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  • 99
    Thermo Fisher sirna treatment
    Inhibition of YAP by <t>siRNA</t> enhanced the cytotoxicity of erlotinib to H1975 cells A. Western blotting showed that YAP protein expression level decreased after YAP siRNA <t>transfection</t> and erlotinib treatment in H1975 cells. B. YAP mRNA expression significantly decreased in H1975 cells with YAP siRNA transfection after erlotinib treatment (***P
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    Millipore sirna treatment
    Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22 -specific <t>shRNA</t> (A and B), and BMDC from wild-type (WT) or PTPN22 deficient ( ptpn22 −/− ) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin A 1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the western blot pictures show results of densitometric measurements. * = p
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    GraphPad Prism Inc shrna treatments
    Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22 -specific <t>shRNA</t> (A and B), and BMDC from wild-type (WT) or PTPN22 deficient ( ptpn22 −/− ) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin A 1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the western blot pictures show results of densitometric measurements. * = p
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    Thermo Fisher sirna treatment plk
    Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22 -specific <t>shRNA</t> (A and B), and BMDC from wild-type (WT) or PTPN22 deficient ( ptpn22 −/− ) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin A 1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the western blot pictures show results of densitometric measurements. * = p
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    3M Co k603 sirna treatments
    p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control <t>siRNA</t> (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or <t>k603</t> siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t -test: * p
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    Lonza sirna treatment nhbecs
    p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control <t>siRNA</t> (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or <t>k603</t> siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t -test: * p
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    Horizon Discovery sirna treatment sirna duplex oligonucleotides
    p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control <t>siRNA</t> (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or <t>k603</t> siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t -test: * p
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    Millipore shrna lentivirus treatment
    EGFR down-regulation results in RB cell death in a dose and infection time-dependent manner. Weri-Rb-1 cells were (A) infected with ascending doses of <t>shRNA</t> <t>lentiviruses</t> and incubated for 48 hr, or (B) infected with 5 MOI shRNA lentiviruses and incubated for up to 96 hr, and then cell viability was analyzed. Data shown are mean±SD of three independent experiments. *, P
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    Lonza sirna treatment
    Comparative cleavage characteristics of the peptidic substrates ([Cy5-D-R-E-I-M-R] 2 -Rh110 and ([Cy5-D-R-E-I-M-D] 2 -Rh110 in <t>U937</t> cells. The Separase-specific conjugated peptidic substrate (DREIMR) is shown in green, the control substrate lacking the Separase cleavage consensus (DREIMD) is depicted in red. (A) Substrate cleavage in the cell extract-based Separase assay as monitored by released Rh110 fluorescence. (B) FACS measurement of Rh110 positive cells after incubation with substrates according to the standardized protocol (90 min at 37°C). (C) Calculation of mean proteolytic activity per cell as measured by released Rh110 fluorescence in the FACS assay. (D) Changes (Δ-values) of proteolytic activities on substrates DREIMR and DREIMD after 48 h of Espl1 silencing when compared to mock treated U937 cells. Corresponding Western blot immunostaining experiments (below) illustrate the knockdown of the Separase protein levels <t>(siRNA</t> (-), 100%; siRNA (+), 24%). Actin served as loading control. Abbreviations: DREIMD, ([Cy5-D-R-E-I-M-D] 2 -Rh110; DREIMR, ([Cy5-D-R-E-I-M-R] 2 -Rh110.
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    Horizon Discovery sirna treatment
    <t>FOXO1</t> represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after <t>siRNA</t> treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.
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    Roche sirna treatment
    Effects of nucleosome assembly proteins on DNA replication activity of EBNA1. (A) CNE2Z cells were treated with <t>siRNA</t> against NAP1, NAP2, TAF-I, or GFP (negative control) and then <t>transfected</t> with an oriP plasmid expressing EBNA1 or lacking EBNA1 (first
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    Santa Cruz Biotechnology sirna treatment
    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) <t>293T</t> cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 <t>siRNA</t> for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.
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    Promega sirna treatment
    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) <t>293T</t> cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 <t>siRNA</t> for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.
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    GE Healthcare sirna treatments
    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) <t>293T</t> cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 <t>siRNA</t> for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.
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    Cell Signaling Technology Inc sirna treatment
    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) <t>293T</t> cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 <t>siRNA</t> for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.
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    merck kgaa sirna treatment
    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) <t>293T</t> cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 <t>siRNA</t> for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.
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    Inhibition of YAP by siRNA enhanced the cytotoxicity of erlotinib to H1975 cells A. Western blotting showed that YAP protein expression level decreased after YAP siRNA transfection and erlotinib treatment in H1975 cells. B. YAP mRNA expression significantly decreased in H1975 cells with YAP siRNA transfection after erlotinib treatment (***P

    Journal: Oncotarget

    Article Title: YAP promotes erlotinib resistance in human non-small cell lung cancer cells

    doi: 10.18632/oncotarget.10458

    Figure Lengend Snippet: Inhibition of YAP by siRNA enhanced the cytotoxicity of erlotinib to H1975 cells A. Western blotting showed that YAP protein expression level decreased after YAP siRNA transfection and erlotinib treatment in H1975 cells. B. YAP mRNA expression significantly decreased in H1975 cells with YAP siRNA transfection after erlotinib treatment (***P

    Article Snippet: The transfection reagent for SiRNA treatment was Lipofectamine RNAiMAX, and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for small molecule inhibitors.

    Techniques: Inhibition, Western Blot, Expressing, Transfection

    Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22 -specific shRNA (A and B), and BMDC from wild-type (WT) or PTPN22 deficient ( ptpn22 −/− ) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin A 1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the western blot pictures show results of densitometric measurements. * = p

    Journal: Autophagy

    Article Title: PTPN22 regulates NLRP3-mediated IL1B secretion in an autophagy-dependent manner

    doi: 10.1080/15548627.2017.1341453

    Figure Lengend Snippet: Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22 -specific shRNA (A and B), and BMDC from wild-type (WT) or PTPN22 deficient ( ptpn22 −/− ) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin A 1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the western blot pictures show results of densitometric measurements. * = p

    Article Snippet: Inhibitors, shRNA, and siRNA treatment For experiments with inhibitors, cells were plated in serum-free medium 12 h before incubation with 3-MA (10 mM; Sigma-Aldrich, M9281), wortmannin (10 µM; Sigma-Aldrich, 95455), or bafilomycin A1 (100 nM; Sigma-Aldrich, B1793) for 1 h before treatment with MDP (100 ng/ml; InvivoGen, tlrl-mdpc), MSU (150 μg/ml; InvivoGen, tlrl-msu) or ATP (2 mM; InvivoGen, tlrl-atp).

    Techniques: Expressing, shRNA, Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay

    The effect of CHRNA5 siRNA in drug sensitivity. A-C. Western blot results in DMSO, CPT (0.125μM), and DOXO (0.125μM) treated MCF7 cells in the absence or presence of siRNA-1 (A) , siRNA-2, (B) and siRNA-3, (C). D-G. Two-Way ANOVA of densitometry measurements for CHRNA5 (D), BAX/BCL2 (E) , CHEK1 (F) , pCHEK1 (G) . H . DFA of densitometry measurements for control and treatment groups in A-C. I. Vector weights of variables and their correlation with LD1 and LD2 of DFA analysis. (n = 3 for DMSO siRNA-CN (50nM) and siRNA-2 and -3; n = 2 for other groups). (+: p

    Journal: PLoS ONE

    Article Title: Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer

    doi: 10.1371/journal.pone.0208982

    Figure Lengend Snippet: The effect of CHRNA5 siRNA in drug sensitivity. A-C. Western blot results in DMSO, CPT (0.125μM), and DOXO (0.125μM) treated MCF7 cells in the absence or presence of siRNA-1 (A) , siRNA-2, (B) and siRNA-3, (C). D-G. Two-Way ANOVA of densitometry measurements for CHRNA5 (D), BAX/BCL2 (E) , CHEK1 (F) , pCHEK1 (G) . H . DFA of densitometry measurements for control and treatment groups in A-C. I. Vector weights of variables and their correlation with LD1 and LD2 of DFA analysis. (n = 3 for DMSO siRNA-CN (50nM) and siRNA-2 and -3; n = 2 for other groups). (+: p

    Article Snippet: BrdU assay After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol.

    Techniques: Western Blot, Cycling Probe Technology, Direct Fluorescent Antibody Test, Plasmid Preparation

    Association between CHRNA RNAi and topoisomerase inhibitors. A. Comparison of log fold changes (logFC) in response to DOXO and SN38, siRNA-1 and SN38, siRNA-1 and DOXO treatment in MCF7 (from left to right) and the corresponding statistical analysis results (Fisher’s Exact Test counts and p-value; bottom). Genes significant in both categories are colored black; genes insignificant (or significant in one type of data) are shown in gray; and TP53 targets are shown in red. B-E Relative cell viability for CPT and siRNA-1 (B), DOXO and siRNA-1 (C), CPT and siRNA-2 (D), and DOXO and siRNA-2 (E) treatments at increasing concentrations of CPT (B, D) and DOXO (C, E). One-Way ANOVA followed by Tukey’s multiple test correction were used for statistical analysis of MTT assays (*: p

    Journal: PLoS ONE

    Article Title: Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer

    doi: 10.1371/journal.pone.0208982

    Figure Lengend Snippet: Association between CHRNA RNAi and topoisomerase inhibitors. A. Comparison of log fold changes (logFC) in response to DOXO and SN38, siRNA-1 and SN38, siRNA-1 and DOXO treatment in MCF7 (from left to right) and the corresponding statistical analysis results (Fisher’s Exact Test counts and p-value; bottom). Genes significant in both categories are colored black; genes insignificant (or significant in one type of data) are shown in gray; and TP53 targets are shown in red. B-E Relative cell viability for CPT and siRNA-1 (B), DOXO and siRNA-1 (C), CPT and siRNA-2 (D), and DOXO and siRNA-2 (E) treatments at increasing concentrations of CPT (B, D) and DOXO (C, E). One-Way ANOVA followed by Tukey’s multiple test correction were used for statistical analysis of MTT assays (*: p

    Article Snippet: BrdU assay After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol.

    Techniques: Cycling Probe Technology, MTT Assay

    RT-qPCR validation of CHRNA5 RNAi microarray data. A. RT-qPCR validation of selected genes modulated by siRNA-1 in MCF7 cells. B. RT-qPCR validation of selected genes in siRNA-2 and siRNA-3 treated MCF7 cells. C. Heatmap of RT-qPCR analysis results of selected genes in MCF7, BT20 and MDA-MB-231 in the presence of siRNA-CN or siRNA-1. For siRNA-1, and siRNA-2 and -3 treatment RT-qPCR results, student’s t-tests in comparison with their corresponding siRNA-CN group, i.e., 10nM for siRNA-1 and 50nM for siRNA-2 and -3, and for microarray data limma were used. (*: p

    Journal: PLoS ONE

    Article Title: Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer

    doi: 10.1371/journal.pone.0208982

    Figure Lengend Snippet: RT-qPCR validation of CHRNA5 RNAi microarray data. A. RT-qPCR validation of selected genes modulated by siRNA-1 in MCF7 cells. B. RT-qPCR validation of selected genes in siRNA-2 and siRNA-3 treated MCF7 cells. C. Heatmap of RT-qPCR analysis results of selected genes in MCF7, BT20 and MDA-MB-231 in the presence of siRNA-CN or siRNA-1. For siRNA-1, and siRNA-2 and -3 treatment RT-qPCR results, student’s t-tests in comparison with their corresponding siRNA-CN group, i.e., 10nM for siRNA-1 and 50nM for siRNA-2 and -3, and for microarray data limma were used. (*: p

    Article Snippet: BrdU assay After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol.

    Techniques: Quantitative RT-PCR, Microarray, Multiple Displacement Amplification

    Western blotting and RT-qPCR analyses relevant to effects of RNAi on cell cycle, apoptosis and DDR in MCF7 cells. A-B. Western Blot results for pRB, total CASP7, cleaved CASP7, BCL2, BAX, total CHEK1, pCHEK1, and pH2AX in siRNA-1 (A) and siRNA-2 and siRNA-3 (B) treated MCF7 cells. C-F. Densitometry measurements of pRB (C), BAX/BCL2 ratio (D), total CHEK1 (E), and pCHEK1 (F). One-Way ANOVA was used in comparison with corresponding control groups, siRNA-CN (10nM) vs. siRNA-1 and siRNA-CN (50nM) vs. siRNA-2 and -3. G. DFA plot for control and RNAi treatment groups. H. Vector weights of variables and their correlation with LD1 and LD2 of DFA. (n = 2 per group for siRNA-CN (10nM) and siRNA-1; n = 3 per group for siRNA-CN (50nM) and siRNA-2 and -3). I. RT-qPCR results of FAS, BAX, BCL2, CCND1, CCNE2, and CHEK1 in siRNA-1-3 treated MCF7 cells (n = 2 per group). J. BAX/BCL2 ratio from RT-qPCR results (n = 2 per group). One-way ANOVA followed by Tukey’s multiple test correction was used. (+: p

    Journal: PLoS ONE

    Article Title: Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer

    doi: 10.1371/journal.pone.0208982

    Figure Lengend Snippet: Western blotting and RT-qPCR analyses relevant to effects of RNAi on cell cycle, apoptosis and DDR in MCF7 cells. A-B. Western Blot results for pRB, total CASP7, cleaved CASP7, BCL2, BAX, total CHEK1, pCHEK1, and pH2AX in siRNA-1 (A) and siRNA-2 and siRNA-3 (B) treated MCF7 cells. C-F. Densitometry measurements of pRB (C), BAX/BCL2 ratio (D), total CHEK1 (E), and pCHEK1 (F). One-Way ANOVA was used in comparison with corresponding control groups, siRNA-CN (10nM) vs. siRNA-1 and siRNA-CN (50nM) vs. siRNA-2 and -3. G. DFA plot for control and RNAi treatment groups. H. Vector weights of variables and their correlation with LD1 and LD2 of DFA. (n = 2 per group for siRNA-CN (10nM) and siRNA-1; n = 3 per group for siRNA-CN (50nM) and siRNA-2 and -3). I. RT-qPCR results of FAS, BAX, BCL2, CCND1, CCNE2, and CHEK1 in siRNA-1-3 treated MCF7 cells (n = 2 per group). J. BAX/BCL2 ratio from RT-qPCR results (n = 2 per group). One-way ANOVA followed by Tukey’s multiple test correction was used. (+: p

    Article Snippet: BrdU assay After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol.

    Techniques: Western Blot, Quantitative RT-PCR, Direct Fluorescent Antibody Test, Plasmid Preparation

    Downregulation of CHRNA5 expression by RNAi. A. Schematic representation of target sites of siRNA molecules and primers for CHRNA5 isoforms. B-D. Downregulation of CHRNA5 isoforms in MCF7 cells upon transient transfection with siRNA-1 (10nM) (B), siRNA-2 (50nM) (C), and siRNA-3 (50nM) (D) in comparison with the corresponding siRNA-CN at 72h of treatment (n = 2 per group). Student’s t-test was applied. E-F. Depletion of CHRNA5 expression at protein level (Western Blotting together with densitometry analyses, loading control, GAPDH) with siRNA-1 (10nM) (n = 4 per group; Student’s t-test) (E), siRNA-2 and siRNA-3 (n = 4 for siRNA-CN; n = 3 for siRNA-2 and siRNA-3; One-Way ANOVA followed by Tukey’s HSD tests) (F). (*: p

    Journal: PLoS ONE

    Article Title: Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer

    doi: 10.1371/journal.pone.0208982

    Figure Lengend Snippet: Downregulation of CHRNA5 expression by RNAi. A. Schematic representation of target sites of siRNA molecules and primers for CHRNA5 isoforms. B-D. Downregulation of CHRNA5 isoforms in MCF7 cells upon transient transfection with siRNA-1 (10nM) (B), siRNA-2 (50nM) (C), and siRNA-3 (50nM) (D) in comparison with the corresponding siRNA-CN at 72h of treatment (n = 2 per group). Student’s t-test was applied. E-F. Depletion of CHRNA5 expression at protein level (Western Blotting together with densitometry analyses, loading control, GAPDH) with siRNA-1 (10nM) (n = 4 per group; Student’s t-test) (E), siRNA-2 and siRNA-3 (n = 4 for siRNA-CN; n = 3 for siRNA-2 and siRNA-3; One-Way ANOVA followed by Tukey’s HSD tests) (F). (*: p

    Article Snippet: BrdU assay After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol.

    Techniques: Expressing, Transfection, Western Blot

    eEF1A binds directly and tightly to HIV-1 RT in vitro . ( A ) Biolayer interferometry assays (BLI) of a biotinylated HIV RTp66/p51-loaded biosensor with 60 nM of purified eEF1A, eEF1B, eEF1D, eEF1G or BSA as analytes. ( B ) BLI assays using a biotinylated HIV-1 IN-loaded biosensor as described in ( A ). ( C ) Co-IP of RT with eEF1A. HEK 293T cells were transfected with plasmids to express FLAG-tagged RTp66, RTp51, both RTp51 and RTp66, or Gag. Expression of the proteins in cell lysates was confirmed by western blot using anti-FLAG antibody (upper panel). The cell lysate was used for co-IP analysis with a rabbit anti-eEF1A capture antibody. Capture of RT subunits and Gag was examined by western blot with an anti-FLAG antibody (second panel). Western blot with a mouse anti-eEF1A antibody was used to confirm eEF1A capture (third panel). Nonspecific binding to the beads was excluded by IP using an anti-rabbit IgG capture antibody followed by western blot using an anti-FLAG antibody (lower panel). ( D ) HEK293T cells were treated with two eEF1A siRNAs, one control siRNA or no siRNA for 48 h before preparing a cell lysate. The levels of eEF1A (upper panel) and β tubulin (lower panel) in the cell lysate were determined by western blot. Densitometry analysis of the western blot image was used to calculate the relative levels of eEF1A in each siRNA-treated sample compared to the untreated control ( E ) Sensorgrams of association and dissociation of cell lysates containing various levels of eEF1A to RTp66-biosensors. ( F ) Addition of purified MYC/DDK-eEF1A1 protein to a cell lysate where eEF1A was strongly reduced by siRNA treatment restored a sensograms profile indicative of strong RT interaction. All experiments were performed at least three times and representative results are shown.

    Journal: PLoS Pathogens

    Article Title: Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target

    doi: 10.1371/journal.ppat.1005289

    Figure Lengend Snippet: eEF1A binds directly and tightly to HIV-1 RT in vitro . ( A ) Biolayer interferometry assays (BLI) of a biotinylated HIV RTp66/p51-loaded biosensor with 60 nM of purified eEF1A, eEF1B, eEF1D, eEF1G or BSA as analytes. ( B ) BLI assays using a biotinylated HIV-1 IN-loaded biosensor as described in ( A ). ( C ) Co-IP of RT with eEF1A. HEK 293T cells were transfected with plasmids to express FLAG-tagged RTp66, RTp51, both RTp51 and RTp66, or Gag. Expression of the proteins in cell lysates was confirmed by western blot using anti-FLAG antibody (upper panel). The cell lysate was used for co-IP analysis with a rabbit anti-eEF1A capture antibody. Capture of RT subunits and Gag was examined by western blot with an anti-FLAG antibody (second panel). Western blot with a mouse anti-eEF1A antibody was used to confirm eEF1A capture (third panel). Nonspecific binding to the beads was excluded by IP using an anti-rabbit IgG capture antibody followed by western blot using an anti-FLAG antibody (lower panel). ( D ) HEK293T cells were treated with two eEF1A siRNAs, one control siRNA or no siRNA for 48 h before preparing a cell lysate. The levels of eEF1A (upper panel) and β tubulin (lower panel) in the cell lysate were determined by western blot. Densitometry analysis of the western blot image was used to calculate the relative levels of eEF1A in each siRNA-treated sample compared to the untreated control ( E ) Sensorgrams of association and dissociation of cell lysates containing various levels of eEF1A to RTp66-biosensors. ( F ) Addition of purified MYC/DDK-eEF1A1 protein to a cell lysate where eEF1A was strongly reduced by siRNA treatment restored a sensograms profile indicative of strong RT interaction. All experiments were performed at least three times and representative results are shown.

    Article Snippet: siRNA treatment siRNA (Sigma-Aldrich) targeting eEF1A (siRNA ID: SASI_Hs02_00331772 and 00331773) and control siRNA (SIC001) were applied to HEK293T cells by large-scale reverse transfection using Lipofectamine RNAiMAX according to the manufacturer’s instructions (Invitrogen).

    Techniques: In Vitro, Purification, Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Binding Assay

    p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or k603 siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t -test: * p

    Journal: Frontiers in Oncology

    Article Title: PDCD4 Knockdown Induces Senescence in Hepatoma Cells by Up-Regulating the p21 Expression

    doi: 10.3389/fonc.2018.00661

    Figure Lengend Snippet: p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or k603 siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t -test: * p

    Article Snippet: Significant p-values were not obtained by a t-test between nc and k603 siRNA treatments. (2.3M, TIF)

    Techniques: Negative Control, Cell Culture, Western Blot, Staining

    PDCD4 knockdown induced replicative senescence. (A) β-galactosidase staining patterns in the untreated control cells (ctr), negative control siRNA-treated cells (nc), and PDCD4-specific p2 siRNA-treated cells (p2) in HepG2, Hep3B, and Huh7 cells at 4 day of culture after the siRNA treatments. The cells with arrows are enlarged and show morphological changes and β-galactosidase positivity. (B) The number of β-galactosidase-positive HepG2, Hep3B, and Huh7 cells after PDCD4 knockdown with p2 siRNA (p2), treatment with negative control siRNA (nc) and no treatment (ctr). (C) The number of β-galactosidase-positive HepG2, Hep3B, and Huh7 cells after PDCD4 knockdown with k603 siRNA (k603), treatment with negative control siRNA (nc) and no treatment (ctr). The data represent the average cell number of three different fields, and thousands of cells were counted in each field. These experiments were repeated independently three times, and similar results were obtained each time. A representative result from the experiments is shown in this figure. t -test: * p

    Journal: Frontiers in Oncology

    Article Title: PDCD4 Knockdown Induces Senescence in Hepatoma Cells by Up-Regulating the p21 Expression

    doi: 10.3389/fonc.2018.00661

    Figure Lengend Snippet: PDCD4 knockdown induced replicative senescence. (A) β-galactosidase staining patterns in the untreated control cells (ctr), negative control siRNA-treated cells (nc), and PDCD4-specific p2 siRNA-treated cells (p2) in HepG2, Hep3B, and Huh7 cells at 4 day of culture after the siRNA treatments. The cells with arrows are enlarged and show morphological changes and β-galactosidase positivity. (B) The number of β-galactosidase-positive HepG2, Hep3B, and Huh7 cells after PDCD4 knockdown with p2 siRNA (p2), treatment with negative control siRNA (nc) and no treatment (ctr). (C) The number of β-galactosidase-positive HepG2, Hep3B, and Huh7 cells after PDCD4 knockdown with k603 siRNA (k603), treatment with negative control siRNA (nc) and no treatment (ctr). The data represent the average cell number of three different fields, and thousands of cells were counted in each field. These experiments were repeated independently three times, and similar results were obtained each time. A representative result from the experiments is shown in this figure. t -test: * p

    Article Snippet: Significant p-values were not obtained by a t-test between nc and k603 siRNA treatments. (2.3M, TIF)

    Techniques: Staining, Negative Control

    siRNA-mediated PDCD4 knockdown suppressed cell growth and induced morphological changes. (A) PDCD4 knockdown suppressed cell growth of HepG2 (a,b) , Huh7 (c,d) , Hep3B (e,f) , and HeLa (g) cells. Cells were transfected with three types of PDCD4-specific siRNAs: p1 or p2 (a,c,e,g) and k603 (b,d,f) , and a negative control siRNA (nc). Cell growth was analyzed by counting the cell numbers under a microscope at different time points, as indicated in the figures. (B) Phase-contrast images of the normal (left), negative control siRNA-treated (middle) and p2 siRNA-mediated PDCD4 knockdown HepG2 cells (right) at 48 h after siRNA treatment.

    Journal: Frontiers in Oncology

    Article Title: PDCD4 Knockdown Induces Senescence in Hepatoma Cells by Up-Regulating the p21 Expression

    doi: 10.3389/fonc.2018.00661

    Figure Lengend Snippet: siRNA-mediated PDCD4 knockdown suppressed cell growth and induced morphological changes. (A) PDCD4 knockdown suppressed cell growth of HepG2 (a,b) , Huh7 (c,d) , Hep3B (e,f) , and HeLa (g) cells. Cells were transfected with three types of PDCD4-specific siRNAs: p1 or p2 (a,c,e,g) and k603 (b,d,f) , and a negative control siRNA (nc). Cell growth was analyzed by counting the cell numbers under a microscope at different time points, as indicated in the figures. (B) Phase-contrast images of the normal (left), negative control siRNA-treated (middle) and p2 siRNA-mediated PDCD4 knockdown HepG2 cells (right) at 48 h after siRNA treatment.

    Article Snippet: Significant p-values were not obtained by a t-test between nc and k603 siRNA treatments. (2.3M, TIF)

    Techniques: Transfection, Negative Control, Microscopy

    EGFR down-regulation results in RB cell death in a dose and infection time-dependent manner. Weri-Rb-1 cells were (A) infected with ascending doses of shRNA lentiviruses and incubated for 48 hr, or (B) infected with 5 MOI shRNA lentiviruses and incubated for up to 96 hr, and then cell viability was analyzed. Data shown are mean±SD of three independent experiments. *, P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: A novel treatment approach for retinoblastoma by targeting epithelial growth factor receptor expression with a shRNA lentiviral system

    doi: 10.22038/IJBMS.2017.9003

    Figure Lengend Snippet: EGFR down-regulation results in RB cell death in a dose and infection time-dependent manner. Weri-Rb-1 cells were (A) infected with ascending doses of shRNA lentiviruses and incubated for 48 hr, or (B) infected with 5 MOI shRNA lentiviruses and incubated for up to 96 hr, and then cell viability was analyzed. Data shown are mean±SD of three independent experiments. *, P

    Article Snippet: In brief, Weri-Rb-1 cells with or without shRNA lentivirus treatment were seeded onto the 1 mg/ml Matrigel-coated (Sigma-Aldrich) upper chamber of a Transwell plate (Corning) and incubated at 37°C for 20 hr.

    Techniques: Infection, shRNA, Incubation

    EGFR is specifically down-regulated by EGFR shRNA lentivirus. Weri-Rb-1 cells were mock infected (mock control) or infected with lentivirus bearing negative control shRNA (control shRNA) or EGFR shRNA (EGFR shRNA) at 5 MOI for 48 hr. Then cells were lysed and EGFR expression was determined by Western blot. (A) Representative result is shown. (B) Grayscale analysis. Data shown are mean ± SD of three independent experiments. **, P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: A novel treatment approach for retinoblastoma by targeting epithelial growth factor receptor expression with a shRNA lentiviral system

    doi: 10.22038/IJBMS.2017.9003

    Figure Lengend Snippet: EGFR is specifically down-regulated by EGFR shRNA lentivirus. Weri-Rb-1 cells were mock infected (mock control) or infected with lentivirus bearing negative control shRNA (control shRNA) or EGFR shRNA (EGFR shRNA) at 5 MOI for 48 hr. Then cells were lysed and EGFR expression was determined by Western blot. (A) Representative result is shown. (B) Grayscale analysis. Data shown are mean ± SD of three independent experiments. **, P

    Article Snippet: In brief, Weri-Rb-1 cells with or without shRNA lentivirus treatment were seeded onto the 1 mg/ml Matrigel-coated (Sigma-Aldrich) upper chamber of a Transwell plate (Corning) and incubated at 37°C for 20 hr.

    Techniques: shRNA, Infection, Negative Control, Expressing, Western Blot

    EGFR down-regulation suppresses RB cell invasion. (A and B) Weri-Rb-1 cells were first infected with shRNA lentiviruses and then cell invasion was determined by a Matrigel-based Transwell system. (A) Representative data is shown. (B) Data shown are mean±SD of three independent experiments. **, P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: A novel treatment approach for retinoblastoma by targeting epithelial growth factor receptor expression with a shRNA lentiviral system

    doi: 10.22038/IJBMS.2017.9003

    Figure Lengend Snippet: EGFR down-regulation suppresses RB cell invasion. (A and B) Weri-Rb-1 cells were first infected with shRNA lentiviruses and then cell invasion was determined by a Matrigel-based Transwell system. (A) Representative data is shown. (B) Data shown are mean±SD of three independent experiments. **, P

    Article Snippet: In brief, Weri-Rb-1 cells with or without shRNA lentivirus treatment were seeded onto the 1 mg/ml Matrigel-coated (Sigma-Aldrich) upper chamber of a Transwell plate (Corning) and incubated at 37°C for 20 hr.

    Techniques: Infection, shRNA

    EGFR down-regulation mediated RB inhibition is through the PI3K/AKT/mTOR signaling pathway. Weri-Rb-1 cells were first transfected with shRNA lentiviruses and then the expression of PI3K, p-AKT, AKT, p-mTOR, and mTOR was determined by Western blot. (A) Representative result is shown. (B) Grayscale analysis. Data shown are mean±SD of three independent experiments. *, P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: A novel treatment approach for retinoblastoma by targeting epithelial growth factor receptor expression with a shRNA lentiviral system

    doi: 10.22038/IJBMS.2017.9003

    Figure Lengend Snippet: EGFR down-regulation mediated RB inhibition is through the PI3K/AKT/mTOR signaling pathway. Weri-Rb-1 cells were first transfected with shRNA lentiviruses and then the expression of PI3K, p-AKT, AKT, p-mTOR, and mTOR was determined by Western blot. (A) Representative result is shown. (B) Grayscale analysis. Data shown are mean±SD of three independent experiments. *, P

    Article Snippet: In brief, Weri-Rb-1 cells with or without shRNA lentivirus treatment were seeded onto the 1 mg/ml Matrigel-coated (Sigma-Aldrich) upper chamber of a Transwell plate (Corning) and incubated at 37°C for 20 hr.

    Techniques: Inhibition, Transfection, shRNA, Expressing, Western Blot

    TRIP-Br1 binds to multiple AC isoforms and reduces their protein level and cAMP production. ( a ) GST-TRIP-Br1 captured ACs 2, 4, 5, 6, and 8. ( b ) Stable expression of TRIP-Br1-V5 reduced the protein expression of ACs 1, 2, 4, 5, 6, and 8 that were transiently expressed in HEK293T cells. ( c–d ) Quantification of protein levels of various AC isoforms in panel b and their forskolin-stimulated cAMP production. cAMP production is normalized relative to that of the control of mock cells. *, different from control, p≤0.048; **p≤0.009; ***p=0.001; n = 3–5. ( e ) Sequence alignment of the AC isoforms used in panel b to identify conserved Lys residues (boxed). Yellow highlight: identical residues; blue highlight: highly conserved. DOI: http://dx.doi.org/10.7554/eLife.28021.013

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: TRIP-Br1 binds to multiple AC isoforms and reduces their protein level and cAMP production. ( a ) GST-TRIP-Br1 captured ACs 2, 4, 5, 6, and 8. ( b ) Stable expression of TRIP-Br1-V5 reduced the protein expression of ACs 1, 2, 4, 5, 6, and 8 that were transiently expressed in HEK293T cells. ( c–d ) Quantification of protein levels of various AC isoforms in panel b and their forskolin-stimulated cAMP production. cAMP production is normalized relative to that of the control of mock cells. *, different from control, p≤0.048; **p≤0.009; ***p=0.001; n = 3–5. ( e ) Sequence alignment of the AC isoforms used in panel b to identify conserved Lys residues (boxed). Yellow highlight: identical residues; blue highlight: highly conserved. DOI: http://dx.doi.org/10.7554/eLife.28021.013

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Expressing, Sequencing

    Endocytosis and recycling of HA-tagged wild-type and K1047R AC1 expressed in HeLa cells. ( a ) Upper panel: Effect of TRIP-Br1 and XIAP on the endocytosis of cell-surface wild-type and K1047R AC1. Lower panel: Summary data of 4 independent experiments similar to that shown in the upper panel. ‘Endocytosed AC1’ in the lower panel is the ratio of endocytosed AC to cell-surface AC in the upper panel. All data are normalized relative to data obtained for HA-AC1 alone. *, different from wild-type AC1, p≤0.0232; **p≤0.009. ( b ) Upper panel: Effect of TRIP-Br1 and XIAP on the recycling of endocytosed wild-type and K1047R AC1. Lower panel: Summary data of 3 independent experiments similar to that shown in the upper panel. ‘AC remaining in the cytosol’ is the ratio of remaining AC to ‘input’ (endocytosed AC) in the upper panel. All data are normalized relative to the data obtained for HA-AC1 alone. DOI: http://dx.doi.org/10.7554/eLife.28021.012

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Endocytosis and recycling of HA-tagged wild-type and K1047R AC1 expressed in HeLa cells. ( a ) Upper panel: Effect of TRIP-Br1 and XIAP on the endocytosis of cell-surface wild-type and K1047R AC1. Lower panel: Summary data of 4 independent experiments similar to that shown in the upper panel. ‘Endocytosed AC1’ in the lower panel is the ratio of endocytosed AC to cell-surface AC in the upper panel. All data are normalized relative to data obtained for HA-AC1 alone. *, different from wild-type AC1, p≤0.0232; **p≤0.009. ( b ) Upper panel: Effect of TRIP-Br1 and XIAP on the recycling of endocytosed wild-type and K1047R AC1. Lower panel: Summary data of 3 independent experiments similar to that shown in the upper panel. ‘AC remaining in the cytosol’ is the ratio of remaining AC to ‘input’ (endocytosed AC) in the upper panel. All data are normalized relative to the data obtained for HA-AC1 alone. DOI: http://dx.doi.org/10.7554/eLife.28021.012

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques:

    Knocking out TRIP-Br1 induces the phosphorylation of CREB in the hippocampus and mood disorder (especially despair-like behavior) in mice. ( a ) Immunostaining of phosphorylated CREB (pCREB, red) in the hippocampus, with NeuN (green, neuronal marker) and DAPI (purple). ( b ) Quantitation of pCREB immunoreactivity relative to NeuN, *, different from WT, p=0.024, n = 3 10 wk-old male mice for both WT and KO (TRIP-Br1 knockout). ( c ) Body weight of the mice. ( d ) Muscle strength in wire hanging test. ( e–i ) Mood disorders, especially despair-like behavior, in several tests: ( e ) immobility time in the forced swimming test; ( f ) Nesting score in 24 (left) and 48 (right) h; ( g ) time in open arms in an elevated plus maze; ( h ) latency to enter a light box (left) and time in the light box (right) in the light-dark transition test; ( i ) number of marble buried in the marble burying test. 9 (±1)-wk-old male and female mice used in the experiments in panels c–I , and ++, +/−, and −/− denote: wild-type (n = 9), heterozygous (n = 8), homozygous (n = 11) TRIP-Br1 knockout mice, respectively. Different from +/+: *p≤0.039; **p=0.007; ****p≤0.0005. DOI: http://dx.doi.org/10.7554/eLife.28021.018

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Knocking out TRIP-Br1 induces the phosphorylation of CREB in the hippocampus and mood disorder (especially despair-like behavior) in mice. ( a ) Immunostaining of phosphorylated CREB (pCREB, red) in the hippocampus, with NeuN (green, neuronal marker) and DAPI (purple). ( b ) Quantitation of pCREB immunoreactivity relative to NeuN, *, different from WT, p=0.024, n = 3 10 wk-old male mice for both WT and KO (TRIP-Br1 knockout). ( c ) Body weight of the mice. ( d ) Muscle strength in wire hanging test. ( e–i ) Mood disorders, especially despair-like behavior, in several tests: ( e ) immobility time in the forced swimming test; ( f ) Nesting score in 24 (left) and 48 (right) h; ( g ) time in open arms in an elevated plus maze; ( h ) latency to enter a light box (left) and time in the light box (right) in the light-dark transition test; ( i ) number of marble buried in the marble burying test. 9 (±1)-wk-old male and female mice used in the experiments in panels c–I , and ++, +/−, and −/− denote: wild-type (n = 9), heterozygous (n = 8), homozygous (n = 11) TRIP-Br1 knockout mice, respectively. Different from +/+: *p≤0.039; **p=0.007; ****p≤0.0005. DOI: http://dx.doi.org/10.7554/eLife.28021.018

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Mouse Assay, Immunostaining, Marker, Quantitation Assay, Knock-Out

    A model depicting a general negative-feedback mechanism of ubiquitination and degradation of multiple AC isoforms. The SERTA domain of TRIP-Br1 binds to the C1b catalytic domain of AC1 or other AC isoforms and subsequently recruits XIAP, which, together with the E2 enzyme UbcH5b (or UbcH5a or UbcH5c) ( Nakatani et al., 2013 ; Mace et al., 2008 ), ubiquitinates K1047 in AC1 or an equivalent Lys residue in other AC isoforms. The N-terminus of TRIP-Br1 interacts with XIAP, probably with its BIR2 domain ( Hong et al., 2009 ), and the Ring domain of XIAP interacts with UbcH5b ( Nakatani et al., 2013 ; Mace et al., 2008 ). The ubiquitination of ACs leads to their endocytosis and degradation. Sustained AC activation and cAMP production under catecholamine stress increase XIAP protein expression through PKA, and elevated XIAP protein expression subsequently dstabilizes ACs and eventually lows cAMP production/signaling. Red arrow, stimulatory effect; black dash: inhibitory effect. DOI: http://dx.doi.org/10.7554/eLife.28021.020

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: A model depicting a general negative-feedback mechanism of ubiquitination and degradation of multiple AC isoforms. The SERTA domain of TRIP-Br1 binds to the C1b catalytic domain of AC1 or other AC isoforms and subsequently recruits XIAP, which, together with the E2 enzyme UbcH5b (or UbcH5a or UbcH5c) ( Nakatani et al., 2013 ; Mace et al., 2008 ), ubiquitinates K1047 in AC1 or an equivalent Lys residue in other AC isoforms. The N-terminus of TRIP-Br1 interacts with XIAP, probably with its BIR2 domain ( Hong et al., 2009 ), and the Ring domain of XIAP interacts with UbcH5b ( Nakatani et al., 2013 ; Mace et al., 2008 ). The ubiquitination of ACs leads to their endocytosis and degradation. Sustained AC activation and cAMP production under catecholamine stress increase XIAP protein expression through PKA, and elevated XIAP protein expression subsequently dstabilizes ACs and eventually lows cAMP production/signaling. Red arrow, stimulatory effect; black dash: inhibitory effect. DOI: http://dx.doi.org/10.7554/eLife.28021.020

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Activation Assay, Expressing

    Knocking out TRIP-Br1 in mice increased AC2 expression in the brain. Na/K ATPase: membrane-protein negative control; actin: loading control. Quantification of the western blots is shown at the right: *, different from wild-type (WT), p=0.0138, n = 3 pairs of female littermates. DOI: http://dx.doi.org/10.7554/eLife.28021.016

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Knocking out TRIP-Br1 in mice increased AC2 expression in the brain. Na/K ATPase: membrane-protein negative control; actin: loading control. Quantification of the western blots is shown at the right: *, different from wild-type (WT), p=0.0138, n = 3 pairs of female littermates. DOI: http://dx.doi.org/10.7554/eLife.28021.016

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Mouse Assay, Expressing, Negative Control, Western Blot

    TRIP-Br1 bridges the interaction of AC2 and AC5/6 with XIAP E3 ligase. ( a ) Summary data of total endogenous AC2 and AC5/6 in Figure 7a . *, different from control, p≤0.019; **p≤0.008; *** p

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: TRIP-Br1 bridges the interaction of AC2 and AC5/6 with XIAP E3 ligase. ( a ) Summary data of total endogenous AC2 and AC5/6 in Figure 7a . *, different from control, p≤0.019; **p≤0.008; *** p

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques:

    Colocalization of AC1 with TRIP-Br1. ( a ) Confocal images of HeLa cells transfected with 3HA-AC1. The cells were immunostained with anti-HA and anti-TRIP-Br1 antibodies. ( b–c ) Confocal images of untransfected HeLa cells. Anti-AC1 antibody was delivered into live cells by Lipofectamine2000, and the signal of anti-AC antibody was amplified by Atto 488-conjugated biotin because endogenous AC1 was barely detectable by conventional immunostaining ( b ). ( c ) is a control without anti-AC1 primary antibody for b . ( d ) STORM (stochastic optical reconstruction microscopy) image of non-transfected HeLa cells immuno-stained with anti-AC1 and anti-TRIP-Br1 antibodies. Because the optical depth of the image is 200 nm, endogenous AC1 and TRIP-Br1 probably interacted directly in or near the plasmalemma. Scale bars: 20 µm in a–c , and 500 nm in d . DOI: http://dx.doi.org/10.7554/eLife.28021.003

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Colocalization of AC1 with TRIP-Br1. ( a ) Confocal images of HeLa cells transfected with 3HA-AC1. The cells were immunostained with anti-HA and anti-TRIP-Br1 antibodies. ( b–c ) Confocal images of untransfected HeLa cells. Anti-AC1 antibody was delivered into live cells by Lipofectamine2000, and the signal of anti-AC antibody was amplified by Atto 488-conjugated biotin because endogenous AC1 was barely detectable by conventional immunostaining ( b ). ( c ) is a control without anti-AC1 primary antibody for b . ( d ) STORM (stochastic optical reconstruction microscopy) image of non-transfected HeLa cells immuno-stained with anti-AC1 and anti-TRIP-Br1 antibodies. Because the optical depth of the image is 200 nm, endogenous AC1 and TRIP-Br1 probably interacted directly in or near the plasmalemma. Scale bars: 20 µm in a–c , and 500 nm in d . DOI: http://dx.doi.org/10.7554/eLife.28021.003

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Transfection, Amplification, Immunostaining, Microscopy, Staining

    Change in the heart rate of TRIP-Br-1 knockout mice under deep anesthesia. Heart rates of wild-type (WT) and TRIP-Br1 knockout (KO) mice under basal and isoproterenol-stimulated (iso) conditions; **, different from WT, p=0.002; *p=0.014; n = 12 (WT, basal); 15 (KO, basal); 7 (WT, iso); 7 (KO, iso). Instead of Avertin, the mice in these experiments were anesthetized with ketamine and xylazine, which induce deep anesthesia. DOI: http://dx.doi.org/10.7554/eLife.28021.017

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Change in the heart rate of TRIP-Br-1 knockout mice under deep anesthesia. Heart rates of wild-type (WT) and TRIP-Br1 knockout (KO) mice under basal and isoproterenol-stimulated (iso) conditions; **, different from WT, p=0.002; *p=0.014; n = 12 (WT, basal); 15 (KO, basal); 7 (WT, iso); 7 (KO, iso). Instead of Avertin, the mice in these experiments were anesthetized with ketamine and xylazine, which induce deep anesthesia. DOI: http://dx.doi.org/10.7554/eLife.28021.017

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Knock-Out, Mouse Assay

    TRIP-Br1 bridges the interaction of AC1 with XIAP E3 ligase. ( a ) Full-length TRIP-Br1 fused with GST—but not GST-TRIP-Br1 truncation mutants or GST alone—captured His-XIAP purified from E. coli . ( b ) Three purified FLAG-His-tagged AC1 fragments, AC1-N, AC1-M, and AC1-C2, were used to pull down purified XIAP in the presence or absence of purified TRIP-Br1 (His-tagged at both N and C termini). ( c ) Endogenous AC1 in HeLa cells was IPed with anti-AC1 antibody or control IgG and immunoblotted with anti-AC1, anti-XIAP, and anti-TRIP-Br1 antibodies. DOI: http://dx.doi.org/10.7554/eLife.28021.006

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: TRIP-Br1 bridges the interaction of AC1 with XIAP E3 ligase. ( a ) Full-length TRIP-Br1 fused with GST—but not GST-TRIP-Br1 truncation mutants or GST alone—captured His-XIAP purified from E. coli . ( b ) Three purified FLAG-His-tagged AC1 fragments, AC1-N, AC1-M, and AC1-C2, were used to pull down purified XIAP in the presence or absence of purified TRIP-Br1 (His-tagged at both N and C termini). ( c ) Endogenous AC1 in HeLa cells was IPed with anti-AC1 antibody or control IgG and immunoblotted with anti-AC1, anti-XIAP, and anti-TRIP-Br1 antibodies. DOI: http://dx.doi.org/10.7554/eLife.28021.006

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Purification

    Effect of TRIP-Br1 on the expression of other membrane proteins and AC1 mRNA levels. ( a–b ) Stable expression of TRIP-Br1-V5 did not affect the protein levels of HA-tagged TRPV4 (transient receptor potential vanilloid 4) ( a ) and TRPP2 (transient receptor potential polycystin 2) ( b ) transiently expressed in HEK293T cells. GAPDH, loading control; GFP, transfection efficiency control. ( c ) AC1 mRNA levels showed no change (compared to control) in the heart tissue of TRIP-Br1 knockout mice in a real-time PCR assay (WT n = 5, KO n = 4; p=0.853). The mRNA level of AC1 was normalized relative to that of GAPDH. DOI: http://dx.doi.org/10.7554/eLife.28021.005

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: Effect of TRIP-Br1 on the expression of other membrane proteins and AC1 mRNA levels. ( a–b ) Stable expression of TRIP-Br1-V5 did not affect the protein levels of HA-tagged TRPV4 (transient receptor potential vanilloid 4) ( a ) and TRPP2 (transient receptor potential polycystin 2) ( b ) transiently expressed in HEK293T cells. GAPDH, loading control; GFP, transfection efficiency control. ( c ) AC1 mRNA levels showed no change (compared to control) in the heart tissue of TRIP-Br1 knockout mice in a real-time PCR assay (WT n = 5, KO n = 4; p=0.853). The mRNA level of AC1 was normalized relative to that of GAPDH. DOI: http://dx.doi.org/10.7554/eLife.28021.005

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Expressing, Transfection, Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    cAMP agonists promote XIAP upregulation and AC1 degradation in a PKA-dependent manner in the macrophages of wild-type but not TRIP-Br1 knockout mice. Changes in AC1, XIAP, and TRIP-Br1 expression with or without 6 hr forskolin (FSK, 10 μM, in the presence of 100 μM IBMX) or isoproterenol (ISO,10 μM) treatment in the microphages of wild-type ( a–b ) and TRIP-Br1 knockout ( c–d ) mice. H89 (10 μM), PKA inhibitor; β-actin, loading control. ( b and d ), Summary data of panels a and b , respectively, relative to β-actin. Different from control (CTRL), *p≤0.0427; **p≤0.0076, n = 3. DOI: http://dx.doi.org/10.7554/eLife.28021.019

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: cAMP agonists promote XIAP upregulation and AC1 degradation in a PKA-dependent manner in the macrophages of wild-type but not TRIP-Br1 knockout mice. Changes in AC1, XIAP, and TRIP-Br1 expression with or without 6 hr forskolin (FSK, 10 μM, in the presence of 100 μM IBMX) or isoproterenol (ISO,10 μM) treatment in the microphages of wild-type ( a–b ) and TRIP-Br1 knockout ( c–d ) mice. H89 (10 μM), PKA inhibitor; β-actin, loading control. ( b and d ), Summary data of panels a and b , respectively, relative to β-actin. Different from control (CTRL), *p≤0.0427; **p≤0.0076, n = 3. DOI: http://dx.doi.org/10.7554/eLife.28021.019

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Knock-Out, Mouse Assay, Expressing

    TRIP-Br1-FLAG was detected by TRIP-Br1 antibody. HeLa cells were seeded in a 60-mm dish the day before the transfection. 5 ug TRIP-Br1-FLAG or control vector were transfected into the HeLa cells. After 48 h culture, cells were lysed by RIPA buffer and the cell lysate was boiled at 95°C for 10 min and added with 200 mM DTT before subject to western blotting by anti-FLAG and anti-TRIP-Br1 antibodies separately. 12% gel was used for better separation of endogenous (arrowhead) and FLAG-tagged TRIP-Br1.

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: TRIP-Br1-FLAG was detected by TRIP-Br1 antibody. HeLa cells were seeded in a 60-mm dish the day before the transfection. 5 ug TRIP-Br1-FLAG or control vector were transfected into the HeLa cells. After 48 h culture, cells were lysed by RIPA buffer and the cell lysate was boiled at 95°C for 10 min and added with 200 mM DTT before subject to western blotting by anti-FLAG and anti-TRIP-Br1 antibodies separately. 12% gel was used for better separation of endogenous (arrowhead) and FLAG-tagged TRIP-Br1.

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Transfection, Plasmid Preparation, Western Blot

    cIAP1 and cIAP2 do not affect AC1 expression. ( a ) GST-TRIP-Br1 fusion protein or GST alone was used to pull down endogenous XIAP, cIAP1, and cIAP2 in Hela cells. ( b ) Overexpression of XIAP but not cIAP1 or cIAP2 degraded endogenous AC1 in Hela cells. DOI: http://dx.doi.org/10.7554/eLife.28021.009

    Journal: eLife

    Article Title: The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms

    doi: 10.7554/eLife.28021

    Figure Lengend Snippet: cIAP1 and cIAP2 do not affect AC1 expression. ( a ) GST-TRIP-Br1 fusion protein or GST alone was used to pull down endogenous XIAP, cIAP1, and cIAP2 in Hela cells. ( b ) Overexpression of XIAP but not cIAP1 or cIAP2 degraded endogenous AC1 in Hela cells. DOI: http://dx.doi.org/10.7554/eLife.28021.009

    Article Snippet: Cycloheximide experiments HeLa cells with or without TRIP-Br1 shRNA treatment were treated with 200 μM CHX (Sigma) for various times.

    Techniques: Expressing, Over Expression

    Comparative cleavage characteristics of the peptidic substrates ([Cy5-D-R-E-I-M-R] 2 -Rh110 and ([Cy5-D-R-E-I-M-D] 2 -Rh110 in U937 cells. The Separase-specific conjugated peptidic substrate (DREIMR) is shown in green, the control substrate lacking the Separase cleavage consensus (DREIMD) is depicted in red. (A) Substrate cleavage in the cell extract-based Separase assay as monitored by released Rh110 fluorescence. (B) FACS measurement of Rh110 positive cells after incubation with substrates according to the standardized protocol (90 min at 37°C). (C) Calculation of mean proteolytic activity per cell as measured by released Rh110 fluorescence in the FACS assay. (D) Changes (Δ-values) of proteolytic activities on substrates DREIMR and DREIMD after 48 h of Espl1 silencing when compared to mock treated U937 cells. Corresponding Western blot immunostaining experiments (below) illustrate the knockdown of the Separase protein levels (siRNA (-), 100%; siRNA (+), 24%). Actin served as loading control. Abbreviations: DREIMD, ([Cy5-D-R-E-I-M-D] 2 -Rh110; DREIMR, ([Cy5-D-R-E-I-M-R] 2 -Rh110.

    Journal: PLoS ONE

    Article Title: Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    doi: 10.1371/journal.pone.0133769

    Figure Lengend Snippet: Comparative cleavage characteristics of the peptidic substrates ([Cy5-D-R-E-I-M-R] 2 -Rh110 and ([Cy5-D-R-E-I-M-D] 2 -Rh110 in U937 cells. The Separase-specific conjugated peptidic substrate (DREIMR) is shown in green, the control substrate lacking the Separase cleavage consensus (DREIMD) is depicted in red. (A) Substrate cleavage in the cell extract-based Separase assay as monitored by released Rh110 fluorescence. (B) FACS measurement of Rh110 positive cells after incubation with substrates according to the standardized protocol (90 min at 37°C). (C) Calculation of mean proteolytic activity per cell as measured by released Rh110 fluorescence in the FACS assay. (D) Changes (Δ-values) of proteolytic activities on substrates DREIMR and DREIMD after 48 h of Espl1 silencing when compared to mock treated U937 cells. Corresponding Western blot immunostaining experiments (below) illustrate the knockdown of the Separase protein levels (siRNA (-), 100%; siRNA (+), 24%). Actin served as loading control. Abbreviations: DREIMD, ([Cy5-D-R-E-I-M-D] 2 -Rh110; DREIMR, ([Cy5-D-R-E-I-M-R] 2 -Rh110.

    Article Snippet: For siRNA treatment, 5x106 U937 cells were resuspended in 100 μl Cell Line Nucleofector Solution (Lonza) containing 18 μl Supplement S Solution (Lonza).

    Techniques: Fluorescence, FACS, Incubation, Activity Assay, Western Blot, Immunostaining

    FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Article Snippet: Inhibition of FOXO1 and FOXO3 by siRNA Treatment A pool of 8 ACCELL siRNAs specific for Foxo1 and Foxo3 (4 siRNAs each, Dharmacon) was used to decrease the expression of Foxo1 and Foxo3 mRNAs.

    Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Activation Assay, Plasmid Preparation, Cell Culture, Transduction, Staining, Flow Cytometry, Cytometry

    Effects of nucleosome assembly proteins on DNA replication activity of EBNA1. (A) CNE2Z cells were treated with siRNA against NAP1, NAP2, TAF-I, or GFP (negative control) and then transfected with an oriP plasmid expressing EBNA1 or lacking EBNA1 (first

    Journal: Journal of Virology

    Article Title: Nucleosome Assembly Proteins Bind to Epstein-Barr Virus Nuclear Antigen 1 and Affect Its Functions in DNA Replication and Transcriptional Activation ▿

    doi: 10.1128/JVI.00931-09

    Figure Lengend Snippet: Effects of nucleosome assembly proteins on DNA replication activity of EBNA1. (A) CNE2Z cells were treated with siRNA against NAP1, NAP2, TAF-I, or GFP (negative control) and then transfected with an oriP plasmid expressing EBNA1 or lacking EBNA1 (first

    Article Snippet: Forty-eight hours after the first siRNA treatment, cells were transfected with 1.0 μg of pFRTKCAT reporter plasmid , 5 ng of pc3OriPEBNA1 or pc3OriP , and 0.5 μg of plasmid CMVPLAP expressing secreted alkaline phosphatase (SEAP) , using Fugene HD (Roche).

    Techniques: Activity Assay, Negative Control, Transfection, Plasmid Preparation, Expressing

    CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 siRNA for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.

    Journal: Scientific Reports

    Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in ClockΔ19/+ mice contributes to improved glucose homeostasis

    doi: 10.1038/srep12801

    Figure Lengend Snippet: CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. ( a , b ) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). ( c ) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 ΔUBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ19, and Myc-p62 (WT or ΔUBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). ( d ) Protein lysates were prepared from p62 +/+ (WT) and p62 − / − MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. ( e ) MEFs ( p62 +/+ or p62 − / − ) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). ( f ) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 siRNA for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.

    Article Snippet: For plasmids transfection, the cells were transfected with indicated plasmids using either iMFectin Poly DNA transfection reagent (GenDEPOT) or Lipofectamine 2000 (Invitrogen) for 24 h. For siRNA treatment, cultured 293T cells were transfected with p62/SQSTM1 siRNA (Santa Cruz) or a RNA interference negative control (Santa Cruz).

    Techniques: Transfection, Expressing, Construct, Staining, Negative Control