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  • 99
    Millipore shrna sirna knockdown experiments shrnas
    NOTCH inhibition suppresses p21 without reducing p53 activity a , qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2 . b , Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g . c , qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2 -transduced human keratinocytes. d , The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4 , and CMYC -transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e , γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC and treatment with DAPT, p53DD, or p53 <t>shRNA.</t> Scale bars = 50 μm. f , The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC . g , The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value
    Shrna Sirna Knockdown Experiments Shrnas, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery shrna mkp1 sirna
    Regulation of the MAPK response to pore-formation is <t>MKP1</t> independent. (A) A549 cells were transfected with 2.5 µg of MKP1 or scrambled <t>siRNA</t> per 1×10 6 cells and stimulated 24 hrs post-transfection with 200 ng/ml of purified Ply for the indicated times. Transfection with MKP1 siRNA inhibited MKP1 expression but did not have an effect on Ply-induced MAPK phosphorylation.
    Shrna Mkp1 Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Arraystar hg19 pirna array
    Regulation of the MAPK response to pore-formation is <t>MKP1</t> independent. (A) A549 cells were transfected with 2.5 µg of MKP1 or scrambled <t>siRNA</t> per 1×10 6 cells and stimulated 24 hrs post-transfection with 200 ng/ml of purified Ply for the indicated times. Transfection with MKP1 siRNA inhibited MKP1 expression but did not have an effect on Ply-induced MAPK phosphorylation.
    Hg19 Pirna Array, supplied by Arraystar, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery shrna sirna smart pools
    Accelerated Cell-Cycle Progression in Cks2 −/− MEFs (A) Time-lapse microscopy of asynchronously growing wild-type, Cks2 −/− and Cks1 −/− MEFs. MEFs were immortalized using <t>shRNA</t> against p53. Wild-type MEFs were infected with a retrovirus expressing histone H2B-GFP, Cks2 −/− and Cks1 −/− MEFs expressed histone H2B-mCherry. Wild-type and knockout MEFs were mixed and imaged. The time between two successive anaphases was recorded for at least 50 cells of each genotype; the mean is shown. An example of a wild-type and Cks2 −/− cell is shown. Alternatively, MEFs were immortalized using the 3T3 protocol, and cell cycle duration was measured as the duration from one cytokinesis to the next for at least 50 cells per genotype. (B) FACS analysis of DNA content to generate cell cycle profiles of asynchronously proliferating wild-type, Cks2 −/− and Cks1 −/− MEFs. (C) Quantification of the cell cycle populations by FACS. (D) Experimental design and example of the FACS pattern of wild-type MEFs at time 0, depicting the G1, BrdU-positive and G2 gates. The ratio of BrdU-positive cells with a G1 DNA content (labeled “early S”) over unlabeled G1 cells was followed over a 24 hr chase.
    Shrna Sirna Smart Pools, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sirna shnr2e3
    NR2E3 regulates ESR1 function in breast cancer cells A-B. MCF-7 cells were stably transfected with <t>shNR2E3</t> or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe. C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes. E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody. F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. G-H. MCF-7 cells were transiently transfected with indicated <t>siRNA,</t> and cell lysates were used for Western blot ( G ) or for qRT-PCR ( H ). All results are shown as mean plus standard deviation (SD) from three-independent replicates (* p
    Sirna Shnr2e3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GenePharma Company shrnas targeting tmprss4
    NR2E3 regulates ESR1 function in breast cancer cells A-B. MCF-7 cells were stably transfected with <t>shNR2E3</t> or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe. C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes. E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody. F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. G-H. MCF-7 cells were transiently transfected with indicated <t>siRNA,</t> and cell lysates were used for Western blot ( G ) or for qRT-PCR ( H ). All results are shown as mean plus standard deviation (SD) from three-independent replicates (* p
    Shrnas Targeting Tmprss4, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore shrnas
    hVps34 and <t>PLD1</t> regulate cell size. (A) HEK293 cells were transduced with lentiviruses expressing <t>shRNAs</t> for raptor, hVps34, or PLD1, puromycin selected, and then subjected to cell size measurement of median forward scatter-height. The result of overnight treatment with 100 nM rapamycin is included as a control. Representative histograms are also shown, with cell counts in arbitrary units. (B) Cells were transfected with wt- or ΔPX-PLD1 together in pCDNA3 (vector), selected with G418 for 3 d, and then subjected to cell size measurement as described in A. A one-sample t test was performed to compare each data with the control. Three independent experiments were performed, and the results of mean ± SD are shown in the graphs. *, P
    Shrnas, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3945 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company sirna shrna
    PD-L2 knockdown-induced inhibition of autophagy attenuates migration and invasion of osteosarcoma cells. a Representative TEM images depict ultrastructures present during autophagy in KHOS and U2OS cells transfected with PD-L2 <t>shRNA</t> or shNC. Images show autophagic vacuoles (arrows) observed in control cells (the picture in the right is a zoom of the picture in the left). No or few autophagic vacuoles were observed in PD-L2 knockdown cells. b Cells after PD-L2 knockdown exhibited a punctate pattern of LC3-II fluorescence, with reduced LC3-II compared with autophagosomes. KHOS and U2OS cells were incubated with or without CQ. c Expression of LC3, p62, and beclin-1 was evaluated by western blot. KHOS and U2OS cells were incubated in the presence or absence of CQ
    Sirna Shrna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Waters Corporation pirna biogenesis
    PD-L2 knockdown-induced inhibition of autophagy attenuates migration and invasion of osteosarcoma cells. a Representative TEM images depict ultrastructures present during autophagy in KHOS and U2OS cells transfected with PD-L2 <t>shRNA</t> or shNC. Images show autophagic vacuoles (arrows) observed in control cells (the picture in the right is a zoom of the picture in the left). No or few autophagic vacuoles were observed in PD-L2 knockdown cells. b Cells after PD-L2 knockdown exhibited a punctate pattern of LC3-II fluorescence, with reduced LC3-II compared with autophagosomes. KHOS and U2OS cells were incubated with or without CQ. c Expression of LC3, p62, and beclin-1 was evaluated by western blot. KHOS and U2OS cells were incubated in the presence or absence of CQ
    Pirna Biogenesis, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sirna gene silencing shrnas
    PD-L2 knockdown-induced inhibition of autophagy attenuates migration and invasion of osteosarcoma cells. a Representative TEM images depict ultrastructures present during autophagy in KHOS and U2OS cells transfected with PD-L2 <t>shRNA</t> or shNC. Images show autophagic vacuoles (arrows) observed in control cells (the picture in the right is a zoom of the picture in the left). No or few autophagic vacuoles were observed in PD-L2 knockdown cells. b Cells after PD-L2 knockdown exhibited a punctate pattern of LC3-II fluorescence, with reduced LC3-II compared with autophagosomes. KHOS and U2OS cells were incubated with or without CQ. c Expression of LC3, p62, and beclin-1 was evaluated by western blot. KHOS and U2OS cells were incubated in the presence or absence of CQ
    Sirna Gene Silencing Shrnas, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NOTCH inhibition suppresses p21 without reducing p53 activity a , qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2 . b , Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g . c , qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2 -transduced human keratinocytes. d , The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4 , and CMYC -transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e , γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC and treatment with DAPT, p53DD, or p53 shRNA. Scale bars = 50 μm. f , The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC . g , The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value

    Journal: Nature chemical biology

    Article Title: Notch inhibition allows oncogene independent generation of iPS cells

    doi: 10.1038/nchembio.1552

    Figure Lengend Snippet: NOTCH inhibition suppresses p21 without reducing p53 activity a , qPCR analysis of p53-dependent genes in human neonatal keratinocytes 3 days after transduction of GFP or OCT4 and SOX2 . b , Western blot of p53 levels in human neonatal keratinocytes with DMSO or 10 μM DAPT treatment for 3 days. Full blot shown in Supplementary Figure 7g . c , qPCR analysis of p53-dependent genes after 10 μM DAPT or 2 μM DBZ treatment for 3 days in OCT4, SOX2 -transduced human keratinocytes. d , The efficiency of NANOG+/TRA-1-81+ iPSC generation in OCT4, SOX2, KLF4 , and CMYC -transduced human neonatal keratinocytes transduced with p53DD or GFP with or without exposure to UV irradiation. e , γH2AX immunostaining in human neonatal keratinocytes 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC and treatment with DAPT, p53DD, or p53 shRNA. Scale bars = 50 μm. f , The percentage of TUNEL-positive cells in human neonatal keratinocyte reprogramming cultures with active or inactive p53 (p53DD expression) 10 days after transduction with OCT4, SOX2, KLF4 , and CMYC . g , The number of insertions or deletions (indels) per iPSC line derived under normal, DAPT, or p53DD conditions, as determined by array CGH. For all experiments, error bars represent the standard deviation between two biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test. * denotes significance p-value

    Article Snippet: shRNA/siRNA knockdown experiments shRNAs and siRNAs were purchased from Sigma and added to reprogramming cultures within 1 day after addition of the reprogramming retroviruses. shRNAs (TRCN0000003753, p53 and TRCN0000287021, p21) were expressed in the pLKO.1 lentiviral backbone. siRNAs were used at 80nM and were transfected into reprogramming cultures using RNAiMAX (Life Technologies).

    Techniques: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction, Transduction, Western Blot, Irradiation, Immunostaining, shRNA, TUNEL Assay, Expressing, Derivative Assay, Standard Deviation, Two Tailed Test

    Notch inhibition promotes keratinocyte reprogramming by suppressing p21 a , Schematic of the DAPT treatment time course on human neonatal keratinocytes. b , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, SOX2, KLF4 , and CMYC and treated with intervals of 10 μM DAPT or c , 2 μM DBZ. d , Western blot for p21 in human neonatal keratinocytes transduced with OCT4 and SOX2 and treated with DMSO or 10 μM DAPT. Full blot shown in Supplementary Figure 7c . e , Western blot for INVOLUCRIN in human neonatal keratinocytes treated with DMSO, 10 μM DAPT, or 1.2 mM calcium chloride for 6 days. Calcium was used as a positive control to induce keratinocyte differentiation. Full blot shown in Supplementary Figure 7d . f , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2 , and CMYC and a scrambled shRNA or a p21 shRNA at day 0 of reprogramming. DAPT was added at 10 μM. g , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4 and SOX2 and a scrambled shRNA control or a p21 shRNA at day 0 of reprogramming. DAPT was added at 2.5 μM. h , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4 , and CMYC and GFP or p21 and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

    Journal: Nature chemical biology

    Article Title: Notch inhibition allows oncogene independent generation of iPS cells

    doi: 10.1038/nchembio.1552

    Figure Lengend Snippet: Notch inhibition promotes keratinocyte reprogramming by suppressing p21 a , Schematic of the DAPT treatment time course on human neonatal keratinocytes. b , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, SOX2, KLF4 , and CMYC and treated with intervals of 10 μM DAPT or c , 2 μM DBZ. d , Western blot for p21 in human neonatal keratinocytes transduced with OCT4 and SOX2 and treated with DMSO or 10 μM DAPT. Full blot shown in Supplementary Figure 7c . e , Western blot for INVOLUCRIN in human neonatal keratinocytes treated with DMSO, 10 μM DAPT, or 1.2 mM calcium chloride for 6 days. Calcium was used as a positive control to induce keratinocyte differentiation. Full blot shown in Supplementary Figure 7d . f , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4, KLF4, SOX2 , and CMYC and a scrambled shRNA or a p21 shRNA at day 0 of reprogramming. DAPT was added at 10 μM. g , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT4 and SOX2 and a scrambled shRNA control or a p21 shRNA at day 0 of reprogramming. DAPT was added at 2.5 μM. h , Efficiency of NANOG+/TRA-1-81+ iPSC generation from human neonatal keratinocytes transduced with OCT, SOX2, KLF4 , and CMYC and GFP or p21 and treated with DMSO or 10 μM DAPT from days 1-18 post-transduction. For all experiments, error bars represent the standard deviation between two-three biological replicates and statistical significance was determined using a two-tailed homoscedastic Student's t-test.

    Article Snippet: shRNA/siRNA knockdown experiments shRNAs and siRNAs were purchased from Sigma and added to reprogramming cultures within 1 day after addition of the reprogramming retroviruses. shRNAs (TRCN0000003753, p53 and TRCN0000287021, p21) were expressed in the pLKO.1 lentiviral backbone. siRNAs were used at 80nM and were transfected into reprogramming cultures using RNAiMAX (Life Technologies).

    Techniques: Inhibition, Transduction, Western Blot, Positive Control, shRNA, Standard Deviation, Two Tailed Test

    Establishment of an ADAM17-knockdown cell line using a lentiviral expression system ( A ) ADAM17 mRNA expression in Huh7 CD133+ and Huh7 CD133− cells at 24 h determined by RT-PCR following transfection of ADAM17 siRNA. ( B ) Transwell migration assays of Huh7 CD133+ , Huh7 CD133− , scram siRNA Huh7 CD133+ and ADAM17 siRNA Huh7 CD133+ cells after 15- Gy irradiation. Images were captured at 48 h under × 40 magnification. The numbers of migrated Huh7 CD133+ , Huh7 CD133− , scram siRNA Huh7 CD133+ and ADAM17 siRNA Huh7 CD133+ cells were compared. ** P

    Journal: Oncotarget

    Article Title: Role of ADAM17 in invasion and migration of CD133-expressing liver cancer stem cells after irradiation

    doi: 10.18632/oncotarget.8112

    Figure Lengend Snippet: Establishment of an ADAM17-knockdown cell line using a lentiviral expression system ( A ) ADAM17 mRNA expression in Huh7 CD133+ and Huh7 CD133− cells at 24 h determined by RT-PCR following transfection of ADAM17 siRNA. ( B ) Transwell migration assays of Huh7 CD133+ , Huh7 CD133− , scram siRNA Huh7 CD133+ and ADAM17 siRNA Huh7 CD133+ cells after 15- Gy irradiation. Images were captured at 48 h under × 40 magnification. The numbers of migrated Huh7 CD133+ , Huh7 CD133− , scram siRNA Huh7 CD133+ and ADAM17 siRNA Huh7 CD133+ cells were compared. ** P

    Article Snippet: ADAM17 knockdown with stable short-hairpin siRNA (shRNA) To establish a stable Huh7 cell line depleted of ADAM17 expression, cells were infected using a short-hairpin RNA (shRNA)-lentiviral infection system (Sigma-Aldrich Co.).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Migration, Irradiation

    IKKβ-mediated IκBα degradation is blocked by S298A AEG-1. ( a ) MCF-7 cells were transfected with AEG-1 siRNA. Forty-eight hours later, cells were starved for 12 h and fixed in 4% PFA for 15 min at room temperature. Cells were immunostained using anti-AEG-1 and anti-IκBα antibodies. ( b ) Cells stained in a were imaged using epi-fluorescence microscope and cell borders were marked using ImageJ. Staining intensity was analysed using ImageJ and normalized to the cell area. Plots show the mean values of at least nine cells per condition from two independent experiments±s.d. Data were analysed by Student's t -test, ** P

    Journal: Nature Communications

    Article Title: Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate

    doi: 10.1038/ncomms7658

    Figure Lengend Snippet: IKKβ-mediated IκBα degradation is blocked by S298A AEG-1. ( a ) MCF-7 cells were transfected with AEG-1 siRNA. Forty-eight hours later, cells were starved for 12 h and fixed in 4% PFA for 15 min at room temperature. Cells were immunostained using anti-AEG-1 and anti-IκBα antibodies. ( b ) Cells stained in a were imaged using epi-fluorescence microscope and cell borders were marked using ImageJ. Staining intensity was analysed using ImageJ and normalized to the cell area. Plots show the mean values of at least nine cells per condition from two independent experiments±s.d. Data were analysed by Student's t -test, ** P

    Article Snippet: shRNA/siRNA/primers Human AEG-1 shRNAs (TRCN0000350650, TRCN0000322947, TRCN0000322949, TRCN0000322872 and TRCN0000151467) and mouse AEG-1 shRNAs (TRCN0000313386, TRCN0000312351, TRCN0000312352, TRCN0000312360 and TRCN0000312291 were purchased from SIGMA, siRNAs (SI00625793, SI04315605, SI05006421 and SI05006428) were purchased from QIAGEN.

    Techniques: Transfection, Staining, Fluorescence, Microscopy

    VEGF-C regulates canonical VEGFR2 survival signaling. (A) WB analysis of downstream VEGFR2 signaling in CPH017 glioblastoma cells starved for 24 h followed by stimulation with VEGF-A (40 ng/mL) or VEGF-C (0.2 µg/mL). (B) Quantitative RT-PCR analysis of VEGF-C expression in glioblastoma cells 48 h following transfection with either siCtrl (nontargeting control) or siVEGF-C (VEGF-C targeting siRNA). Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (C) WB for VEGF-C and tubulin in CPH017 cells 72 h following siCtrl or siVEGF-C transfection. (D) Quantitative RT-PCR analysis of VEGF-A expression in CPH017 and IN1123 cells 48 h following siCtrl or siVEGF-C transfection. Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (E) ELISA quantification of VEGF-A in conditioned media from CPH017 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 2. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. (F) Viability of CPH017 and IN1123 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 3–5. Significance determined by 2-way ANOVA with Bonferroni post-test correction. (G) WB of pro- and cleaved (Cl.) caspase-3 and tubulin in CPH017 and IN1123 glioblastoma cells 72 h following siCtrl or siVEGF-C transfection. (H) Viability of CPH017 and IN1123 glioblastoma cells 5 days after transfection with siCtrl or siVEGF-C alone and combined treatment with 0.5 mg/mL bevacizumab (Bev). Data are represented as mean ± SEM; n = 3. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

    Journal: Neuro-Oncology

    Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

    doi: 10.1093/neuonc/noy103

    Figure Lengend Snippet: VEGF-C regulates canonical VEGFR2 survival signaling. (A) WB analysis of downstream VEGFR2 signaling in CPH017 glioblastoma cells starved for 24 h followed by stimulation with VEGF-A (40 ng/mL) or VEGF-C (0.2 µg/mL). (B) Quantitative RT-PCR analysis of VEGF-C expression in glioblastoma cells 48 h following transfection with either siCtrl (nontargeting control) or siVEGF-C (VEGF-C targeting siRNA). Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (C) WB for VEGF-C and tubulin in CPH017 cells 72 h following siCtrl or siVEGF-C transfection. (D) Quantitative RT-PCR analysis of VEGF-A expression in CPH017 and IN1123 cells 48 h following siCtrl or siVEGF-C transfection. Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (E) ELISA quantification of VEGF-A in conditioned media from CPH017 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 2. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. (F) Viability of CPH017 and IN1123 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 3–5. Significance determined by 2-way ANOVA with Bonferroni post-test correction. (G) WB of pro- and cleaved (Cl.) caspase-3 and tubulin in CPH017 and IN1123 glioblastoma cells 72 h following siCtrl or siVEGF-C transfection. (H) Viability of CPH017 and IN1123 glioblastoma cells 5 days after transfection with siCtrl or siVEGF-C alone and combined treatment with 0.5 mg/mL bevacizumab (Bev). Data are represented as mean ± SEM; n = 3. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

    Article Snippet: SiRNA Transfection/shRNA Transduction Cells were transfected using 75 pmol of either VEGF-C–small interfering (si)RNA (siVEGF-C-1: esiRNA pool/EHU013781, Sigma-Aldrich; siVEGF-C-2: single siRNA/HSS111277, ThermoFisher Scientific) or scrambled control siRNA (siCtrl: Stealth RNAi Negative Control Duplex Med CG, ThermoFisher Scientific).

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay

    Positive regulation of STAT3 in the proliferation of Caco-2 cells. (a) Growth curve of blank Caco-2 cells (as blank control), Caco-2 cells which were transfected with sh-STAT3 (STAT3 (KD)), or control shRNA (control (KD)), and the cell number was counted in each group after an incubation of 24, 48, or 72 hours; (b) colony forming assay of blank, STAT3 (KD), and control (KD) Caco-2 cells after 48-hour incubation; (c) colony counting of the three groups of Caco-2 cells. Results were repeated in triplicate independently, ∗∗ represented p

    Journal: Gastroenterology Research and Practice

    Article Title: Annexin A2 Coordinates STAT3 to Regulate the Invasion and Migration of Colorectal Cancer Cells In Vitro

    doi: 10.1155/2016/3521453

    Figure Lengend Snippet: Positive regulation of STAT3 in the proliferation of Caco-2 cells. (a) Growth curve of blank Caco-2 cells (as blank control), Caco-2 cells which were transfected with sh-STAT3 (STAT3 (KD)), or control shRNA (control (KD)), and the cell number was counted in each group after an incubation of 24, 48, or 72 hours; (b) colony forming assay of blank, STAT3 (KD), and control (KD) Caco-2 cells after 48-hour incubation; (c) colony counting of the three groups of Caco-2 cells. Results were repeated in triplicate independently, ∗∗ represented p

    Article Snippet: Annexin A2 knockdown and STAT3 knockdown were conducted by using siRNAi-Annexin A2 (5′-CAAGCCCCTGTATTTTGCTGAT-3′) (for Annexin A2 silence) and scramble RNA (as control siRNA, 5′-UUCUCAGAACGUGUGACGU-3′) and shRNA STAT3/Puro (SIGMA-Aldrich) (for STAT3 knockdown, targeting sequence: 5′-GCGTCCAGTTCACTACTAAAG-3′) lentiviral vectors, respectively, with shRNAi MISSION Non-Target shRNA Control/Puro (SIGMA Aldrich, St. Louis, MO, USA) performed as negative control of gene knockdown.

    Techniques: Transfection, shRNA, Incubation

    Effective knockdown of ADAMTS5 in SW982 synovial fibroblasts cells transduced with ADAMTS5 targeting shRNA lentiviral particles. SW982 cells were untransduced (unt), or transduced with non-targeting shRNA (Ctl) or with two distinct ADAMTS5-targeting shRNA (denoted #3 or #5). Post-transduction the cells were treated in the presence or absence of IL-1β (1ng/ml, 16h) to induce ADAMTS5 expression. Subsequently, a western blot of whole cell extracts from these cells were run and probed with anti-ADAMTS5 or anti-GAPDH antibodies as indicated. GAPDH abundance was used as a loading control reference. A representative blot is shown.

    Journal: PLoS ONE

    Article Title: Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts

    doi: 10.1371/journal.pone.0129999

    Figure Lengend Snippet: Effective knockdown of ADAMTS5 in SW982 synovial fibroblasts cells transduced with ADAMTS5 targeting shRNA lentiviral particles. SW982 cells were untransduced (unt), or transduced with non-targeting shRNA (Ctl) or with two distinct ADAMTS5-targeting shRNA (denoted #3 or #5). Post-transduction the cells were treated in the presence or absence of IL-1β (1ng/ml, 16h) to induce ADAMTS5 expression. Subsequently, a western blot of whole cell extracts from these cells were run and probed with anti-ADAMTS5 or anti-GAPDH antibodies as indicated. GAPDH abundance was used as a loading control reference. A representative blot is shown.

    Article Snippet: Lentiviral transduction and siRNA transfection shRNA lentiviral particles were purchased from Sigma-Aldrich (St Louis, MO), and siRNAs were purchased from Thermo Scientific (Waltham, MA).

    Techniques: Transduction, shRNA, CTL Assay, Expressing, Western Blot

    Knockdown of Mp1 in E14T ES cells resembles Nanog overexpression or growth in the presence of LIF. (A) Growth curves (left) of ES cells infected with shMp1, the negative control shGFP, or the positive control shMbd3 when grown in LDM for 3 wk. After 3 wk, colonies were stained for AP. Additional positive controls were transfection with pCAG-Nanog or addition of LIF. Knockdown of Mbd3, as measured with qPCR, is shown below the Mbd3 growth curve. (B) AP staining shows that knockdown of Mp1 in two other ES cell lines, E14/Tg2a and F1V6.5, inhibits differentiation when these cells were grown in LDM for 2 wk. Knockdown was performed using the shRNA identified from the library (shMp1-Lib) and an additional shRNA indicated by shMp1-G. Negative controls were shGFP and a shRNA containing a random sequence (shRnd1). Western blot analysis shows the knockdown levels of Mp1 in these cell lines (right). Data represent at least two independent experiments. Error bars, standard deviation. Bar, 100 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation

    doi: 10.1084/jem.20102037

    Figure Lengend Snippet: Knockdown of Mp1 in E14T ES cells resembles Nanog overexpression or growth in the presence of LIF. (A) Growth curves (left) of ES cells infected with shMp1, the negative control shGFP, or the positive control shMbd3 when grown in LDM for 3 wk. After 3 wk, colonies were stained for AP. Additional positive controls were transfection with pCAG-Nanog or addition of LIF. Knockdown of Mbd3, as measured with qPCR, is shown below the Mbd3 growth curve. (B) AP staining shows that knockdown of Mp1 in two other ES cell lines, E14/Tg2a and F1V6.5, inhibits differentiation when these cells were grown in LDM for 2 wk. Knockdown was performed using the shRNA identified from the library (shMp1-Lib) and an additional shRNA indicated by shMp1-G. Negative controls were shGFP and a shRNA containing a random sequence (shRnd1). Western blot analysis shows the knockdown levels of Mp1 in these cell lines (right). Data represent at least two independent experiments. Error bars, standard deviation. Bar, 100 µm.

    Article Snippet: To differentiate NCC-IT cells , which are considered equivalent to a stage intermediate between Sem and embryonal carcinoma, cells were transfected with a stealth siRNA against MP1 or a mock siRNA using Lipofectamine, or cells were infected with an shRNA against MP1 (Sigma-Aldrich) or a mock shRNA vector (SHC002; Sigma-Aldrich).

    Techniques: Over Expression, Infection, Negative Control, Positive Control, Staining, Transfection, Real-time Polymerase Chain Reaction, shRNA, Sequencing, Western Blot, Standard Deviation

    Knockdown of Mp1 inhibits differentiation and stimulates proliferation in ES cells. (A) FACS plot of E14T-Nanog-GFP reporter ES cells containing a randomly targeted Nanog promoter fragment that drives GFP expression. Reporter activity was assayed after growing the cells in LDM for 4 d after knockdown of Mp1 or after using a control shRNA containing a random sequence (shRnd1). Knockdown was performed using the shRNA identified from the library (shMp1-Lib) and an additional shRNA indicated by shMp1-G. Positive control cells were grown in the presence of LIF for 4 d. Knockdown of Mp1 was confirmed by qPCR (bottom) and Western blotting (middle right). Knockdown of Mp1 in the absence of Bmp4 maintains OCT3/4 expression (positive fraction is shown in red) while suppressing Nestin expression (positive fraction is shown in green). Positive controls were: undifferentiated ES cells and subventricular zone neural stem cells. (B) Knockdown of Mp1 gives a proliferation advantage in the absence (top) and presence (middle) of LIF in the total population of seeded cells, but also in Nanog-GFP–positive ES cells that were cultured 4 d without LIF (bottom). Error bars, standard deviation. Data represent two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation

    doi: 10.1084/jem.20102037

    Figure Lengend Snippet: Knockdown of Mp1 inhibits differentiation and stimulates proliferation in ES cells. (A) FACS plot of E14T-Nanog-GFP reporter ES cells containing a randomly targeted Nanog promoter fragment that drives GFP expression. Reporter activity was assayed after growing the cells in LDM for 4 d after knockdown of Mp1 or after using a control shRNA containing a random sequence (shRnd1). Knockdown was performed using the shRNA identified from the library (shMp1-Lib) and an additional shRNA indicated by shMp1-G. Positive control cells were grown in the presence of LIF for 4 d. Knockdown of Mp1 was confirmed by qPCR (bottom) and Western blotting (middle right). Knockdown of Mp1 in the absence of Bmp4 maintains OCT3/4 expression (positive fraction is shown in red) while suppressing Nestin expression (positive fraction is shown in green). Positive controls were: undifferentiated ES cells and subventricular zone neural stem cells. (B) Knockdown of Mp1 gives a proliferation advantage in the absence (top) and presence (middle) of LIF in the total population of seeded cells, but also in Nanog-GFP–positive ES cells that were cultured 4 d without LIF (bottom). Error bars, standard deviation. Data represent two independent experiments.

    Article Snippet: To differentiate NCC-IT cells , which are considered equivalent to a stage intermediate between Sem and embryonal carcinoma, cells were transfected with a stealth siRNA against MP1 or a mock siRNA using Lipofectamine, or cells were infected with an shRNA against MP1 (Sigma-Aldrich) or a mock shRNA vector (SHC002; Sigma-Aldrich).

    Techniques: FACS, Expressing, Activity Assay, shRNA, Sequencing, Positive Control, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Standard Deviation

    Knockdown UBXN1 results in enhanced retroviral vector production and NFκB signaling. (A) Quantification of HIV vector production after co-transfection of third-generation HIV-based vector encoding both anti-UBXN1 shRNA and puro r gene (vs. control, empty vector), along with VSV G and either FG12, HIV-eYFP, or FG12-SV40 into 293T cells. FG12 is a third generation, self-inactivating HIV vector that encodes eGFP driven by the UbiC promote; FG12-SV40 is similar except it has an internal SV40 promoter driving eGFP. Top: puro r titer on HOS targets; Bottom: percentage of eYFP/eGFP+ HOS targets as measured by flow cytometry for each of the three vectors, using two different amounts of indicated vector supernatant; (B) (left) Quantification of NFκB-FFLUC reporter in HEK293 cells after siRNA knockdown of UBXN1, compared to Trilencer-27 Universal scrambled negative control siRNA; (right) corresponding immunoblot of UBXN1 and β-tubulin in presence of either control or anti-UBXN1 siRNA. FFLUC values were normalized to those of co-transfected Renilla luciferase reporter; cells were stimulated for 4 h with 5 ng/mL of TNFα 48h post-transfection. ( C ) Similar to (B) except that either empty HIV vector pLK0.1 or vector encoding anti-UBXN1 shRNA was transfected into HEK293 cells, with corresponding immunoblot shown on right. Data represent mean ± SEM (n = 3). * p

    Journal: PLoS Pathogens

    Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

    doi: 10.1371/journal.ppat.1006187

    Figure Lengend Snippet: Knockdown UBXN1 results in enhanced retroviral vector production and NFκB signaling. (A) Quantification of HIV vector production after co-transfection of third-generation HIV-based vector encoding both anti-UBXN1 shRNA and puro r gene (vs. control, empty vector), along with VSV G and either FG12, HIV-eYFP, or FG12-SV40 into 293T cells. FG12 is a third generation, self-inactivating HIV vector that encodes eGFP driven by the UbiC promote; FG12-SV40 is similar except it has an internal SV40 promoter driving eGFP. Top: puro r titer on HOS targets; Bottom: percentage of eYFP/eGFP+ HOS targets as measured by flow cytometry for each of the three vectors, using two different amounts of indicated vector supernatant; (B) (left) Quantification of NFκB-FFLUC reporter in HEK293 cells after siRNA knockdown of UBXN1, compared to Trilencer-27 Universal scrambled negative control siRNA; (right) corresponding immunoblot of UBXN1 and β-tubulin in presence of either control or anti-UBXN1 siRNA. FFLUC values were normalized to those of co-transfected Renilla luciferase reporter; cells were stimulated for 4 h with 5 ng/mL of TNFα 48h post-transfection. ( C ) Similar to (B) except that either empty HIV vector pLK0.1 or vector encoding anti-UBXN1 shRNA was transfected into HEK293 cells, with corresponding immunoblot shown on right. Data represent mean ± SEM (n = 3). * p

    Article Snippet: In order to generate stable UBXN1 knockdown cell lines, UBXN1-specific shRNA duplex oligonucleotides were designed targeting the same sequence as the siRNA above (#SASI_Hs01_00134629, Sigma-Aldrich), and inserted into pLKO.1 cloning vector between Age1 and EcoR1 sites, and DNA sequence confirmed.

    Techniques: Plasmid Preparation, Cotransfection, shRNA, Flow Cytometry, Cytometry, Negative Control, Transfection, Luciferase

    UBXN1 blocks NFκB signaling and inhibits HIV LTR activity. (A) Left: immunoblot of indicated proteins from HFFs stably transduced with either empty HIV-based vector or vector encoding anti-UBXN1 shRNA, after stimulation with 5 ng/mL TNFα for indicated times; right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot image); (B) Quantification of NFκB (left) and HIV LTR (right) FFLUC reporters in cell lines of (A) , normalized to co-transfected Renilla-LUC plasmid; for NFκB reporter cells were treated for 4 h with 5 ng/mL TNFα 48 h post-transfection; (C) Left: immunoblot of indicated proteins from UBXN1-/- HPRT-/- and control HPRT-/- MEFs, after stimulation with 10 ng/mL TNFα for indicated times (upper) or treated with 1 μM Bortezomib for 5 h and then stimulated with 10 ng/mL TNFα for the indicated times (lower); right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot images); (D) Confocal immunofluorescence microscopy of UBXN1 and Cul1 in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Nuclear DNA stained with TO-PRO-3 (I); UBXN1 (II) and Cul1 (III) were stained using secondary antibodies conjugated to Alexa Fluor 546 and 488, respectively; IV shows merge; Scale bar = 10 μm; (E) Quantification of NFκB and HIV LTR-FFLUC reporters in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Left: NFκB-FFLUC values 48 h post-transfection in the presence of 5 ng/mL TNFα for 4h, normalized to Renilla-LUC reporter; right: HIV LTR-FFLUC values 48 hrs post-transfection, similarly normalized; (F) JLAT10.6 T cells stably transduced with either HIV-based vector encoding anti-UBXN1 shRNA or control empty vector, after stimulating the cells with 5 ng/mL TNFα for the indicated times and measuring % eGFP+ by FACS. Data in (B) and (E) represent mean ± SEM (n = 3). ** p

    Journal: PLoS Pathogens

    Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

    doi: 10.1371/journal.ppat.1006187

    Figure Lengend Snippet: UBXN1 blocks NFκB signaling and inhibits HIV LTR activity. (A) Left: immunoblot of indicated proteins from HFFs stably transduced with either empty HIV-based vector or vector encoding anti-UBXN1 shRNA, after stimulation with 5 ng/mL TNFα for indicated times; right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot image); (B) Quantification of NFκB (left) and HIV LTR (right) FFLUC reporters in cell lines of (A) , normalized to co-transfected Renilla-LUC plasmid; for NFκB reporter cells were treated for 4 h with 5 ng/mL TNFα 48 h post-transfection; (C) Left: immunoblot of indicated proteins from UBXN1-/- HPRT-/- and control HPRT-/- MEFs, after stimulation with 10 ng/mL TNFα for indicated times (upper) or treated with 1 μM Bortezomib for 5 h and then stimulated with 10 ng/mL TNFα for the indicated times (lower); right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot images); (D) Confocal immunofluorescence microscopy of UBXN1 and Cul1 in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Nuclear DNA stained with TO-PRO-3 (I); UBXN1 (II) and Cul1 (III) were stained using secondary antibodies conjugated to Alexa Fluor 546 and 488, respectively; IV shows merge; Scale bar = 10 μm; (E) Quantification of NFκB and HIV LTR-FFLUC reporters in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Left: NFκB-FFLUC values 48 h post-transfection in the presence of 5 ng/mL TNFα for 4h, normalized to Renilla-LUC reporter; right: HIV LTR-FFLUC values 48 hrs post-transfection, similarly normalized; (F) JLAT10.6 T cells stably transduced with either HIV-based vector encoding anti-UBXN1 shRNA or control empty vector, after stimulating the cells with 5 ng/mL TNFα for the indicated times and measuring % eGFP+ by FACS. Data in (B) and (E) represent mean ± SEM (n = 3). ** p

    Article Snippet: In order to generate stable UBXN1 knockdown cell lines, UBXN1-specific shRNA duplex oligonucleotides were designed targeting the same sequence as the siRNA above (#SASI_Hs01_00134629, Sigma-Aldrich), and inserted into pLKO.1 cloning vector between Age1 and EcoR1 sites, and DNA sequence confirmed.

    Techniques: Activity Assay, Stable Transfection, Transduction, Plasmid Preparation, shRNA, Transfection, Immunofluorescence, Microscopy, Staining, FACS

    Regulation of the MAPK response to pore-formation is MKP1 independent. (A) A549 cells were transfected with 2.5 µg of MKP1 or scrambled siRNA per 1×10 6 cells and stimulated 24 hrs post-transfection with 200 ng/ml of purified Ply for the indicated times. Transfection with MKP1 siRNA inhibited MKP1 expression but did not have an effect on Ply-induced MAPK phosphorylation.

    Journal: PLoS ONE

    Article Title: Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores

    doi: 10.1371/journal.pone.0008076

    Figure Lengend Snippet: Regulation of the MAPK response to pore-formation is MKP1 independent. (A) A549 cells were transfected with 2.5 µg of MKP1 or scrambled siRNA per 1×10 6 cells and stimulated 24 hrs post-transfection with 200 ng/ml of purified Ply for the indicated times. Transfection with MKP1 siRNA inhibited MKP1 expression but did not have an effect on Ply-induced MAPK phosphorylation.

    Article Snippet: siRNA and shRNA MKP1 siRNA was from Dharmacon (Lafayette, CO).

    Techniques: Transfection, Purification, Expressing

    PP1 and PP2A mediate inactivation of the epithelial MAPK response to pore-formation. (A)A549 cells were transfected with 2 µg PP1 or scrambled siRNA per 1×10 6 cells and stimulated 54 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP1 siRNA leads to reduced PP1 expression and a corresponding increase in Ply-induced p38 and JNK phosphorylation. (B) A549 cells were transfected with 6 µg PP2Aα/β or control shRNA per 1×10 6 cells and stimulated 60 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP2Aα/β shRNA leads to reduced PP2A expression and a moderate increase in p38 phosphorylation.

    Journal: PLoS ONE

    Article Title: Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores

    doi: 10.1371/journal.pone.0008076

    Figure Lengend Snippet: PP1 and PP2A mediate inactivation of the epithelial MAPK response to pore-formation. (A)A549 cells were transfected with 2 µg PP1 or scrambled siRNA per 1×10 6 cells and stimulated 54 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP1 siRNA leads to reduced PP1 expression and a corresponding increase in Ply-induced p38 and JNK phosphorylation. (B) A549 cells were transfected with 6 µg PP2Aα/β or control shRNA per 1×10 6 cells and stimulated 60 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP2Aα/β shRNA leads to reduced PP2A expression and a moderate increase in p38 phosphorylation.

    Article Snippet: siRNA and shRNA MKP1 siRNA was from Dharmacon (Lafayette, CO).

    Techniques: Transfection, Purification, Expressing, shRNA

    Accelerated Cell-Cycle Progression in Cks2 −/− MEFs (A) Time-lapse microscopy of asynchronously growing wild-type, Cks2 −/− and Cks1 −/− MEFs. MEFs were immortalized using shRNA against p53. Wild-type MEFs were infected with a retrovirus expressing histone H2B-GFP, Cks2 −/− and Cks1 −/− MEFs expressed histone H2B-mCherry. Wild-type and knockout MEFs were mixed and imaged. The time between two successive anaphases was recorded for at least 50 cells of each genotype; the mean is shown. An example of a wild-type and Cks2 −/− cell is shown. Alternatively, MEFs were immortalized using the 3T3 protocol, and cell cycle duration was measured as the duration from one cytokinesis to the next for at least 50 cells per genotype. (B) FACS analysis of DNA content to generate cell cycle profiles of asynchronously proliferating wild-type, Cks2 −/− and Cks1 −/− MEFs. (C) Quantification of the cell cycle populations by FACS. (D) Experimental design and example of the FACS pattern of wild-type MEFs at time 0, depicting the G1, BrdU-positive and G2 gates. The ratio of BrdU-positive cells with a G1 DNA content (labeled “early S”) over unlabeled G1 cells was followed over a 24 hr chase.

    Journal: Developmental Cell

    Article Title: The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

    doi: 10.1016/j.devcel.2012.06.018

    Figure Lengend Snippet: Accelerated Cell-Cycle Progression in Cks2 −/− MEFs (A) Time-lapse microscopy of asynchronously growing wild-type, Cks2 −/− and Cks1 −/− MEFs. MEFs were immortalized using shRNA against p53. Wild-type MEFs were infected with a retrovirus expressing histone H2B-GFP, Cks2 −/− and Cks1 −/− MEFs expressed histone H2B-mCherry. Wild-type and knockout MEFs were mixed and imaged. The time between two successive anaphases was recorded for at least 50 cells of each genotype; the mean is shown. An example of a wild-type and Cks2 −/− cell is shown. Alternatively, MEFs were immortalized using the 3T3 protocol, and cell cycle duration was measured as the duration from one cytokinesis to the next for at least 50 cells per genotype. (B) FACS analysis of DNA content to generate cell cycle profiles of asynchronously proliferating wild-type, Cks2 −/− and Cks1 −/− MEFs. (C) Quantification of the cell cycle populations by FACS. (D) Experimental design and example of the FACS pattern of wild-type MEFs at time 0, depicting the G1, BrdU-positive and G2 gates. The ratio of BrdU-positive cells with a G1 DNA content (labeled “early S”) over unlabeled G1 cells was followed over a 24 hr chase.

    Article Snippet: siRNA and shRNA siRNA Smart pools were purchased from Dharmacon and transfected using Hyperfect (QIAGEN) following the manufacturer's instructions.

    Techniques: Time-lapse Microscopy, shRNA, Infection, Expressing, Knock-Out, FACS, Labeling

    NR2E3 regulates ESR1 function in breast cancer cells A-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe. C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes. E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody. F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot ( G ) or for qRT-PCR ( H ). All results are shown as mean plus standard deviation (SD) from three-independent replicates (* p

    Journal: EMBO Molecular Medicine

    Article Title: Reconstruction of nuclear receptor network reveals that NR2E3 is a novel upstream regulator of ESR1 in breast cancer

    doi: 10.1002/emmm.201100187

    Figure Lengend Snippet: NR2E3 regulates ESR1 function in breast cancer cells A-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe. C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes. E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody. F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot ( G ) or for qRT-PCR ( H ). All results are shown as mean plus standard deviation (SD) from three-independent replicates (* p

    Article Snippet: shRNA and siRNA shNR2E3 (SHCLND-NM_014249) and shControl (SHC002) clones were purchased from Sigma (St. Louis, MO).

    Techniques: Stable Transfection, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Construct, Luciferase, Activity Assay, Standard Deviation

    hVps34 and PLD1 regulate cell size. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs for raptor, hVps34, or PLD1, puromycin selected, and then subjected to cell size measurement of median forward scatter-height. The result of overnight treatment with 100 nM rapamycin is included as a control. Representative histograms are also shown, with cell counts in arbitrary units. (B) Cells were transfected with wt- or ΔPX-PLD1 together in pCDNA3 (vector), selected with G418 for 3 d, and then subjected to cell size measurement as described in A. A one-sample t test was performed to compare each data with the control. Three independent experiments were performed, and the results of mean ± SD are shown in the graphs. *, P

    Journal: The Journal of Cell Biology

    Article Title: Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway

    doi: 10.1083/jcb.201107033

    Figure Lengend Snippet: hVps34 and PLD1 regulate cell size. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs for raptor, hVps34, or PLD1, puromycin selected, and then subjected to cell size measurement of median forward scatter-height. The result of overnight treatment with 100 nM rapamycin is included as a control. Representative histograms are also shown, with cell counts in arbitrary units. (B) Cells were transfected with wt- or ΔPX-PLD1 together in pCDNA3 (vector), selected with G418 for 3 d, and then subjected to cell size measurement as described in A. A one-sample t test was performed to compare each data with the control. Three independent experiments were performed, and the results of mean ± SD are shown in the graphs. *, P

    Article Snippet: The shRNAs for human PLD1 (TRCN0000001011 and TRCN0000010572), mouse PLD1 (TRCN0000076820), and human PLD2 (TRCN0000051149 and TRCN0000051150) were previously reported ( ; ). shRNAs for the following genes were obtained from Sigma-Aldrich based on published information: Rag C (TRCN0000072874), Rag D (TRCN0000059533), and P18 (TRCN0000263628; , ).

    Techniques: Transduction, Expressing, Transfection, Plasmid Preparation

    PLD1 and Rag pathways act in parallel to mediate amino acid activation of mTORC1. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs, selected with puromycin, serum starved, and amino acid (AA) deprived followed by amino acid stimulation for 30 min. Cell lysates were analyzed by Western blotting. scram, scrambled. (B) Cells were treated as in A, and in vivo PLD assays were performed. *, P

    Journal: The Journal of Cell Biology

    Article Title: Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway

    doi: 10.1083/jcb.201107033

    Figure Lengend Snippet: PLD1 and Rag pathways act in parallel to mediate amino acid activation of mTORC1. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs, selected with puromycin, serum starved, and amino acid (AA) deprived followed by amino acid stimulation for 30 min. Cell lysates were analyzed by Western blotting. scram, scrambled. (B) Cells were treated as in A, and in vivo PLD assays were performed. *, P

    Article Snippet: The shRNAs for human PLD1 (TRCN0000001011 and TRCN0000010572), mouse PLD1 (TRCN0000076820), and human PLD2 (TRCN0000051149 and TRCN0000051150) were previously reported ( ; ). shRNAs for the following genes were obtained from Sigma-Aldrich based on published information: Rag C (TRCN0000072874), Rag D (TRCN0000059533), and P18 (TRCN0000263628; , ).

    Techniques: Activated Clotting Time Assay, Activation Assay, Transduction, Expressing, Western Blot, In Vivo

    hVps34 is necessary for amino acid activation of PLD1 upstream of mTORC1. HEK293 cells were treated as described below, and in vivo PLD assays and Western analysis of cell lysates were performed in parallel. (A) Serum-starved cells were subjected to amino acid withdrawal for 2 h and were then stimulated with amino acids for 30 min. 10 mM 3-MA and 100 nM rapamycin were added 60 and 30 min before stimulation, respectively, where indicated. (B and C) Cells were transduced with lentiviruses expressing two independent shRNAs against hVps34 and a scrambled (scram) sequence as a negative control, selected with puromycin, serum starved overnight, and amino acid deprived for 2 h followed by 30 min of amino acid (AA) stimulation (B) or insulin (100 nM) stimulation (C). (D) Cells transduced with lentiviruses expressing PLD1-shRNA or scramble control and selected with puromycin were transiently transfected with an Myc-hVps34/V5-hVps15 bicistronic construct or empty vector. The cells were then serum starved overnight and amino acid deprived for 2 h followed by amino acid stimulation for 30 min. Cell lysates were subjected to Western analysis. (E) Cells were transduced with lentiviruses expressing hVps34-shRNA or scramble control, selected with puromycin, serum starved overnight, and then stimulated with 300 µM PA for 30 min. Cell lysates were subjected to Western analysis. (F) Cells were treated as in E but amino acid deprived for 2 h followed by amino acid stimulation, PA (300 µM) stimulation, or both for 30 min. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein. (A–C) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P

    Journal: The Journal of Cell Biology

    Article Title: Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway

    doi: 10.1083/jcb.201107033

    Figure Lengend Snippet: hVps34 is necessary for amino acid activation of PLD1 upstream of mTORC1. HEK293 cells were treated as described below, and in vivo PLD assays and Western analysis of cell lysates were performed in parallel. (A) Serum-starved cells were subjected to amino acid withdrawal for 2 h and were then stimulated with amino acids for 30 min. 10 mM 3-MA and 100 nM rapamycin were added 60 and 30 min before stimulation, respectively, where indicated. (B and C) Cells were transduced with lentiviruses expressing two independent shRNAs against hVps34 and a scrambled (scram) sequence as a negative control, selected with puromycin, serum starved overnight, and amino acid deprived for 2 h followed by 30 min of amino acid (AA) stimulation (B) or insulin (100 nM) stimulation (C). (D) Cells transduced with lentiviruses expressing PLD1-shRNA or scramble control and selected with puromycin were transiently transfected with an Myc-hVps34/V5-hVps15 bicistronic construct or empty vector. The cells were then serum starved overnight and amino acid deprived for 2 h followed by amino acid stimulation for 30 min. Cell lysates were subjected to Western analysis. (E) Cells were transduced with lentiviruses expressing hVps34-shRNA or scramble control, selected with puromycin, serum starved overnight, and then stimulated with 300 µM PA for 30 min. Cell lysates were subjected to Western analysis. (F) Cells were treated as in E but amino acid deprived for 2 h followed by amino acid stimulation, PA (300 µM) stimulation, or both for 30 min. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein. (A–C) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P

    Article Snippet: The shRNAs for human PLD1 (TRCN0000001011 and TRCN0000010572), mouse PLD1 (TRCN0000076820), and human PLD2 (TRCN0000051149 and TRCN0000051150) were previously reported ( ; ). shRNAs for the following genes were obtained from Sigma-Aldrich based on published information: Rag C (TRCN0000072874), Rag D (TRCN0000059533), and P18 (TRCN0000263628; , ).

    Techniques: Activation Assay, In Vivo, Western Blot, Transduction, Expressing, Sequencing, Negative Control, shRNA, Transfection, Construct, Plasmid Preparation, SDS Page

    PD-L2 knockdown-induced inhibition of autophagy attenuates migration and invasion of osteosarcoma cells. a Representative TEM images depict ultrastructures present during autophagy in KHOS and U2OS cells transfected with PD-L2 shRNA or shNC. Images show autophagic vacuoles (arrows) observed in control cells (the picture in the right is a zoom of the picture in the left). No or few autophagic vacuoles were observed in PD-L2 knockdown cells. b Cells after PD-L2 knockdown exhibited a punctate pattern of LC3-II fluorescence, with reduced LC3-II compared with autophagosomes. KHOS and U2OS cells were incubated with or without CQ. c Expression of LC3, p62, and beclin-1 was evaluated by western blot. KHOS and U2OS cells were incubated in the presence or absence of CQ

    Journal: Cell Death & Disease

    Article Title: Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways

    doi: 10.1038/s41419-019-1497-1

    Figure Lengend Snippet: PD-L2 knockdown-induced inhibition of autophagy attenuates migration and invasion of osteosarcoma cells. a Representative TEM images depict ultrastructures present during autophagy in KHOS and U2OS cells transfected with PD-L2 shRNA or shNC. Images show autophagic vacuoles (arrows) observed in control cells (the picture in the right is a zoom of the picture in the left). No or few autophagic vacuoles were observed in PD-L2 knockdown cells. b Cells after PD-L2 knockdown exhibited a punctate pattern of LC3-II fluorescence, with reduced LC3-II compared with autophagosomes. KHOS and U2OS cells were incubated with or without CQ. c Expression of LC3, p62, and beclin-1 was evaluated by western blot. KHOS and U2OS cells were incubated in the presence or absence of CQ

    Article Snippet: Gene knockdown with siRNA/shRNA and overexpression with adenovirus Lentiviruses targeting PD-L2 and BMPR2 were obtained from GenePharma (Suzhou, China).

    Techniques: Inhibition, Migration, Transmission Electron Microscopy, Transfection, shRNA, Fluorescence, Incubation, Expressing, Western Blot

    Autophagy promotes migration and invasion of osteosarcoma cells through targeting the RhoA-ROCK-LIMK2 pathway. ( a ) Three beclin-1 siRNA sequences were used to downregulate beclin-1 in KHOS cells (top). The migration and invasion of osteosarcoma cells after beclin-1 knockdown were analyzed by transwell and wound-healing assays (bottom). ( b and c ) Bioinformatics prediction indicated there may be co-expression between beclin-1 and RhoA, and western blot validated the relationship between them. Beclin-1 knockdown decreased p-LIMK and p-cofilin expressions ( b ) as well as RhoA activation ( c ). d The expression levels of autophagy markers (LC3, p62, Beclin1) in the primary and metastatic osteosarcoma tumors were evaluated by IHC (magnification 200X). All experiments were repeated three times. Data are presented as the mean ± S.D. ***P

    Journal: Cell Death & Disease

    Article Title: Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways

    doi: 10.1038/s41419-019-1497-1

    Figure Lengend Snippet: Autophagy promotes migration and invasion of osteosarcoma cells through targeting the RhoA-ROCK-LIMK2 pathway. ( a ) Three beclin-1 siRNA sequences were used to downregulate beclin-1 in KHOS cells (top). The migration and invasion of osteosarcoma cells after beclin-1 knockdown were analyzed by transwell and wound-healing assays (bottom). ( b and c ) Bioinformatics prediction indicated there may be co-expression between beclin-1 and RhoA, and western blot validated the relationship between them. Beclin-1 knockdown decreased p-LIMK and p-cofilin expressions ( b ) as well as RhoA activation ( c ). d The expression levels of autophagy markers (LC3, p62, Beclin1) in the primary and metastatic osteosarcoma tumors were evaluated by IHC (magnification 200X). All experiments were repeated three times. Data are presented as the mean ± S.D. ***P

    Article Snippet: Gene knockdown with siRNA/shRNA and overexpression with adenovirus Lentiviruses targeting PD-L2 and BMPR2 were obtained from GenePharma (Suzhou, China).

    Techniques: Migration, Expressing, Western Blot, Activation Assay, Immunohistochemistry