. A , MonoMac6 cells were transfected with siRNA against STAT3 or control siRNA. Expression of STAT3 and actin were examined 72 h post-transfection. B , MonoMac6 cells transfected with control siRNA or siRNA directed against STAT3 or Akt were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat or IL-10 (both at 100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. Data are representative of 3 independent experiments presented as means ± SEM; ***p≤0.001, ∧p≥0.05.

    Journal: PLoS ONE

    Article Title: HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3

    doi: 10.1371/journal.pone.0011733

    Figure Lengend Snippet: STAT3 and Akt are required for HIV-1 and IL-10 to inhibit autophagy. A , MonoMac6 cells were transfected with siRNA against STAT3 or control siRNA. Expression of STAT3 and actin were examined 72 h post-transfection. B , MonoMac6 cells transfected with control siRNA or siRNA directed against STAT3 or Akt were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat or IL-10 (both at 100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3 + structures. Data are representative of 3 independent experiments presented as means ± SEM; ***p≤0.001, ∧p≥0.05.

    Article Snippet: Assessment of Autophagy MonoMac6 cells were transfected with LC3-eGFP or double transfected with LC3-eGFP and siRNA for Akt1 , Src, or STAT3 by Amaxa Nucleofection (Amaxa) per manufacturer's protocol.

    Techniques: Transfection, Expressing, Incubation