lentiviral packaging Search Results


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  • 99
    Thermo Fisher virapower lentivirus packaging vectors
    Construction of apoptin-modified MSCs. (A) <t>Lentiviral</t> vector pLV/Final-puro-EF1α-apoptin was identified by PCR. Lane 1/3, negative control; lane 2, amplification product of fragment of apoptin; lane 4, amplification product of fragment of EF1α promoter. (B) Expression of apoptin was confirmed by western blot analysis. Lane 1, negative control; lane 2, MSCs APOPTIN 1; lane 3, MSCs APOPTIN 2. (C) Morphology of MSCs hrGFP and MSCs APOPTIN. (D) Adipogenesis and osteogenesis differentiation of MSCs APOPTIN. Magnification, ×200. MSCs APOPTIN, mesenchymal stem cells expressing apoptin; PCR, polymerase chain reaction; hrGFP, humanized Renilla green fluorescence protein.
    Virapower Lentivirus Packaging Vectors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore shrna containing lentiviral particles
    Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1 G93A mice. Lumbar spinal cords, from 12- and 15-week-old Tg SOD1 G93A , WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, <t>MLKL,</t> and p-MLKL. A , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA from 12-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1 G93A compared to Tg SOD1 WT ( p = 0.0021) and NTg ( p = 0.0076) at 12 weeks. B , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA in spinal cords of 15-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk1 ( p = 0.0009, vs Tg SOD1 WT ; p = 0.0020, vs NTg) and Ripk3 ( p = 0.0115; vs Tg SOD1 WT , p = 0.0067; vs NTg) but not for Mlkl in Tg SOD1 G93A compared to Tg SOD1 WT and NTg mice at 15 weeks. C , Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1 WT , and Tg SOD1 G93A 15-week-old mice. β-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral <t>shRNA</t> in mouse PMN cultures (mPMNs). D , Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1 G93A samples compared to Tg SOD1 WT ( p = 0.0279) and NTg ( p = 0.0033) mice. Results are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey’s post hoc analysis; n = 3 biological replicates per genotype. E , Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Non-specific band at 47 kDa is designated as an asterisk (*). NTg spleen: positive control tissue, Ripk3 −/− spleen and Tg SOD1 G93A ; Ripk3 −/− spinal cord: negative control tissue. F , Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Ripk3 −/− spinal cord: negative control tissue. G , Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H , Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). NIH 3T3: positive control cell lysate. I , Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.
    Shrna Containing Lentiviral Particles, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa lentiviral packaging
    SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1 + normal tissues in the presence of circulating and bone marrow-resident CD19 + ROR1 + Raji tumors. ( A) Map of <t>lentiviral</t> constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p
    Lentiviral Packaging, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Biosettia lentiviral packaging
    SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1 + normal tissues in the presence of circulating and bone marrow-resident CD19 + ROR1 + Raji tumors. ( A) Map of <t>lentiviral</t> constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p
    Lentiviral Packaging, supplied by Biosettia, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Shanghai Generay Biotech lentiviral packaging
    SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1 + normal tissues in the presence of circulating and bone marrow-resident CD19 + ROR1 + Raji tumors. ( A) Map of <t>lentiviral</t> constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p
    Lentiviral Packaging, supplied by Shanghai Generay Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mission lentivaral packaging mix
    Effects of shRNA on FMDV-IRES activity. a The shRNA-expressing <t>lentiviral</t> vector targeting the conserved region of FMDV-IRES (Lenti-FMDV-sh) or control vector (pLL3.7 alone) was used to infect HEK293 cells at various MOIs. After 14 days, firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values between cells with and without shRNA (** p
    Mission Lentivaral Packaging Mix, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher lentivirus packaging mix
    Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY <t>lentivirus-infected</t> E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”
    Lentivirus Packaging Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cyagen Biosciences lentiviruses packaging
    Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY <t>lentivirus-infected</t> E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”
    Lentiviruses Packaging, supplied by Cyagen Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore lentiviral packaging protocol
    Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY <t>lentivirus-infected</t> E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”
    Lentiviral Packaging Protocol, supplied by Millipore, 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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    96
    Genechem lentiviral packaging kit
    Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY <t>lentivirus-infected</t> E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”
    Lentiviral Packaging Kit, supplied by Genechem, used in various techniques. Bioz Stars score: 96/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai Genechem lentiviral packaging kit
    Inhibitory effect of miR-223 on IGF1R/PI3K/Akt signaling pathway in vitro . (A) The GV259-miR-223 <t>lentiviral</t> vector was packaged and delivered to PC9/ER cells. Transduction efficiency was evaluated based on the fluorescent GFP signal. Fluorescent GFP signal was stably observed 72 h after infection. EV for control vector-infected cells (right panel) and miR-223 for GV259-miR-223-infected cells (left panel) are shown. Magnification, ×100. (B) miR-223 was overexpressed in PC9/ER cells as confirmed by real-time RT-PCR analysis. (C) Downregulation of IGF1R expression in PC9/ER-miR-223 cells was confirmed by real-time RT-PCR analysis. (D) IGF1R/Akt/p70S6K pathway was suppressed in the miR-223-overexpressing cells. Phosphorylation of Akt and p70S6K was significantly inhibited, according to western blot analysis. (E) miR-223 mediated inhibition of IGF1R/PI3K/Akt was reversed by agonist (IGF1) of IGF1R in miR-223 transfected cells. p-IGF1R and p-Atk were increased as shown in western blots. (F) The cytotoxicity and IC 50 values in PC9, PC9/ER, PC9/ER-EV, and PC9/ER-miR-223 cells. The cells were treated with the indicated concentrations of erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC 50 values were determined using the CCK8 assay.
    Lentiviral Packaging Kit, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 93/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher lentiviral packaging system
    EZH2 is a direct target of miR-141. ( a ) Putative binding site of miR-141 in EZH2 3′-UTR. ( b ) Luc reporter assays in DU145 cells co-transfected with either wild-type EZH2 3′-UTR or the 3′-UTR with the predicted miR-141-binding site mutated and miR-141 or control oligos. ( c ) qPCR analysis of EZH2 mRNA levels in CD44 + PCa cells. ( d ) qPCR of EZH2 in Du145 cells overexpressing miR-141 using either oligo transfection or <t>lentiviral</t> infection. ( e ) WB of EZH2 and SUZ12 in PC3 and Du145 cells overexpressing miR-141. Relative protein levels were measured by densitometry and normalized to GAPDH levels (indicated below). ( f ) Invasion assay in DU145 cells treated with the EZH2 inhibitor DZNep. Inset, WB showing reduced EZH2 proteins by DZNep. ( g ) Invasion assays in Du145 cells co-transfected with 30 nM NC or miR-141 oligos together with empty vector or pCMV-EZH2 construct. ( h ) qPCR of EZH2 target genes including FBN1 , RAD51C , KIAA0101 and CDKN2A in DU145 cells infected with lenti-Ctrl or lenti-141. ( i ) WB analysis of H3K27me3 in DU145 cells overexpressing miR-141 using either lentiviral vectors or oligo transfection. Total H3 and actin were included as endogenous controls. ( j ) miR-141 exhibits pleiotropic anti-tumour and anti-metastasis effects in PCa by targeting multiple molecules associated with CSCs, cell cycle and proliferation, and cytoskeleton, cell motility/invasion and metastasis. Values represent mean±s.d. of triplicates from three to five independent experiments. * P
    Lentiviral Packaging System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa lentiviral packaging system
    Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with <t>lentiviral</t> particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g ×
    Lentiviral Packaging System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    OriGene lentiviral packaging kit
    Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with <t>lentiviral</t> particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g ×
    Lentiviral Packaging Kit, supplied by OriGene, used in various techniques. Bioz Stars score: 96/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Macrogen lentiviral packaging service
    Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with <t>lentiviral</t> particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g ×
    Lentiviral Packaging Service, supplied by Macrogen, used in various techniques. Bioz Stars score: 87/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher lentiviral packaging kit
    MGAT5 restoration counteracts the suppressive effects of miR-124 on BC cell proliferation and migration. MDA-MB-231 and MCF-7 cells were transfected with miR-124 inhibitor, sh-MGAT5, sh-NC, and miR-124 inhibitor+sh-MGAT5 <t>lentiviral</t> vectors respectively to culture stably cell lines which followed by puromycin selection. A: Cell viability was measured using CCK-8 assays. B: EDU staining was performed and the percentage of EDU-positive cells were counted. Scale bar 100 µm. C: Representative photographs of the colony formation of MDA-MB-231 and MCF-7 cells, and related quantitative analysis. D: Cell migrations were determined using transwell. Scale bars, 50 µm. E: Cell migrations were determined using wound healing assay and the percentage of migrated was calculated. All data are shown as means ± SD of three separate experiments. * P
    Lentiviral Packaging Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Addgene inc lentiviral packaging vectors
    shRNA constructs and production of <t>lentiviral</t> particles.
    Lentiviral Packaging Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 97/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai Genechem lentiviral packaging protocol
    shRNA constructs and production of <t>lentiviral</t> particles.
    Lentiviral Packaging Protocol, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Biowit Technologies lentiviral packaging kit
    shRNA constructs and production of <t>lentiviral</t> particles.
    Lentiviral Packaging Kit, supplied by Biowit Technologies, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenePharma Company lentiviral packaging kit
    shRNA constructs and production of <t>lentiviral</t> particles.
    Lentiviral Packaging Kit, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Genecopoeia lentiviral packaging vectors
    Overexpression of eIF5A1 upregulates neurofilament expression, promotes neuronal survival and improves functional recovery in vivo. ( A,B ) eIF5A mRNA ( A ) and protein levels ( B ) were measured 28 days after <t>lentiviral</t> injections (described below) and SCT with β-actin as an internal control. ( C ) Expression of control lentiviral constructs in the anterior horn (AH) cells of the spinal cord. ( D ) Levels of neurofilament (NF+) (green) and NeuN + neurons (red) were measured 28 days after injection of lentiviral constructs caudal to SCT. Nuclei were visualized by DAPI (blue). Bar (NeuN + ), 100 μm. Bar (NF+), 30 μm. Quantifications are shown in part panels ( E–G ) BBB scores were used to assay hind limb motor function. The uninjured sham controls rats had BBB scores of 21 ± 0.00. BBB scores of rats overexpressing eIF5A1 were higher than control rats while rats with reduced eIF5A1 had lower BBB scores. S, sham group; C, SCT, control lentiviral construct; F+, SCT, eIF5A1 overexpressing lentiviral construct; F–, SCT, shRNA-eIF5A1 expressing lentiviral construct; Data were presented as mean ± SEM. *P
    Lentiviral Packaging Vectors, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    System Biosciences Inc lentiviral packaging systems
    In vivo inhibitors do not alter A-type lamin protein levels but do alter their subnuclear localization and assembly state. (A) Confocal images of wild-type U2OS cells and cells that stably expressed a scrambled small interfering (si)RNA (scrambled RNAi), an siRNA for LMNA silencing ( LMNA RNAi, LMNA in B, LMNA kd in C), the empty <t>lentiviral</t> plasmid (mock), lamin A (lamin A OE), lamin C (lamin C OE), the control DARPin E3_5, or the indicated different lamin A-specific DARPins (LaA_x, where x is the DARPin identifier). Cells were immunostained with antibodies against lamin A/C, lamin B1 and emerin. Scale bars: 20 µm. (B) Western blot analysis of the supernatant (SN) and pellet fractions (P) of wild-type and modified U2OS cells shown in A after extraction with a buffer containing 0.2% NP40 and using antibodies against lamin A/C, lamin B1 and lamin B2. Note that the supernatant and pellet were loaded in a 10:1 ratio. (C) Statistical analysis of the ratio of extracted to total lamin A and lamin C in different U2OS cell lines. The average of three individual experiments±s.e.m. is shown. Statistical significance was calculated using a one-way ANOVA test followed by Bonferroni's multiple comparison test, where * P ≤0.05, ** P ≤0.01 and *** P ≤0.001. (D) Western blot analysis of lamin and lamin-binding protein levels in the wild-type and modified U2OS cells shown in A. The levels of exogenous lamin A, lamin C, and of DARPin expression is reflected by levels of bicistronically expressed copGFP, a green fluorescent protein cloned from Pontellina plumata . β-actin was used as loading control.
    Lentiviral Packaging Systems, supplied by System Biosciences Inc, 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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    94
    GE Healthcare lentiviral packaging systems
    miR-221 Down-Regulates transcripts involved in apoptosis, including Pro-Apoptotic protein BIM, and protects DJ-1 knock-down cells from cell death. (A) Compared to cells transfected with negative control pre-miR (pre-miR-NC), SH-SY5Y cells transfected with pre-miR-221 showed a decrease of mRNA transcripts implicated in apoptosis, including BIM, BMF, BNIP3L, and FOXO3A. (B) All three isoforms of BIM protein (BIM EL , BIM L , BIM S ) were decreased by over-expression of miR-221 and (C) increased by the inhibition of miR-221 using anti-miR-221. (D) Pre-miR-221 transfected cells were treated with 250 uM H 2 O 2 for 24 h. Compared to control, over-expression of miR-221 prevented the oxidative stress mediated induction of pro-apoptotic BIM transcript. (E) Cells were made to stably express non-targeting control (Ctrl) short hairpin RNA (shRNA) or pooled DJ-1 shRNA. Cells were then transduced with either <t>lentiviral</t> control miR (Ctrl miR) or miR-221 for 24 h. Stable DJ-1 KD decreases the levels of mature miR-221, while transduction with lentiviral miR-221 robustly increases its levels. (F) Cells were then treated with the indicated concentrations of MPP + for 24 h, and cell death was assessed using LDH assay. Compared to control KD cells, stable DJ-1 KD leads to increased cell death, which is rescued when miR-221 is over-expressed. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (A, D, F) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test. (B, C) analyzed using two-tailed student's t -test. (E) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.
    Lentiviral Packaging Systems, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genecopoeia lentivirus packaging
    PKM2 overexpression increased resistance of EC109 to shikonin. EC109/PKM2 stable cell was established by transducing <t>Lentivirus</t> plasmid according to previous methods. RT-PCR(A) and Western blot(B) showed that the expression of PKM2 was higher in EC109/PKM2 cell than EC109 cell. C. Cell viability was detected by CCK-8 assay. The results showed cell viability and resistance of EC109/PKM2 cells to shikonin increased evidently compared to EC109 cells. D. Shikonin decreased the expression of PKM2 and cyclinD1 in EC109 and Ec109/pkm2 cells. *p
    Lentivirus Packaging, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher packaging plasmids
    PKM2 overexpression increased resistance of EC109 to shikonin. EC109/PKM2 stable cell was established by transducing <t>Lentivirus</t> plasmid according to previous methods. RT-PCR(A) and Western blot(B) showed that the expression of PKM2 was higher in EC109/PKM2 cell than EC109 cell. C. Cell viability was detected by CCK-8 assay. The results showed cell viability and resistance of EC109/PKM2 cells to shikonin increased evidently compared to EC109 cells. D. Shikonin decreased the expression of PKM2 and cyclinD1 in EC109 and Ec109/pkm2 cells. *p
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    92
    Biosettia lentivirus packaging
    Silencing of Nrf2 decreases usnic acid-induced γ-GCSc upregulation. HepG2 cells stably infected with <t>lentivirus</t> carrying doxycycline-inducible sh-Nrf2 or SC (scramble control). The two established cell lines were incubated with 1 μg/ml doxycycline for 72 h followed by continued culture for another 24 h without doxycycline; then, the efficiency of Nrf2 knockdown was determined by real-time PCR ( a ) and Western blotting ( b , c ). Bar graphs are mean ± SD of three individual experiments. *p
    Lentivirus Packaging, supplied by Biosettia, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore lentivirus packaging
    Silencing of Nrf2 decreases usnic acid-induced γ-GCSc upregulation. HepG2 cells stably infected with <t>lentivirus</t> carrying doxycycline-inducible sh-Nrf2 or SC (scramble control). The two established cell lines were incubated with 1 μg/ml doxycycline for 72 h followed by continued culture for another 24 h without doxycycline; then, the efficiency of Nrf2 knockdown was determined by real-time PCR ( a ) and Western blotting ( b , c ). Bar graphs are mean ± SD of three individual experiments. *p
    Lentivirus Packaging, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Horizon Discovery trans lentiviral packaging system
    Sorafenib induces phosphorylation of IRF7 by reducing its inhibitor ATF4 (a, b) Quantification of ATF4 (western blot) normalized to β-actin (fold change with respect to DMSO treated controls) in mouse Ba/F3-ITD (a) or human FLT3-ITD mutant MV4-11 (b) leukemia cells exposed to sorafenib as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled; a: n =6 biologically independent samples per group, b: n =4 biologically independent samples per group. The P- values were calculated using a two-sided Mann-Whitney U test. (c) . (d) Quantification of pIRF7/tIRF7 normalized to β-actin (fold change with respect to DMSO treated controls) in Ba/F3-ITD cells treated as described. The experiment was repeated four times and the results (mean ± s.e.m.) were pooled, n =5 biologically independent samples per group. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated using a two-sided Mann-Whitney U test. (e) A representative Western blot showing the expression of pIRF7, tIRF7 and loading control (β-actin) in MV4-11 cells treated with the indicated sorafenib concentrations for 24 hours. (f) Fold change of IL-15 (MFI) in Ba/F3-ITD cells or Ba/F3-ITD cells transfected with a <t>lentiviral</t> vector overexpressing mouse ATF4 and when indicated treated with sorafenib (0.1 μM) for 24 hours. n =9 biologically independent cell culture samples. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated by using a two-sided Mann-Whitney U test. (g) Percentage survival of BALB/c recipients receiving C57BL/6 BM and ATF4-overexpressing or ATF4-wildtype Ba/F3-ITD cells (500 cells) with additional C57BL/6 Tc (Tc, 2×10 5 cells, given on day 2) and treated with vehicle or sorafenib. The experiment was performed twice and the results were pooled; n= 10, biologically independent animals per group. P -values were calculated using the two-sided Mantel-Cox test. (h) Quantification of IL-15 mRNA by qPCR in MOLM-13 (human FLT3-ITD + AML cell line) cells containing a non-silencing vector (MOLM-13 NS shRNA ) or an IRF7 knockdown vector (MOLM-13 IRF7 shRNA ). The MOLM-13 cells were exposed to the indicated concentrations of sorafenib. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n =6 biologically independent samples per group. The P -values were calculated using a two-sided Mann-Whitney U test. (i) Percentage survival of Rag2 −/− Il2rγ −/− recipient mice receiving MOLM-13 NS shRNA and MOLM-13 IRF7 shRNA as indicated. The experiment was performed twice and the results were pooled, n =12 biologically independent animals per group. P- values were calculated using a two-sided Mantel-Cox test. (j) Proposed mechanism as to how sorafenib leads to increased IL-15 transcription. Sorafenib inhibits FLT3 receptor tyrosine kinase signaling which normally leads to ATF4 production. Reduced ATF4 levels lead to less inhibition of IRF7 phosphorylation and activation. Active pIRF7 can translocate to the nucleus where it activates IL-15 transcription.
    Trans Lentiviral Packaging System, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 96/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene lentiviral packaging mix
    Sorafenib induces phosphorylation of IRF7 by reducing its inhibitor ATF4 (a, b) Quantification of ATF4 (western blot) normalized to β-actin (fold change with respect to DMSO treated controls) in mouse Ba/F3-ITD (a) or human FLT3-ITD mutant MV4-11 (b) leukemia cells exposed to sorafenib as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled; a: n =6 biologically independent samples per group, b: n =4 biologically independent samples per group. The P- values were calculated using a two-sided Mann-Whitney U test. (c) . (d) Quantification of pIRF7/tIRF7 normalized to β-actin (fold change with respect to DMSO treated controls) in Ba/F3-ITD cells treated as described. The experiment was repeated four times and the results (mean ± s.e.m.) were pooled, n =5 biologically independent samples per group. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated using a two-sided Mann-Whitney U test. (e) A representative Western blot showing the expression of pIRF7, tIRF7 and loading control (β-actin) in MV4-11 cells treated with the indicated sorafenib concentrations for 24 hours. (f) Fold change of IL-15 (MFI) in Ba/F3-ITD cells or Ba/F3-ITD cells transfected with a <t>lentiviral</t> vector overexpressing mouse ATF4 and when indicated treated with sorafenib (0.1 μM) for 24 hours. n =9 biologically independent cell culture samples. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated by using a two-sided Mann-Whitney U test. (g) Percentage survival of BALB/c recipients receiving C57BL/6 BM and ATF4-overexpressing or ATF4-wildtype Ba/F3-ITD cells (500 cells) with additional C57BL/6 Tc (Tc, 2×10 5 cells, given on day 2) and treated with vehicle or sorafenib. The experiment was performed twice and the results were pooled; n= 10, biologically independent animals per group. P -values were calculated using the two-sided Mantel-Cox test. (h) Quantification of IL-15 mRNA by qPCR in MOLM-13 (human FLT3-ITD + AML cell line) cells containing a non-silencing vector (MOLM-13 NS shRNA ) or an IRF7 knockdown vector (MOLM-13 IRF7 shRNA ). The MOLM-13 cells were exposed to the indicated concentrations of sorafenib. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n =6 biologically independent samples per group. The P -values were calculated using a two-sided Mann-Whitney U test. (i) Percentage survival of Rag2 −/− Il2rγ −/− recipient mice receiving MOLM-13 NS shRNA and MOLM-13 IRF7 shRNA as indicated. The experiment was performed twice and the results were pooled, n =12 biologically independent animals per group. P- values were calculated using a two-sided Mantel-Cox test. (j) Proposed mechanism as to how sorafenib leads to increased IL-15 transcription. Sorafenib inhibits FLT3 receptor tyrosine kinase signaling which normally leads to ATF4 production. Reduced ATF4 levels lead to less inhibition of IRF7 phosphorylation and activation. Active pIRF7 can translocate to the nucleus where it activates IL-15 transcription.
    Lentiviral Packaging Mix, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher lentiviral packaging plasmids
    Effects of NRF-1 on the viability and mRNA expression levels of HIF-1α, NRF-1 and NRF-2 of H9C2 cells. CoCl 2 -treated H9C2 cells were cultured in DMEM plus 10% FBS containing 200 or 400 µM CoCl 2 for 6 or 24 h. (A) GFP expression in <t>lentiviral</t> vector-transfected H9C2 cells, as observed by fluorescence microscopy (left); western blot analysis of NRF-1 expression levels in pLenti-H9C2 and NRF1-H9C2 cells (right). (B) CCK-8 assays were used to detect cell viability following treatment of pLenti-H9C2 and NRF1-H9C2 cells with 200 or 400 µM CoCl 2 for 6 or 24 h. (C) Reverse transcription-quantitative polymerase chain reaction was used to analyze HIF-1α, NRF-1 and NRF-2 mRNA expression levels in pLenti-H9C2 and NRF1-H9C2 cells. **P
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    Construction of apoptin-modified MSCs. (A) Lentiviral vector pLV/Final-puro-EF1α-apoptin was identified by PCR. Lane 1/3, negative control; lane 2, amplification product of fragment of apoptin; lane 4, amplification product of fragment of EF1α promoter. (B) Expression of apoptin was confirmed by western blot analysis. Lane 1, negative control; lane 2, MSCs APOPTIN 1; lane 3, MSCs APOPTIN 2. (C) Morphology of MSCs hrGFP and MSCs APOPTIN. (D) Adipogenesis and osteogenesis differentiation of MSCs APOPTIN. Magnification, ×200. MSCs APOPTIN, mesenchymal stem cells expressing apoptin; PCR, polymerase chain reaction; hrGFP, humanized Renilla green fluorescence protein.

    Journal: Molecular Medicine Reports

    Article Title: Apoptin-modified human mesenchymal stem cells inhibit growth of lung carcinoma in nude mice

    doi: 10.3892/mmr.2015.3501

    Figure Lengend Snippet: Construction of apoptin-modified MSCs. (A) Lentiviral vector pLV/Final-puro-EF1α-apoptin was identified by PCR. Lane 1/3, negative control; lane 2, amplification product of fragment of apoptin; lane 4, amplification product of fragment of EF1α promoter. (B) Expression of apoptin was confirmed by western blot analysis. Lane 1, negative control; lane 2, MSCs APOPTIN 1; lane 3, MSCs APOPTIN 2. (C) Morphology of MSCs hrGFP and MSCs APOPTIN. (D) Adipogenesis and osteogenesis differentiation of MSCs APOPTIN. Magnification, ×200. MSCs APOPTIN, mesenchymal stem cells expressing apoptin; PCR, polymerase chain reaction; hrGFP, humanized Renilla green fluorescence protein.

    Article Snippet: Lentivirus construction and transduction of MSCs Lentiviral particles carrying apoptin gene were prepared by transient co-transduction of pLV/Final-puro-EF1α-apoptin (Invitrogen Life Technologies) and a lentiviral packaging mix (Invitrogen Life Technologies) into 293FT cells (Invitrogen Life Technologies) using Lipofectamine 2000 (Invitrogen Life Technologies), according to manufacturer’s instructions.

    Techniques: Modification, Plasmid Preparation, Polymerase Chain Reaction, Negative Control, Amplification, Expressing, Western Blot, Fluorescence

    Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1 G93A mice. Lumbar spinal cords, from 12- and 15-week-old Tg SOD1 G93A , WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, MLKL, and p-MLKL. A , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA from 12-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1 G93A compared to Tg SOD1 WT ( p = 0.0021) and NTg ( p = 0.0076) at 12 weeks. B , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA in spinal cords of 15-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk1 ( p = 0.0009, vs Tg SOD1 WT ; p = 0.0020, vs NTg) and Ripk3 ( p = 0.0115; vs Tg SOD1 WT , p = 0.0067; vs NTg) but not for Mlkl in Tg SOD1 G93A compared to Tg SOD1 WT and NTg mice at 15 weeks. C , Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1 WT , and Tg SOD1 G93A 15-week-old mice. β-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral shRNA in mouse PMN cultures (mPMNs). D , Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1 G93A samples compared to Tg SOD1 WT ( p = 0.0279) and NTg ( p = 0.0033) mice. Results are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey’s post hoc analysis; n = 3 biological replicates per genotype. E , Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Non-specific band at 47 kDa is designated as an asterisk (*). NTg spleen: positive control tissue, Ripk3 −/− spleen and Tg SOD1 G93A ; Ripk3 −/− spinal cord: negative control tissue. F , Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Ripk3 −/− spinal cord: negative control tissue. G , Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H , Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). NIH 3T3: positive control cell lysate. I , Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.

    Journal: eNeuro

    Article Title: Deletion of Ripk3 Prevents Motor Neuron Death In Vitro but not In Vivo

    doi: 10.1523/ENEURO.0308-18.2018

    Figure Lengend Snippet: Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1 G93A mice. Lumbar spinal cords, from 12- and 15-week-old Tg SOD1 G93A , WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, MLKL, and p-MLKL. A , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA from 12-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1 G93A compared to Tg SOD1 WT ( p = 0.0021) and NTg ( p = 0.0076) at 12 weeks. B , Quantification of Ripk1 , Ripk3 , and Mlkl mRNA in spinal cords of 15-week-old mice . Gapdh : housekeeping gene. A significant increase was detected for Ripk1 ( p = 0.0009, vs Tg SOD1 WT ; p = 0.0020, vs NTg) and Ripk3 ( p = 0.0115; vs Tg SOD1 WT , p = 0.0067; vs NTg) but not for Mlkl in Tg SOD1 G93A compared to Tg SOD1 WT and NTg mice at 15 weeks. C , Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1 WT , and Tg SOD1 G93A 15-week-old mice. β-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral shRNA in mouse PMN cultures (mPMNs). D , Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1 G93A samples compared to Tg SOD1 WT ( p = 0.0279) and NTg ( p = 0.0033) mice. Results are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey’s post hoc analysis; n = 3 biological replicates per genotype. E , Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Non-specific band at 47 kDa is designated as an asterisk (*). NTg spleen: positive control tissue, Ripk3 −/− spleen and Tg SOD1 G93A ; Ripk3 −/− spinal cord: negative control tissue. F , Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Ripk3 −/− spinal cord: negative control tissue. G , Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H , Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). NIH 3T3: positive control cell lysate. I , Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.

    Article Snippet: For mixed-lineage kinase domain-like (Mlkl) silencing, L929 cells were infected with lentivirus-containing shRNA against Mlkl (360819; Sigma Mission) at a multiplicity of infection (MOI) 100.

    Techniques: Mouse Assay, Isolation, Expressing, Western Blot, shRNA, Positive Control, Negative Control

    Pdp2 -shRNA–transfected Th17 cells exacerbate disease activity in an adoptive cell transfer EAE model. ( A and B ) Naïve CD4 + T cells from 2D2 mice were cultured under Th17-polarizing conditions. Pdp2 -shRNA or control shRNA containing lentiviral particles were infected on day 1. On day 3 of culture, harvested those cells were transferred to recipient Rag1 -deficient mice i.v. ( A ) The clinical scores of recipient mice are shown. Cumulative results of 10 mice per group are shown (mean ± SEM). ( B ) Absolute cell numbers of spinal cord-infiltrated CD4 + T cells and IL-17A–producing CD4 + T cells from recipient mice were evaluated by flow cytometry on day 14. Cumulative data are shown (mean ± SEM); n = 8. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Pyruvate dehydrogenase phosphatase catalytic subunit 2 limits Th17 differentiation

    doi: 10.1073/pnas.1805717115

    Figure Lengend Snippet: Pdp2 -shRNA–transfected Th17 cells exacerbate disease activity in an adoptive cell transfer EAE model. ( A and B ) Naïve CD4 + T cells from 2D2 mice were cultured under Th17-polarizing conditions. Pdp2 -shRNA or control shRNA containing lentiviral particles were infected on day 1. On day 3 of culture, harvested those cells were transferred to recipient Rag1 -deficient mice i.v. ( A ) The clinical scores of recipient mice are shown. Cumulative results of 10 mice per group are shown (mean ± SEM). ( B ) Absolute cell numbers of spinal cord-infiltrated CD4 + T cells and IL-17A–producing CD4 + T cells from recipient mice were evaluated by flow cytometry on day 14. Cumulative data are shown (mean ± SEM); n = 8. * P

    Article Snippet: On day 1 of culture, Pdp2 -shRNA or control shRNA-containing lentiviral particles were added to media with polybrene infection/transfection reagent (Sigma-Aldrich).

    Techniques: shRNA, Transfection, Activity Assay, Mouse Assay, Cell Culture, Infection, Flow Cytometry, Cytometry

    PDP2 decreases Th17 differentiation by limiting the glycolysis and lactate production in in vitro Th17-polarized T cells. ( A and B ) Naïve CD4 + T cells were cultured under Th17-polarizing conditions. Empty vector (Empty) or PDP2 plasmids were transfected into cultured T cells on day 1. ( A ) Glycolysis and glycolytic capacity were assessed by measuring ECAR by extracellular flux analyzer on day 3. Cumulative data of ECAR were shown (mean ± SEM); n = 3. ( B ) Percentage of IL-17A–positive cells was measured by flow cytometry. Cumulative data were shown (mean ± SEM); n = 5. ( C and D ) Naïve CD4 + T cells were cultured under Th17-polarizing conditions. Two types of Pdp2 -shRNA (shRNA1 and shRNA2) or control shRNA (Control) containing lentiviral particles were infected on day 1. ( C ) ECAR was measured by extracellular flux analyzer on day 3. Cumulative data of ECAR are shown (mean ± SEM); n = 3. ( D ) Percentage of IL-17A–positive cells was estimated by flow cytometry. Cumulative data are shown (mean ± SEM); n . * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Pyruvate dehydrogenase phosphatase catalytic subunit 2 limits Th17 differentiation

    doi: 10.1073/pnas.1805717115

    Figure Lengend Snippet: PDP2 decreases Th17 differentiation by limiting the glycolysis and lactate production in in vitro Th17-polarized T cells. ( A and B ) Naïve CD4 + T cells were cultured under Th17-polarizing conditions. Empty vector (Empty) or PDP2 plasmids were transfected into cultured T cells on day 1. ( A ) Glycolysis and glycolytic capacity were assessed by measuring ECAR by extracellular flux analyzer on day 3. Cumulative data of ECAR were shown (mean ± SEM); n = 3. ( B ) Percentage of IL-17A–positive cells was measured by flow cytometry. Cumulative data were shown (mean ± SEM); n = 5. ( C and D ) Naïve CD4 + T cells were cultured under Th17-polarizing conditions. Two types of Pdp2 -shRNA (shRNA1 and shRNA2) or control shRNA (Control) containing lentiviral particles were infected on day 1. ( C ) ECAR was measured by extracellular flux analyzer on day 3. Cumulative data of ECAR are shown (mean ± SEM); n = 3. ( D ) Percentage of IL-17A–positive cells was estimated by flow cytometry. Cumulative data are shown (mean ± SEM); n . * P

    Article Snippet: On day 1 of culture, Pdp2 -shRNA or control shRNA-containing lentiviral particles were added to media with polybrene infection/transfection reagent (Sigma-Aldrich).

    Techniques: In Vitro, Cell Culture, Plasmid Preparation, Transfection, Flow Cytometry, Cytometry, shRNA, Infection

    Increasing A3G levels in A3.01 cells induces a non-permissive phenotype whereas silencing of A3G produces a fully permissive phenotype. (A) A3.01 cells were transduced with lentiviral vectors encoding untagged wild type A3G (lane 4) or a deaminase-defective mutant (A3G C288S, C291A) (lane 5). Cells transduced with empty vector were analyzed in parallel (lane 3). Untreated H9 cells (lane 1) and A3.01 cells (lane 2) were analyzed in parallel. After transduction, cells were cultured for 48 h prior to puromycin selection. Puromycin-resistant A3.01 cells were amplified and A3G expression was determined by immunoblotting using untreated parental A3.01 cells and endogenous tubulin (tub) as reference. Protein gels were quantified by densitometry. (B) Transduced A3.01 cells were infected with wild type and Vif-null virus and replication profiles were determined by measuring supernatant reverse transcriptase levels over a 15-day period. Data are presented as mean +/− SD calculated from triplicate infections. (C) A3G was knocked down by transducing A3.01 cells with A3G shRNA encoding lentiviral vectors (lane 3). As a control, cells were treated with a vector encoding a non-silencing shRNA (lane 2). Puromycin-resistant cells were selected as described for panel A. Successful knockdown of A3G was verified by immunoblotting using an A3G specific polyclonal antibody. Parental A3.01 cells (untreated) served as specificity control (lane 1). The blot was reprobed with antibodies to α-tubulin to control for sample loading. (D) Puromycin-resistant knock-down cells and control cells were infected with wild type or NL4-3 Vif-null and replication profiles were determined as in panel B. Data are presented as mean +/− SD calculated from triplicate infections.

    Journal: Virology

    Article Title: Long-term passage of Vif-null HIV-1 in CD4+ T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

    doi: 10.1016/j.virol.2017.01.016

    Figure Lengend Snippet: Increasing A3G levels in A3.01 cells induces a non-permissive phenotype whereas silencing of A3G produces a fully permissive phenotype. (A) A3.01 cells were transduced with lentiviral vectors encoding untagged wild type A3G (lane 4) or a deaminase-defective mutant (A3G C288S, C291A) (lane 5). Cells transduced with empty vector were analyzed in parallel (lane 3). Untreated H9 cells (lane 1) and A3.01 cells (lane 2) were analyzed in parallel. After transduction, cells were cultured for 48 h prior to puromycin selection. Puromycin-resistant A3.01 cells were amplified and A3G expression was determined by immunoblotting using untreated parental A3.01 cells and endogenous tubulin (tub) as reference. Protein gels were quantified by densitometry. (B) Transduced A3.01 cells were infected with wild type and Vif-null virus and replication profiles were determined by measuring supernatant reverse transcriptase levels over a 15-day period. Data are presented as mean +/− SD calculated from triplicate infections. (C) A3G was knocked down by transducing A3.01 cells with A3G shRNA encoding lentiviral vectors (lane 3). As a control, cells were treated with a vector encoding a non-silencing shRNA (lane 2). Puromycin-resistant cells were selected as described for panel A. Successful knockdown of A3G was verified by immunoblotting using an A3G specific polyclonal antibody. Parental A3.01 cells (untreated) served as specificity control (lane 1). The blot was reprobed with antibodies to α-tubulin to control for sample loading. (D) Puromycin-resistant knock-down cells and control cells were infected with wild type or NL4-3 Vif-null and replication profiles were determined as in panel B. Data are presented as mean +/− SD calculated from triplicate infections.

    Article Snippet: For production of A3G shRNA-containing lentiviral particles, 1 μg of A3G shRNA plasmid was mixed with 10 μl of packaging mix (Sigma-Aldrich, Inc., St. Louis MO; Cat # SHP001) and added to 293TN cells.

    Techniques: Transduction, Mutagenesis, Plasmid Preparation, Cell Culture, Selection, Amplification, Expressing, Infection, shRNA

    Suppression of Tip60 by shRNA knockdown blocks HPV genome amplification and late gene expression upon keratinocyte differentiation. HPV31-positive CIN612 cells were infected with lentiviruses expressing TRC1 scrambled shRNA or Tip60 shRNA and incubated

    Journal: Journal of Virology

    Article Title: The Acetyltransferase Tip60 Is a Critical Regulator of the Differentiation-Dependent Amplification of Human Papillomaviruses

    doi: 10.1128/JVI.03455-14

    Figure Lengend Snippet: Suppression of Tip60 by shRNA knockdown blocks HPV genome amplification and late gene expression upon keratinocyte differentiation. HPV31-positive CIN612 cells were infected with lentiviruses expressing TRC1 scrambled shRNA or Tip60 shRNA and incubated

    Article Snippet: CIN612 cells were incubated with 5 ml fresh medium including concentrated Tip60 shRNA or scrambled shRNA control lentivirus-containing supernatant in the presence of 4 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St. Louis, MO) overnight at 37°C.

    Techniques: shRNA, Amplification, Expressing, Infection, Incubation

    SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1 + normal tissues in the presence of circulating and bone marrow-resident CD19 + ROR1 + Raji tumors. ( A) Map of lentiviral constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p

    Journal: Cancer cell

    Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

    doi: 10.1016/j.ccell.2019.02.003

    Figure Lengend Snippet: SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1 + normal tissues in the presence of circulating and bone marrow-resident CD19 + ROR1 + Raji tumors. ( A) Map of lentiviral constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p

    Article Snippet: Lenti-X cells for lentiviral packaging were purchased from Clontech.

    Techniques: Construct, Mouse Assay, Irradiation

    SKP2 overexpression suppresses apoptosis through p53 in GH3 cells. (A) GH3 cells infected with a lentiviral vector stably expressing the rat SKP2-inducible system. SKP2 was induced by 1 μg/mL doxycycline for 24 hours, and cells were then lysed and collected for immunoblotting analysis. (B, C). Twenty-four hours after inducing SKP2, the cells were treated with 5 μM nutlin-3 for 48 hours, and then lysed and collected either for immunoblotting analysis using the antibodies as indicated (B) or for quantitative real-time PCR analysis (C). (D) Twenty-four hours after inducing SKP2 expression, a reporter containing a synthetic p53-binding site (p53-luc) or reporter containing a mutation on the p53-binding site (p53MUT-luc) was transfected into the GH3 cells, together with a control reporter containing renilla luciferase. Twelve hours after transfection, the cells were treated with different concentrations of nutlin-3, as indicated for 48 hours, and the cells were then lysed and collected for luciferase activity analysis. (E) Twenty-four hours after SKP2 induction, the cells were treated with 10 μM nutlin-3 for 48 hours; apoptosis was determined and is shown by measuring the relative caspase 3/7 activity. SKP2, S-phase kinase associated protein 2; PCR, polymerase chain reaction; Dox, doxycycline. * p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Inhibition of SKP2 Sensitizes Bromocriptine-Induced Apoptosis in Human Prolactinoma Cells

    doi: 10.4143/crt.2016.017

    Figure Lengend Snippet: SKP2 overexpression suppresses apoptosis through p53 in GH3 cells. (A) GH3 cells infected with a lentiviral vector stably expressing the rat SKP2-inducible system. SKP2 was induced by 1 μg/mL doxycycline for 24 hours, and cells were then lysed and collected for immunoblotting analysis. (B, C). Twenty-four hours after inducing SKP2, the cells were treated with 5 μM nutlin-3 for 48 hours, and then lysed and collected either for immunoblotting analysis using the antibodies as indicated (B) or for quantitative real-time PCR analysis (C). (D) Twenty-four hours after inducing SKP2 expression, a reporter containing a synthetic p53-binding site (p53-luc) or reporter containing a mutation on the p53-binding site (p53MUT-luc) was transfected into the GH3 cells, together with a control reporter containing renilla luciferase. Twelve hours after transfection, the cells were treated with different concentrations of nutlin-3, as indicated for 48 hours, and the cells were then lysed and collected for luciferase activity analysis. (E) Twenty-four hours after SKP2 induction, the cells were treated with 10 μM nutlin-3 for 48 hours; apoptosis was determined and is shown by measuring the relative caspase 3/7 activity. SKP2, S-phase kinase associated protein 2; PCR, polymerase chain reaction; Dox, doxycycline. * p

    Article Snippet: A lentivirus was then generated using lentiviral packaging systems (Clontech) and the GH3 cells were infected with the p53 knockdown lentivirus.

    Techniques: Over Expression, Infection, Plasmid Preparation, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Transfection, Luciferase, Activity Assay, Polymerase Chain Reaction

    Effects of shRNA on FMDV-IRES activity. a The shRNA-expressing lentiviral vector targeting the conserved region of FMDV-IRES (Lenti-FMDV-sh) or control vector (pLL3.7 alone) was used to infect HEK293 cells at various MOIs. After 14 days, firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values between cells with and without shRNA (** p

    Journal: Virus Genes

    Article Title: Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes

    doi: 10.1007/s11262-019-01696-6

    Figure Lengend Snippet: Effects of shRNA on FMDV-IRES activity. a The shRNA-expressing lentiviral vector targeting the conserved region of FMDV-IRES (Lenti-FMDV-sh) or control vector (pLL3.7 alone) was used to infect HEK293 cells at various MOIs. After 14 days, firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values between cells with and without shRNA (** p

    Article Snippet: Lentiviral packaging was performed using MISSION Lentiviral Packaging Mix (Sigma-Aldrich, St. Louis, MO, USA), and infection with lentivirus was performed according to the manufacturer’s instructions.

    Techniques: shRNA, Activity Assay, Expressing, Plasmid Preparation, Luciferase

    Chronic GCN5L1 KD attenuated mitochondrial mass and protein levels via autophagic degradation. (A) Representative immunoblot of mitochondrial protein acetylation and the accumulation of autophagy mediators in isolated mitochondria following chronic lentiviral

    Journal: Journal of Cell Science

    Article Title: Restricted mitochondrial protein acetylation initiates mitochondrial autophagy

    doi: 10.1242/jcs.131300

    Figure Lengend Snippet: Chronic GCN5L1 KD attenuated mitochondrial mass and protein levels via autophagic degradation. (A) Representative immunoblot of mitochondrial protein acetylation and the accumulation of autophagy mediators in isolated mitochondria following chronic lentiviral

    Article Snippet: Lentiviral shRNA were generated in HEK293T cells using lentiviral Packaging Mix (Sigma).

    Techniques: Isolation

    Establishment of NRF2 overexpression and knockdown stable cell lines. (A and B) A2780 cells were transfected with NRF2 plasmid or NRF2 shRNA by lentiviral packaging system to generate the stable cell lines. NRF2 and NQO1 protein levels were detected in A2780, A2780-NRF2, A2780cp, and A2780cp-NRF2-shRNA by immunoblot analysis. (C and D) Cell viability assay. A2780-NRF2 and A2780 cells (C) and A2780cp-NRF2-shRNA and A2780cp cells (D) were treated with the indicated doses of cisplatin for 48 h to determine cell viability. (E and F) Analysis of apoptosis. A2780-NRF2 and A2780 cells were treated with 1 μg/ml cisplatin (E) and A2780cp-NRF2-shRNA and A2780cp cells were treated with 10 μg/ml cisplatin (F) for 24 h to determine apoptosis by TUNEL assay. All data are presented as mean ± SD from three independent experiments, n=3. * P

    Journal: Molecular carcinogenesis

    Article Title: ABCF2, an Nrf2 target gene, contributes to cisplatin resistance in ovarian cancer cells

    doi: 10.1002/mc.22615

    Figure Lengend Snippet: Establishment of NRF2 overexpression and knockdown stable cell lines. (A and B) A2780 cells were transfected with NRF2 plasmid or NRF2 shRNA by lentiviral packaging system to generate the stable cell lines. NRF2 and NQO1 protein levels were detected in A2780, A2780-NRF2, A2780cp, and A2780cp-NRF2-shRNA by immunoblot analysis. (C and D) Cell viability assay. A2780-NRF2 and A2780 cells (C) and A2780cp-NRF2-shRNA and A2780cp cells (D) were treated with the indicated doses of cisplatin for 48 h to determine cell viability. (E and F) Analysis of apoptosis. A2780-NRF2 and A2780 cells were treated with 1 μg/ml cisplatin (E) and A2780cp-NRF2-shRNA and A2780cp cells were treated with 10 μg/ml cisplatin (F) for 24 h to determine apoptosis by TUNEL assay. All data are presented as mean ± SD from three independent experiments, n=3. * P

    Article Snippet: Lentiviral particles packaged with either vector, or the vector with NRF2 were produced by cotransfecting HEK293T cells with the appropriate plasmid and the Mission Lentiviral Packaging Mix (Sigma-Aldrich, MO, USA).

    Techniques: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Viability Assay, TUNEL Assay

    Schematic representation of the genome-wide shRNA screening workflow. HeLa CD4++ cells were transduced with a pooled shRNA lentiviral library. Productively transduced cells were selected with puromycin and transduced with a retroviral vector for the expression of HIV-1 Nef and eGFP as a reporter gene. Cells that maintained high CD4 surface levels despite the expression of Nef (Nef(eGFP) + CD4 hi , red box) and the Nef(eGFP) + CD4 low population (green box) were sorted by FACS. The shRNA hairpins present in the cDNA obtained from the two sorted populations were amplified by nested PCR with biotinylated primers. The samples obtained were hybridized on different Affymetrix U133 v2-plus microarrays and the results compared. shRNA sequences enriched in the Nef(eGFP) + CD4 hi cells were selected and further analyzed with pathway analysis tools to obtain a final list of 55 genes to be further validated together with 20 genes selected from literature.

    Journal: Retrovirology

    Article Title: Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

    doi: 10.1186/s12977-014-0118-4

    Figure Lengend Snippet: Schematic representation of the genome-wide shRNA screening workflow. HeLa CD4++ cells were transduced with a pooled shRNA lentiviral library. Productively transduced cells were selected with puromycin and transduced with a retroviral vector for the expression of HIV-1 Nef and eGFP as a reporter gene. Cells that maintained high CD4 surface levels despite the expression of Nef (Nef(eGFP) + CD4 hi , red box) and the Nef(eGFP) + CD4 low population (green box) were sorted by FACS. The shRNA hairpins present in the cDNA obtained from the two sorted populations were amplified by nested PCR with biotinylated primers. The samples obtained were hybridized on different Affymetrix U133 v2-plus microarrays and the results compared. shRNA sequences enriched in the Nef(eGFP) + CD4 hi cells were selected and further analyzed with pathway analysis tools to obtain a final list of 55 genes to be further validated together with 20 genes selected from literature.

    Article Snippet: MISSION® Lentiviral Packaging Mix containing lentiviral packaging plasmid and the VSV envelope plasmid were purchased as a pre-made mix from Sigma Aldrich.

    Techniques: Genome Wide, shRNA, Transduction, Plasmid Preparation, Expressing, FACS, Amplification, Nested PCR

    Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY lentivirus-infected E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

    doi: 10.1073/pnas.1210271109

    Figure Lengend Snippet: Intein splicing kinetics. ( A ) Autoradiogram after SDS/PAGE and anti-HA immunoprecipitation of lysates from GFP-RecA-HY lentivirus-infected E36 cells pretreated with epoxomicin (5 μM, 2 h) subjected to [ 35 S]-Met/Cys pulse label followed by a “cold”

    Article Snippet: Vesicular stomatitis virus G protein (VSV-G) pseudotyped lentivirus particles were generated by cotransfecting the lentiviral vector with the Open Biosystems lentivirus packaging mix into LentiX 293T cells (Clontech).

    Techniques: SDS Page, Immunoprecipitation, Infection

    Inhibitory effect of miR-223 on IGF1R/PI3K/Akt signaling pathway in vitro . (A) The GV259-miR-223 lentiviral vector was packaged and delivered to PC9/ER cells. Transduction efficiency was evaluated based on the fluorescent GFP signal. Fluorescent GFP signal was stably observed 72 h after infection. EV for control vector-infected cells (right panel) and miR-223 for GV259-miR-223-infected cells (left panel) are shown. Magnification, ×100. (B) miR-223 was overexpressed in PC9/ER cells as confirmed by real-time RT-PCR analysis. (C) Downregulation of IGF1R expression in PC9/ER-miR-223 cells was confirmed by real-time RT-PCR analysis. (D) IGF1R/Akt/p70S6K pathway was suppressed in the miR-223-overexpressing cells. Phosphorylation of Akt and p70S6K was significantly inhibited, according to western blot analysis. (E) miR-223 mediated inhibition of IGF1R/PI3K/Akt was reversed by agonist (IGF1) of IGF1R in miR-223 transfected cells. p-IGF1R and p-Atk were increased as shown in western blots. (F) The cytotoxicity and IC 50 values in PC9, PC9/ER, PC9/ER-EV, and PC9/ER-miR-223 cells. The cells were treated with the indicated concentrations of erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC 50 values were determined using the CCK8 assay.

    Journal: International Journal of Oncology

    Article Title: miR-223 reverses the resistance of EGFR-TKIs through IGF1R/PI3K/Akt signaling pathway

    doi: 10.3892/ijo.2016.3401

    Figure Lengend Snippet: Inhibitory effect of miR-223 on IGF1R/PI3K/Akt signaling pathway in vitro . (A) The GV259-miR-223 lentiviral vector was packaged and delivered to PC9/ER cells. Transduction efficiency was evaluated based on the fluorescent GFP signal. Fluorescent GFP signal was stably observed 72 h after infection. EV for control vector-infected cells (right panel) and miR-223 for GV259-miR-223-infected cells (left panel) are shown. Magnification, ×100. (B) miR-223 was overexpressed in PC9/ER cells as confirmed by real-time RT-PCR analysis. (C) Downregulation of IGF1R expression in PC9/ER-miR-223 cells was confirmed by real-time RT-PCR analysis. (D) IGF1R/Akt/p70S6K pathway was suppressed in the miR-223-overexpressing cells. Phosphorylation of Akt and p70S6K was significantly inhibited, according to western blot analysis. (E) miR-223 mediated inhibition of IGF1R/PI3K/Akt was reversed by agonist (IGF1) of IGF1R in miR-223 transfected cells. p-IGF1R and p-Atk were increased as shown in western blots. (F) The cytotoxicity and IC 50 values in PC9, PC9/ER, PC9/ER-EV, and PC9/ER-miR-223 cells. The cells were treated with the indicated concentrations of erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC 50 values were determined using the CCK8 assay.

    Article Snippet: Construction of stable cell lines with overexpressed miR-223 To stably upregulate miR-223 expression in PC9/ER cells or PC9/CD133+ , lentivirus carrying the miR-223 or negative control vector (empty viral vector; EV) was packaged using the lentiviral packaging kit (Shanghai Genechem Co., Ltd., Shanghai, China), according to the manufacturer's instructions.

    Techniques: In Vitro, Plasmid Preparation, Transduction, Stable Transfection, Infection, Quantitative RT-PCR, Expressing, Western Blot, Inhibition, Transfection, CCK-8 Assay

    miR-223 inhibits the IGF1R/PI3K/Akt signaling pathway in CD133 + subpopulation cells. (A) The GV259-miR-223 lentiviral vector was packaged and delivered to PC9/CD133 + cells. Transduction efficiency was evaluated based on fluorescent GFP signal. Fluorescent GFP signal was stably observed 72 h after infection. EV for control vector-infected cells (a) and miR-223 for GV259-miR-223-infected cells (b). Magnification, ×100. (B) Transduction efficiency was evaluated by real-time RT-PCR analysis, and expression of miR-223 in PC9/CD133 + -miR-223 cells was 12.5-fold that in PC9/CD133 + cells. (C) Cytotoxicity and IC 50 values of erlotinib in PC9,PC9/CD133 + , PC9/CD133 + -miR-223, and PC9/CD133 + -EV cells. Cells were treated with indicated concentrations of erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC 50 values in the treated cells were determined using the CCK8 assay. (D) Western blot analysis of the key molecules of IGF1R/PI3K/Akt signaling pathway in the erlotinib-treated (0.01 μM for 72 h) PC9, PC9/CD133 + , PC9/CD133 + -miR-223, and PC9/CD133 + -EV cells.

    Journal: International Journal of Oncology

    Article Title: miR-223 reverses the resistance of EGFR-TKIs through IGF1R/PI3K/Akt signaling pathway

    doi: 10.3892/ijo.2016.3401

    Figure Lengend Snippet: miR-223 inhibits the IGF1R/PI3K/Akt signaling pathway in CD133 + subpopulation cells. (A) The GV259-miR-223 lentiviral vector was packaged and delivered to PC9/CD133 + cells. Transduction efficiency was evaluated based on fluorescent GFP signal. Fluorescent GFP signal was stably observed 72 h after infection. EV for control vector-infected cells (a) and miR-223 for GV259-miR-223-infected cells (b). Magnification, ×100. (B) Transduction efficiency was evaluated by real-time RT-PCR analysis, and expression of miR-223 in PC9/CD133 + -miR-223 cells was 12.5-fold that in PC9/CD133 + cells. (C) Cytotoxicity and IC 50 values of erlotinib in PC9,PC9/CD133 + , PC9/CD133 + -miR-223, and PC9/CD133 + -EV cells. Cells were treated with indicated concentrations of erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC 50 values in the treated cells were determined using the CCK8 assay. (D) Western blot analysis of the key molecules of IGF1R/PI3K/Akt signaling pathway in the erlotinib-treated (0.01 μM for 72 h) PC9, PC9/CD133 + , PC9/CD133 + -miR-223, and PC9/CD133 + -EV cells.

    Article Snippet: Construction of stable cell lines with overexpressed miR-223 To stably upregulate miR-223 expression in PC9/ER cells or PC9/CD133+ , lentivirus carrying the miR-223 or negative control vector (empty viral vector; EV) was packaged using the lentiviral packaging kit (Shanghai Genechem Co., Ltd., Shanghai, China), according to the manufacturer's instructions.

    Techniques: Plasmid Preparation, Transduction, Stable Transfection, Infection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Western Blot

    EZH2 is a direct target of miR-141. ( a ) Putative binding site of miR-141 in EZH2 3′-UTR. ( b ) Luc reporter assays in DU145 cells co-transfected with either wild-type EZH2 3′-UTR or the 3′-UTR with the predicted miR-141-binding site mutated and miR-141 or control oligos. ( c ) qPCR analysis of EZH2 mRNA levels in CD44 + PCa cells. ( d ) qPCR of EZH2 in Du145 cells overexpressing miR-141 using either oligo transfection or lentiviral infection. ( e ) WB of EZH2 and SUZ12 in PC3 and Du145 cells overexpressing miR-141. Relative protein levels were measured by densitometry and normalized to GAPDH levels (indicated below). ( f ) Invasion assay in DU145 cells treated with the EZH2 inhibitor DZNep. Inset, WB showing reduced EZH2 proteins by DZNep. ( g ) Invasion assays in Du145 cells co-transfected with 30 nM NC or miR-141 oligos together with empty vector or pCMV-EZH2 construct. ( h ) qPCR of EZH2 target genes including FBN1 , RAD51C , KIAA0101 and CDKN2A in DU145 cells infected with lenti-Ctrl or lenti-141. ( i ) WB analysis of H3K27me3 in DU145 cells overexpressing miR-141 using either lentiviral vectors or oligo transfection. Total H3 and actin were included as endogenous controls. ( j ) miR-141 exhibits pleiotropic anti-tumour and anti-metastasis effects in PCa by targeting multiple molecules associated with CSCs, cell cycle and proliferation, and cytoskeleton, cell motility/invasion and metastasis. Values represent mean±s.d. of triplicates from three to five independent experiments. * P

    Journal: Nature Communications

    Article Title: MicroRNA-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes

    doi: 10.1038/ncomms14270

    Figure Lengend Snippet: EZH2 is a direct target of miR-141. ( a ) Putative binding site of miR-141 in EZH2 3′-UTR. ( b ) Luc reporter assays in DU145 cells co-transfected with either wild-type EZH2 3′-UTR or the 3′-UTR with the predicted miR-141-binding site mutated and miR-141 or control oligos. ( c ) qPCR analysis of EZH2 mRNA levels in CD44 + PCa cells. ( d ) qPCR of EZH2 in Du145 cells overexpressing miR-141 using either oligo transfection or lentiviral infection. ( e ) WB of EZH2 and SUZ12 in PC3 and Du145 cells overexpressing miR-141. Relative protein levels were measured by densitometry and normalized to GAPDH levels (indicated below). ( f ) Invasion assay in DU145 cells treated with the EZH2 inhibitor DZNep. Inset, WB showing reduced EZH2 proteins by DZNep. ( g ) Invasion assays in Du145 cells co-transfected with 30 nM NC or miR-141 oligos together with empty vector or pCMV-EZH2 construct. ( h ) qPCR of EZH2 target genes including FBN1 , RAD51C , KIAA0101 and CDKN2A in DU145 cells infected with lenti-Ctrl or lenti-141. ( i ) WB analysis of H3K27me3 in DU145 cells overexpressing miR-141 using either lentiviral vectors or oligo transfection. Total H3 and actin were included as endogenous controls. ( j ) miR-141 exhibits pleiotropic anti-tumour and anti-metastasis effects in PCa by targeting multiple molecules associated with CSCs, cell cycle and proliferation, and cytoskeleton, cell motility/invasion and metastasis. Values represent mean±s.d. of triplicates from three to five independent experiments. * P

    Article Snippet: Lentiviral-mediated overexpression of miR-141 Three different lentiviral vectors over-expressing miR-141 were generated in our lab, that is, Lenti-miR-141 (based on Lenti-miR over-expression system from System Biosciences, Mountain View, CA; ), pGIPZ-miR-141 (based on pGIPZ lentiviral backbone from Open Biosystems, pGIPZ-NS as the control; ) and pTRIPZ-141 (based on pTRIPZ-inducible lentiviral backbone from Open Biosystems, pTRIPZ-NS as the control; ). pGIPZ and pTRIPZ lentiviral vectors were produced in HEK293T packaging cells with Trans -Lentiviral packaging system (Open Biosystems).

    Techniques: Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Western Blot, Invasion Assay, Plasmid Preparation, Construct

    Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with lentiviral particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g ×

    Journal: Journal of Alzheimer's disease : JAD

    Article Title: Decreased Level of Olfactory Receptors in Blood Cells Following Traumatic Brain Injury and Potential Association with Tauopathy

    doi: 10.3233/JAD-121894

    Figure Lengend Snippet: Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with lentiviral particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g ×

    Article Snippet: For lentivirus packaging, we transfected Lenti-X 293T cells with either pLVX-OR4M1-IRES-ZsGreen or pLVX-IRES-ZsGreen using Lentiviral Packaging System (Clontech).

    Techniques: Activation Assay, Transduction, Infection

    MGAT5 restoration counteracts the suppressive effects of miR-124 on BC cell proliferation and migration. MDA-MB-231 and MCF-7 cells were transfected with miR-124 inhibitor, sh-MGAT5, sh-NC, and miR-124 inhibitor+sh-MGAT5 lentiviral vectors respectively to culture stably cell lines which followed by puromycin selection. A: Cell viability was measured using CCK-8 assays. B: EDU staining was performed and the percentage of EDU-positive cells were counted. Scale bar 100 µm. C: Representative photographs of the colony formation of MDA-MB-231 and MCF-7 cells, and related quantitative analysis. D: Cell migrations were determined using transwell. Scale bars, 50 µm. E: Cell migrations were determined using wound healing assay and the percentage of migrated was calculated. All data are shown as means ± SD of three separate experiments. * P

    Journal: American Journal of Cancer Research

    Article Title: Decreased miR-124-3p promoted breast cancer proliferation and metastasis by targeting MGAT5

    doi:

    Figure Lengend Snippet: MGAT5 restoration counteracts the suppressive effects of miR-124 on BC cell proliferation and migration. MDA-MB-231 and MCF-7 cells were transfected with miR-124 inhibitor, sh-MGAT5, sh-NC, and miR-124 inhibitor+sh-MGAT5 lentiviral vectors respectively to culture stably cell lines which followed by puromycin selection. A: Cell viability was measured using CCK-8 assays. B: EDU staining was performed and the percentage of EDU-positive cells were counted. Scale bar 100 µm. C: Representative photographs of the colony formation of MDA-MB-231 and MCF-7 cells, and related quantitative analysis. D: Cell migrations were determined using transwell. Scale bars, 50 µm. E: Cell migrations were determined using wound healing assay and the percentage of migrated was calculated. All data are shown as means ± SD of three separate experiments. * P

    Article Snippet: The lentiviral packaging kit was purchased from Open Biosystems (Genomeditech).

    Techniques: Migration, Multiple Displacement Amplification, Transfection, Stable Transfection, Selection, CCK-8 Assay, Staining, Wound Healing Assay

    MGAT5 restoration counteracts the suppressive effects of miR-124 on BC cell proliferation and migration. MDA-MB-231 and MCF-7 cells were transfected with miR-124, MGAT5, NC, and miR-124+MGAT5 lentiviral vectors respectively to culture stably cell lines which followed by puromycin selection. A: Cell viability was measured using CCK-8 assays. B: EDU staining was performed and the percentage of EDU-positive cells were counted. Scale bar 100 µm. C: Representative photographs of the colony formation of MDA-MB-231 and MCF-7 cells, and related quantitative analysis. D: Cell migrations were determined using transwell. Scale bars, 50 µm. E: Cell migrations were determined using wound healing assay and the percentage of migrated was calculated. All data are shown as means ± SD of three separate experiments. * P

    Journal: American Journal of Cancer Research

    Article Title: Decreased miR-124-3p promoted breast cancer proliferation and metastasis by targeting MGAT5

    doi:

    Figure Lengend Snippet: MGAT5 restoration counteracts the suppressive effects of miR-124 on BC cell proliferation and migration. MDA-MB-231 and MCF-7 cells were transfected with miR-124, MGAT5, NC, and miR-124+MGAT5 lentiviral vectors respectively to culture stably cell lines which followed by puromycin selection. A: Cell viability was measured using CCK-8 assays. B: EDU staining was performed and the percentage of EDU-positive cells were counted. Scale bar 100 µm. C: Representative photographs of the colony formation of MDA-MB-231 and MCF-7 cells, and related quantitative analysis. D: Cell migrations were determined using transwell. Scale bars, 50 µm. E: Cell migrations were determined using wound healing assay and the percentage of migrated was calculated. All data are shown as means ± SD of three separate experiments. * P

    Article Snippet: The lentiviral packaging kit was purchased from Open Biosystems (Genomeditech).

    Techniques: Migration, Multiple Displacement Amplification, Transfection, Stable Transfection, Selection, CCK-8 Assay, Staining, Wound Healing Assay

    TMPRSS2-ERG regulates androgen biosynthetic enzyme (ABE) expression in PCa cells. A.) RT-qPCR analysis of ERG in VCaP shScrambled (shScr) and VCaP shERG lentiviral cells. Western blot analysis of ERG in VCaP shScr and shERG cells. MOI = multiplicity of

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: ERG/AKR1C3/AR constitutes a feed-forward loop for AR signaling in prostate cancer cells

    doi: 10.1158/1078-0432.CCR-14-2352

    Figure Lengend Snippet: TMPRSS2-ERG regulates androgen biosynthetic enzyme (ABE) expression in PCa cells. A.) RT-qPCR analysis of ERG in VCaP shScrambled (shScr) and VCaP shERG lentiviral cells. Western blot analysis of ERG in VCaP shScr and shERG cells. MOI = multiplicity of

    Article Snippet: Lentiviral particles were generated in HEK293T cells using a Trans-Lentiviral Packaging Kit (Thermo Fisher Scientific, Waltham, MA, TLP5918).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    shRNA constructs and production of lentiviral particles.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic Regulation of AP-1 Transcriptional Complexes Directs Trophoblast Differentiation

    doi: 10.1128/MCB.00118-15

    Figure Lengend Snippet: shRNA constructs and production of lentiviral particles.

    Article Snippet: Lentiviral packaging vectors (Addgene) were used to produce lentiviral particles.

    Techniques: shRNA, Construct

    Rb-Deficient HMECs are Incapable of G0 Arrest and Fail to Upregulate Elafin (A) 76NE6, 76NF2V, and 76NE7 immortalized HMECs were cultured with GFs (+GFs) or without GFs (−GFs) for 48 hours and examined by immunofluorescence staining for Ki67 expression. Scale bars are 50 μm. (B) Western blot of pRb S807/811, total Rb, p53, and elafin in HMECs cultured with GFs (+GFs) for 24 hours or without GFs (−GFs) for 24 and 48 hours. Actin, loading control. (C) HMECS were cultured in growth factor containing medium (asynchronous; Asy), in the absence of growth factors (arrested in G0), with 10 μM lovastatin (arrested in G1), with 10 μM aphidicolin (arrested in S phase), and with 5 μM nocodazole (arrested in M phase). Western blot of pRb S807/811, total Rb, and elafin expression. Actin, loading control. (D,E) 76NF2V and 76NE6 cells stably infected with pGIPZ lentiviral vectors containing control and two unique, RB-specific shRNAs were cultured with GFs (+GFs) or without GFs (−GFs) for 24 hours. (D) Cell cycle distribution was determined DNA content analysis. (C) Western blot of phosphorylated Rb (S807/811) and elafin in 76NE6, 76NF2V, AND 76NE7

    Journal: Oncogene

    Article Title: The Serine Protease Inhibitor Elafin Maintains Normal Growth Control by Opposing the Mitogenic Effects of Neutrophil Elastase

    doi: 10.1038/onc.2014.284

    Figure Lengend Snippet: Rb-Deficient HMECs are Incapable of G0 Arrest and Fail to Upregulate Elafin (A) 76NE6, 76NF2V, and 76NE7 immortalized HMECs were cultured with GFs (+GFs) or without GFs (−GFs) for 48 hours and examined by immunofluorescence staining for Ki67 expression. Scale bars are 50 μm. (B) Western blot of pRb S807/811, total Rb, p53, and elafin in HMECs cultured with GFs (+GFs) for 24 hours or without GFs (−GFs) for 24 and 48 hours. Actin, loading control. (C) HMECS were cultured in growth factor containing medium (asynchronous; Asy), in the absence of growth factors (arrested in G0), with 10 μM lovastatin (arrested in G1), with 10 μM aphidicolin (arrested in S phase), and with 5 μM nocodazole (arrested in M phase). Western blot of pRb S807/811, total Rb, and elafin expression. Actin, loading control. (D,E) 76NF2V and 76NE6 cells stably infected with pGIPZ lentiviral vectors containing control and two unique, RB-specific shRNAs were cultured with GFs (+GFs) or without GFs (−GFs) for 24 hours. (D) Cell cycle distribution was determined DNA content analysis. (C) Western blot of phosphorylated Rb (S807/811) and elafin in 76NE6, 76NF2V, AND 76NE7

    Article Snippet: Lentiviral packaging was performed in HEK-293T cells using the pCMV deltaR8.2 and pMD2.G vectors produced by the Didier Trono laboratory and made available through the Addgene repository.

    Techniques: Cell Culture, Immunofluorescence, Staining, Expressing, Western Blot, Stable Transfection, Infection

    Overexpression of eIF5A1 upregulates neurofilament expression, promotes neuronal survival and improves functional recovery in vivo. ( A,B ) eIF5A mRNA ( A ) and protein levels ( B ) were measured 28 days after lentiviral injections (described below) and SCT with β-actin as an internal control. ( C ) Expression of control lentiviral constructs in the anterior horn (AH) cells of the spinal cord. ( D ) Levels of neurofilament (NF+) (green) and NeuN + neurons (red) were measured 28 days after injection of lentiviral constructs caudal to SCT. Nuclei were visualized by DAPI (blue). Bar (NeuN + ), 100 μm. Bar (NF+), 30 μm. Quantifications are shown in part panels ( E–G ) BBB scores were used to assay hind limb motor function. The uninjured sham controls rats had BBB scores of 21 ± 0.00. BBB scores of rats overexpressing eIF5A1 were higher than control rats while rats with reduced eIF5A1 had lower BBB scores. S, sham group; C, SCT, control lentiviral construct; F+, SCT, eIF5A1 overexpressing lentiviral construct; F–, SCT, shRNA-eIF5A1 expressing lentiviral construct; Data were presented as mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    doi: 10.1038/srep16911

    Figure Lengend Snippet: Overexpression of eIF5A1 upregulates neurofilament expression, promotes neuronal survival and improves functional recovery in vivo. ( A,B ) eIF5A mRNA ( A ) and protein levels ( B ) were measured 28 days after lentiviral injections (described below) and SCT with β-actin as an internal control. ( C ) Expression of control lentiviral constructs in the anterior horn (AH) cells of the spinal cord. ( D ) Levels of neurofilament (NF+) (green) and NeuN + neurons (red) were measured 28 days after injection of lentiviral constructs caudal to SCT. Nuclei were visualized by DAPI (blue). Bar (NeuN + ), 100 μm. Bar (NF+), 30 μm. Quantifications are shown in part panels ( E–G ) BBB scores were used to assay hind limb motor function. The uninjured sham controls rats had BBB scores of 21 ± 0.00. BBB scores of rats overexpressing eIF5A1 were higher than control rats while rats with reduced eIF5A1 had lower BBB scores. S, sham group; C, SCT, control lentiviral construct; F+, SCT, eIF5A1 overexpressing lentiviral construct; F–, SCT, shRNA-eIF5A1 expressing lentiviral construct; Data were presented as mean ± SEM. *P

    Article Snippet: As described previously , the lentiviral packaging was done according to HIV Expression Packaging Kit User Manual of GeneCopoeia.

    Techniques: Over Expression, Expressing, Functional Assay, In Vivo, Construct, Injection, shRNA

    eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury was identified using a proteomics approach. The restoration of neurological functions after CNS injury is limited. Such limited functional recovery is observed in rodents and primates as well 7 34 . Here, we seek to identify possible mechanisms through which such spontaneous function recovery occurs, and to explore the new potential therapeutic targets for SCI and other CNS injuries. Using a proteomics approach we detected 20 proteins expressed differentially in the spinal cord caudal the epicenter of the injury. Of these proteins, we focus on eIF5A1 and RhoGDIα 41 42 . eIF5A1 is a protein with function in cell proliferation and survival while RhoGDIα is involved in cytoskeleton dynamics and promotes axonal growth. Lentiviral vectors were used to manipulate their expression in vivo and in vitro in order to study their roles in neuroplasticity and functional recovery after SCT. Consequently, we found that eIF5A1 regulates RhoGDIα to promote neuroplasticity and functional recovery. This pathway might play a pivotal role in enhancing the level of functional recovery and in turn improve patients’ quality of life. Finally, Fig. 8 was drawn by one of our co-authors, Dr. Fei-Fei Shang.

    Journal: Scientific Reports

    Article Title: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    doi: 10.1038/srep16911

    Figure Lengend Snippet: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury was identified using a proteomics approach. The restoration of neurological functions after CNS injury is limited. Such limited functional recovery is observed in rodents and primates as well 7 34 . Here, we seek to identify possible mechanisms through which such spontaneous function recovery occurs, and to explore the new potential therapeutic targets for SCI and other CNS injuries. Using a proteomics approach we detected 20 proteins expressed differentially in the spinal cord caudal the epicenter of the injury. Of these proteins, we focus on eIF5A1 and RhoGDIα 41 42 . eIF5A1 is a protein with function in cell proliferation and survival while RhoGDIα is involved in cytoskeleton dynamics and promotes axonal growth. Lentiviral vectors were used to manipulate their expression in vivo and in vitro in order to study their roles in neuroplasticity and functional recovery after SCT. Consequently, we found that eIF5A1 regulates RhoGDIα to promote neuroplasticity and functional recovery. This pathway might play a pivotal role in enhancing the level of functional recovery and in turn improve patients’ quality of life. Finally, Fig. 8 was drawn by one of our co-authors, Dr. Fei-Fei Shang.

    Article Snippet: As described previously , the lentiviral packaging was done according to HIV Expression Packaging Kit User Manual of GeneCopoeia.

    Techniques: Functional Assay, Expressing, In Vivo, In Vitro

    Overexpression of eIF5A1 promotes neuronal survival and axonal growth in vitro . ( A ) Primary neurons were cultured for 2 and 4 days (d) after transduction with lentiviral construct described in Fig. 3 . Bar, 50 μm. ( B ) Expression of control lentiviral construct (green) in cultured primary cord neurons. Numbers of neurons and neurite length from panel A were quantified by Leica LAS AF software. And all groups were normalized to normal (N) group at 2d. ( C ) After transduction of lentiviral construct described in Fig. 3 in cultured primary neurons, mRNA level of 20 proteins identified in Fig. 1 were quantified using qPCR. The RhoGDIα (spot 5) was significantly upregulated in eIF5A1 overexpressed group, while it was significantly downregulated in eIF5A1 reduced group. Albumin (spot 1) was not detected. Data were presented as mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    doi: 10.1038/srep16911

    Figure Lengend Snippet: Overexpression of eIF5A1 promotes neuronal survival and axonal growth in vitro . ( A ) Primary neurons were cultured for 2 and 4 days (d) after transduction with lentiviral construct described in Fig. 3 . Bar, 50 μm. ( B ) Expression of control lentiviral construct (green) in cultured primary cord neurons. Numbers of neurons and neurite length from panel A were quantified by Leica LAS AF software. And all groups were normalized to normal (N) group at 2d. ( C ) After transduction of lentiviral construct described in Fig. 3 in cultured primary neurons, mRNA level of 20 proteins identified in Fig. 1 were quantified using qPCR. The RhoGDIα (spot 5) was significantly upregulated in eIF5A1 overexpressed group, while it was significantly downregulated in eIF5A1 reduced group. Albumin (spot 1) was not detected. Data were presented as mean ± SEM. *P

    Article Snippet: As described previously , the lentiviral packaging was done according to HIV Expression Packaging Kit User Manual of GeneCopoeia.

    Techniques: Over Expression, In Vitro, Cell Culture, Transduction, Construct, Expressing, Software, Real-time Polymerase Chain Reaction

    eIF5A1 upregulates RhoGDIα to promote neuroplasticity caudal to the SCT and functional recovery after SCT. ( A,B ) eIF5A1 and RhoGDIα mRNA ( A ) and protein levels ( B ) were measured 28 days after lentiviral injections (described below) and SCT with β-actin as an internal control. ( C ) Levels of NF (green) and number of NeuN + neurons (red) were measured 28 days after injection of lentiviral constructs caudal to SCT. Nuclei were visualized by DAPI (blue). Bar (NeuN + ), 100 μm. Bar (NF +), 30 μm. Quantifications are shown in part panels ( D–F ) BBB scores were used to assay hind limb motor function. The uninjured sham controls rats had BBB scores of 21 ± 0.00. BBB scores of rats in group F + G- where lower than the control but recovered in the F-G + group. Indeed, BBB scores were statistically indistinguishable to the control group. S, sham group; C, SCT, control lentiviral construct; F + G-, SCT, eIF5A1 overexpressing and shRNA-RhoGDIα expressing lentiviral constructs; F-G + , SCT, shRNA-eIF5A1 expressing and RhoGDIα overexpressing lentiviral constructs. Data were presented as mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    doi: 10.1038/srep16911

    Figure Lengend Snippet: eIF5A1 upregulates RhoGDIα to promote neuroplasticity caudal to the SCT and functional recovery after SCT. ( A,B ) eIF5A1 and RhoGDIα mRNA ( A ) and protein levels ( B ) were measured 28 days after lentiviral injections (described below) and SCT with β-actin as an internal control. ( C ) Levels of NF (green) and number of NeuN + neurons (red) were measured 28 days after injection of lentiviral constructs caudal to SCT. Nuclei were visualized by DAPI (blue). Bar (NeuN + ), 100 μm. Bar (NF +), 30 μm. Quantifications are shown in part panels ( D–F ) BBB scores were used to assay hind limb motor function. The uninjured sham controls rats had BBB scores of 21 ± 0.00. BBB scores of rats in group F + G- where lower than the control but recovered in the F-G + group. Indeed, BBB scores were statistically indistinguishable to the control group. S, sham group; C, SCT, control lentiviral construct; F + G-, SCT, eIF5A1 overexpressing and shRNA-RhoGDIα expressing lentiviral constructs; F-G + , SCT, shRNA-eIF5A1 expressing and RhoGDIα overexpressing lentiviral constructs. Data were presented as mean ± SEM. *P

    Article Snippet: As described previously , the lentiviral packaging was done according to HIV Expression Packaging Kit User Manual of GeneCopoeia.

    Techniques: Functional Assay, Injection, Construct, shRNA, Expressing

    eIF5A1 upregulates RhoGDIα in neurons to promote neuronal survival and axonal growth in vitro. ( A ) Primary neurons were cultured for 2 and 4 d after transduction with lentiviral construct described in Fig. 6 . Bar, 50 μm. ( B ) Numbers of neurons and neurite length from panel A were quantified by Leica LAS AF software. And all groups were normalized to normal (N) group at 2d. Data were presented as mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    doi: 10.1038/srep16911

    Figure Lengend Snippet: eIF5A1 upregulates RhoGDIα in neurons to promote neuronal survival and axonal growth in vitro. ( A ) Primary neurons were cultured for 2 and 4 d after transduction with lentiviral construct described in Fig. 6 . Bar, 50 μm. ( B ) Numbers of neurons and neurite length from panel A were quantified by Leica LAS AF software. And all groups were normalized to normal (N) group at 2d. Data were presented as mean ± SEM. *P

    Article Snippet: As described previously , the lentiviral packaging was done according to HIV Expression Packaging Kit User Manual of GeneCopoeia.

    Techniques: In Vitro, Cell Culture, Transduction, Construct, Software

    In vivo inhibitors do not alter A-type lamin protein levels but do alter their subnuclear localization and assembly state. (A) Confocal images of wild-type U2OS cells and cells that stably expressed a scrambled small interfering (si)RNA (scrambled RNAi), an siRNA for LMNA silencing ( LMNA RNAi, LMNA in B, LMNA kd in C), the empty lentiviral plasmid (mock), lamin A (lamin A OE), lamin C (lamin C OE), the control DARPin E3_5, or the indicated different lamin A-specific DARPins (LaA_x, where x is the DARPin identifier). Cells were immunostained with antibodies against lamin A/C, lamin B1 and emerin. Scale bars: 20 µm. (B) Western blot analysis of the supernatant (SN) and pellet fractions (P) of wild-type and modified U2OS cells shown in A after extraction with a buffer containing 0.2% NP40 and using antibodies against lamin A/C, lamin B1 and lamin B2. Note that the supernatant and pellet were loaded in a 10:1 ratio. (C) Statistical analysis of the ratio of extracted to total lamin A and lamin C in different U2OS cell lines. The average of three individual experiments±s.e.m. is shown. Statistical significance was calculated using a one-way ANOVA test followed by Bonferroni's multiple comparison test, where * P ≤0.05, ** P ≤0.01 and *** P ≤0.001. (D) Western blot analysis of lamin and lamin-binding protein levels in the wild-type and modified U2OS cells shown in A. The levels of exogenous lamin A, lamin C, and of DARPin expression is reflected by levels of bicistronically expressed copGFP, a green fluorescent protein cloned from Pontellina plumata . β-actin was used as loading control.

    Journal: Journal of Cell Science

    Article Title: Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

    doi: 10.1242/jcs.171843

    Figure Lengend Snippet: In vivo inhibitors do not alter A-type lamin protein levels but do alter their subnuclear localization and assembly state. (A) Confocal images of wild-type U2OS cells and cells that stably expressed a scrambled small interfering (si)RNA (scrambled RNAi), an siRNA for LMNA silencing ( LMNA RNAi, LMNA in B, LMNA kd in C), the empty lentiviral plasmid (mock), lamin A (lamin A OE), lamin C (lamin C OE), the control DARPin E3_5, or the indicated different lamin A-specific DARPins (LaA_x, where x is the DARPin identifier). Cells were immunostained with antibodies against lamin A/C, lamin B1 and emerin. Scale bars: 20 µm. (B) Western blot analysis of the supernatant (SN) and pellet fractions (P) of wild-type and modified U2OS cells shown in A after extraction with a buffer containing 0.2% NP40 and using antibodies against lamin A/C, lamin B1 and lamin B2. Note that the supernatant and pellet were loaded in a 10:1 ratio. (C) Statistical analysis of the ratio of extracted to total lamin A and lamin C in different U2OS cell lines. The average of three individual experiments±s.e.m. is shown. Statistical significance was calculated using a one-way ANOVA test followed by Bonferroni's multiple comparison test, where * P ≤0.05, ** P ≤0.01 and *** P ≤0.001. (D) Western blot analysis of lamin and lamin-binding protein levels in the wild-type and modified U2OS cells shown in A. The levels of exogenous lamin A, lamin C, and of DARPin expression is reflected by levels of bicistronically expressed copGFP, a green fluorescent protein cloned from Pontellina plumata . β-actin was used as loading control.

    Article Snippet: U2OS cells, HDFs and HeLa-K cells were modified through lentiviral infection [in the case of HeLa-K, cells were modified for stable knockdown of lamin A/C (Sigma mission, clone number .1-752s1c1) and for the respective scrambled control] using HEK-293T cells as packaging cells and the packaging vectors dMD2.G and psPAX2 (Addgene), and following the Purefection protocol for Lentiviral packaging (System Biosciences).

    Techniques: In Vivo, Stable Transfection, Plasmid Preparation, Western Blot, Modification, Binding Assay, Expressing, Clone Assay

    HDFs with A-type lamin delocalization display gross nuclear envelope and chromatin alterations. (A) Confocal images of wild-type HDFs, HDFs homozygous for the mutation Y259X, which completely lack A-type lamins, and HDFs that stably expressed the empty lentiviral plasmid (mock), the control DARPin E3_5, or the indicated different lamin-A-specific DARPins. Cells were immunostained with antibodies against lamin A/C, lamin B1, emerin, Lap2β or nuclear pore complexes (NPCs). Lentiviral vectors contain an internal ribosome entry site (IRES) site followed by the coding sequence for copGFP for bicistronic expression. Consequently, the green copGFP signal directly correlates with DARPin expression levels. Arrows indicate nuclear poles in which the nuclear envelope composition appears to be grossly altered. Scale bar: 20 µm. (B) Statistical analysis of the percentage of nuclei displaying disrupted nuclear envelope organization at one (light grey for E3_5 control, green or turquoise for DARPins that do or do not cause A-type lamin delocalization, respectively, bottom of columns) or both poles (dark grey, and green or turquoise, top of columns), as determined by staining for lamin B1, and at different passages (P) after viral transduction. At least 300 cells were analyzed per data point. (C) Statistical analysis of the percentage of nuclei displaying disrupted nuclear envelope organization (as determined after staining for lamin B1) among cells in which A-type lamins delocalized to the nuclear interior (as determined by staining for lamin A/C) at passages 1 and 10. NA, not applicable.

    Journal: Journal of Cell Science

    Article Title: Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

    doi: 10.1242/jcs.171843

    Figure Lengend Snippet: HDFs with A-type lamin delocalization display gross nuclear envelope and chromatin alterations. (A) Confocal images of wild-type HDFs, HDFs homozygous for the mutation Y259X, which completely lack A-type lamins, and HDFs that stably expressed the empty lentiviral plasmid (mock), the control DARPin E3_5, or the indicated different lamin-A-specific DARPins. Cells were immunostained with antibodies against lamin A/C, lamin B1, emerin, Lap2β or nuclear pore complexes (NPCs). Lentiviral vectors contain an internal ribosome entry site (IRES) site followed by the coding sequence for copGFP for bicistronic expression. Consequently, the green copGFP signal directly correlates with DARPin expression levels. Arrows indicate nuclear poles in which the nuclear envelope composition appears to be grossly altered. Scale bar: 20 µm. (B) Statistical analysis of the percentage of nuclei displaying disrupted nuclear envelope organization at one (light grey for E3_5 control, green or turquoise for DARPins that do or do not cause A-type lamin delocalization, respectively, bottom of columns) or both poles (dark grey, and green or turquoise, top of columns), as determined by staining for lamin B1, and at different passages (P) after viral transduction. At least 300 cells were analyzed per data point. (C) Statistical analysis of the percentage of nuclei displaying disrupted nuclear envelope organization (as determined after staining for lamin B1) among cells in which A-type lamins delocalized to the nuclear interior (as determined by staining for lamin A/C) at passages 1 and 10. NA, not applicable.

    Article Snippet: U2OS cells, HDFs and HeLa-K cells were modified through lentiviral infection [in the case of HeLa-K, cells were modified for stable knockdown of lamin A/C (Sigma mission, clone number .1-752s1c1) and for the respective scrambled control] using HEK-293T cells as packaging cells and the packaging vectors dMD2.G and psPAX2 (Addgene), and following the Purefection protocol for Lentiviral packaging (System Biosciences).

    Techniques: Mutagenesis, Stable Transfection, Plasmid Preparation, Sequencing, Expressing, Staining, Transduction

    miR-221 Down-Regulates transcripts involved in apoptosis, including Pro-Apoptotic protein BIM, and protects DJ-1 knock-down cells from cell death. (A) Compared to cells transfected with negative control pre-miR (pre-miR-NC), SH-SY5Y cells transfected with pre-miR-221 showed a decrease of mRNA transcripts implicated in apoptosis, including BIM, BMF, BNIP3L, and FOXO3A. (B) All three isoforms of BIM protein (BIM EL , BIM L , BIM S ) were decreased by over-expression of miR-221 and (C) increased by the inhibition of miR-221 using anti-miR-221. (D) Pre-miR-221 transfected cells were treated with 250 uM H 2 O 2 for 24 h. Compared to control, over-expression of miR-221 prevented the oxidative stress mediated induction of pro-apoptotic BIM transcript. (E) Cells were made to stably express non-targeting control (Ctrl) short hairpin RNA (shRNA) or pooled DJ-1 shRNA. Cells were then transduced with either lentiviral control miR (Ctrl miR) or miR-221 for 24 h. Stable DJ-1 KD decreases the levels of mature miR-221, while transduction with lentiviral miR-221 robustly increases its levels. (F) Cells were then treated with the indicated concentrations of MPP + for 24 h, and cell death was assessed using LDH assay. Compared to control KD cells, stable DJ-1 KD leads to increased cell death, which is rescued when miR-221 is over-expressed. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (A, D, F) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test. (B, C) analyzed using two-tailed student's t -test. (E) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

    Journal: Redox Biology

    Article Title: The Parkinson's disease gene product DJ-1 modulates miR-221 to promote neuronal survival against oxidative stress

    doi: 10.1016/j.redox.2018.07.021

    Figure Lengend Snippet: miR-221 Down-Regulates transcripts involved in apoptosis, including Pro-Apoptotic protein BIM, and protects DJ-1 knock-down cells from cell death. (A) Compared to cells transfected with negative control pre-miR (pre-miR-NC), SH-SY5Y cells transfected with pre-miR-221 showed a decrease of mRNA transcripts implicated in apoptosis, including BIM, BMF, BNIP3L, and FOXO3A. (B) All three isoforms of BIM protein (BIM EL , BIM L , BIM S ) were decreased by over-expression of miR-221 and (C) increased by the inhibition of miR-221 using anti-miR-221. (D) Pre-miR-221 transfected cells were treated with 250 uM H 2 O 2 for 24 h. Compared to control, over-expression of miR-221 prevented the oxidative stress mediated induction of pro-apoptotic BIM transcript. (E) Cells were made to stably express non-targeting control (Ctrl) short hairpin RNA (shRNA) or pooled DJ-1 shRNA. Cells were then transduced with either lentiviral control miR (Ctrl miR) or miR-221 for 24 h. Stable DJ-1 KD decreases the levels of mature miR-221, while transduction with lentiviral miR-221 robustly increases its levels. (F) Cells were then treated with the indicated concentrations of MPP + for 24 h, and cell death was assessed using LDH assay. Compared to control KD cells, stable DJ-1 KD leads to increased cell death, which is rescued when miR-221 is over-expressed. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (A, D, F) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test. (B, C) analyzed using two-tailed student's t -test. (E) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

    Article Snippet: 4.7 Lentiviral production and transduction DJ-1 short hairpin RNA (shRNA) and control non-silencing shRNA expressing plasmids (pGIPZ) and corresponding lentiviral packaging systems were purchased from GE Healthcare/Dharmacon.

    Techniques: Transfection, Negative Control, Over Expression, Inhibition, Stable Transfection, shRNA, Transduction, Lactate Dehydrogenase Assay, Two Tailed Test

    PKM2 overexpression increased resistance of EC109 to shikonin. EC109/PKM2 stable cell was established by transducing Lentivirus plasmid according to previous methods. RT-PCR(A) and Western blot(B) showed that the expression of PKM2 was higher in EC109/PKM2 cell than EC109 cell. C. Cell viability was detected by CCK-8 assay. The results showed cell viability and resistance of EC109/PKM2 cells to shikonin increased evidently compared to EC109 cells. D. Shikonin decreased the expression of PKM2 and cyclinD1 in EC109 and Ec109/pkm2 cells. *p

    Journal: Journal of Cancer

    Article Title: Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo

    doi: 10.7150/jca.21224

    Figure Lengend Snippet: PKM2 overexpression increased resistance of EC109 to shikonin. EC109/PKM2 stable cell was established by transducing Lentivirus plasmid according to previous methods. RT-PCR(A) and Western blot(B) showed that the expression of PKM2 was higher in EC109/PKM2 cell than EC109 cell. C. Cell viability was detected by CCK-8 assay. The results showed cell viability and resistance of EC109/PKM2 cells to shikonin increased evidently compared to EC109 cells. D. Shikonin decreased the expression of PKM2 and cyclinD1 in EC109 and Ec109/pkm2 cells. *p

    Article Snippet: The plasmid pLV-201-CMV-EGFP/PKM2 constructed and Lentivirus packaging (p-LV201-200, pLV201-050) were performed by GeneCopoeia company (Guang Zhou, China).

    Techniques: Over Expression, Stable Transfection, Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Expressing, CCK-8 Assay

    Silencing of Nrf2 decreases usnic acid-induced γ-GCSc upregulation. HepG2 cells stably infected with lentivirus carrying doxycycline-inducible sh-Nrf2 or SC (scramble control). The two established cell lines were incubated with 1 μg/ml doxycycline for 72 h followed by continued culture for another 24 h without doxycycline; then, the efficiency of Nrf2 knockdown was determined by real-time PCR ( a ) and Western blotting ( b , c ). Bar graphs are mean ± SD of three individual experiments. *p

    Journal: Archives of toxicology

    Article Title: Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells

    doi: 10.1007/s00204-016-1775-y

    Figure Lengend Snippet: Silencing of Nrf2 decreases usnic acid-induced γ-GCSc upregulation. HepG2 cells stably infected with lentivirus carrying doxycycline-inducible sh-Nrf2 or SC (scramble control). The two established cell lines were incubated with 1 μg/ml doxycycline for 72 h followed by continued culture for another 24 h without doxycycline; then, the efficiency of Nrf2 knockdown was determined by real-time PCR ( a ) and Western blotting ( b , c ). Bar graphs are mean ± SD of three individual experiments. *p

    Article Snippet: The 293T cell line used for lentivirus packaging was obtained from Biosettia (San Diego, CA) and maintained in DMEM supplemented with 10 % FBS, 1 mM sodium pyruvate and 0.1 mM nonessential amino acids.).

    Techniques: Stable Transfection, Infection, Incubation, Real-time Polymerase Chain Reaction, Western Blot

    Sorafenib induces phosphorylation of IRF7 by reducing its inhibitor ATF4 (a, b) Quantification of ATF4 (western blot) normalized to β-actin (fold change with respect to DMSO treated controls) in mouse Ba/F3-ITD (a) or human FLT3-ITD mutant MV4-11 (b) leukemia cells exposed to sorafenib as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled; a: n =6 biologically independent samples per group, b: n =4 biologically independent samples per group. The P- values were calculated using a two-sided Mann-Whitney U test. (c) . (d) Quantification of pIRF7/tIRF7 normalized to β-actin (fold change with respect to DMSO treated controls) in Ba/F3-ITD cells treated as described. The experiment was repeated four times and the results (mean ± s.e.m.) were pooled, n =5 biologically independent samples per group. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated using a two-sided Mann-Whitney U test. (e) A representative Western blot showing the expression of pIRF7, tIRF7 and loading control (β-actin) in MV4-11 cells treated with the indicated sorafenib concentrations for 24 hours. (f) Fold change of IL-15 (MFI) in Ba/F3-ITD cells or Ba/F3-ITD cells transfected with a lentiviral vector overexpressing mouse ATF4 and when indicated treated with sorafenib (0.1 μM) for 24 hours. n =9 biologically independent cell culture samples. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated by using a two-sided Mann-Whitney U test. (g) Percentage survival of BALB/c recipients receiving C57BL/6 BM and ATF4-overexpressing or ATF4-wildtype Ba/F3-ITD cells (500 cells) with additional C57BL/6 Tc (Tc, 2×10 5 cells, given on day 2) and treated with vehicle or sorafenib. The experiment was performed twice and the results were pooled; n= 10, biologically independent animals per group. P -values were calculated using the two-sided Mantel-Cox test. (h) Quantification of IL-15 mRNA by qPCR in MOLM-13 (human FLT3-ITD + AML cell line) cells containing a non-silencing vector (MOLM-13 NS shRNA ) or an IRF7 knockdown vector (MOLM-13 IRF7 shRNA ). The MOLM-13 cells were exposed to the indicated concentrations of sorafenib. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n =6 biologically independent samples per group. The P -values were calculated using a two-sided Mann-Whitney U test. (i) Percentage survival of Rag2 −/− Il2rγ −/− recipient mice receiving MOLM-13 NS shRNA and MOLM-13 IRF7 shRNA as indicated. The experiment was performed twice and the results were pooled, n =12 biologically independent animals per group. P- values were calculated using a two-sided Mantel-Cox test. (j) Proposed mechanism as to how sorafenib leads to increased IL-15 transcription. Sorafenib inhibits FLT3 receptor tyrosine kinase signaling which normally leads to ATF4 production. Reduced ATF4 levels lead to less inhibition of IRF7 phosphorylation and activation. Active pIRF7 can translocate to the nucleus where it activates IL-15 transcription.

    Journal: Nature medicine

    Article Title: Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD mutant leukemia cells

    doi: 10.1038/nm.4484

    Figure Lengend Snippet: Sorafenib induces phosphorylation of IRF7 by reducing its inhibitor ATF4 (a, b) Quantification of ATF4 (western blot) normalized to β-actin (fold change with respect to DMSO treated controls) in mouse Ba/F3-ITD (a) or human FLT3-ITD mutant MV4-11 (b) leukemia cells exposed to sorafenib as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled; a: n =6 biologically independent samples per group, b: n =4 biologically independent samples per group. The P- values were calculated using a two-sided Mann-Whitney U test. (c) . (d) Quantification of pIRF7/tIRF7 normalized to β-actin (fold change with respect to DMSO treated controls) in Ba/F3-ITD cells treated as described. The experiment was repeated four times and the results (mean ± s.e.m.) were pooled, n =5 biologically independent samples per group. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated using a two-sided Mann-Whitney U test. (e) A representative Western blot showing the expression of pIRF7, tIRF7 and loading control (β-actin) in MV4-11 cells treated with the indicated sorafenib concentrations for 24 hours. (f) Fold change of IL-15 (MFI) in Ba/F3-ITD cells or Ba/F3-ITD cells transfected with a lentiviral vector overexpressing mouse ATF4 and when indicated treated with sorafenib (0.1 μM) for 24 hours. n =9 biologically independent cell culture samples. Each data point represents an individual sample of one independent cell culture experiment. The P- values were calculated by using a two-sided Mann-Whitney U test. (g) Percentage survival of BALB/c recipients receiving C57BL/6 BM and ATF4-overexpressing or ATF4-wildtype Ba/F3-ITD cells (500 cells) with additional C57BL/6 Tc (Tc, 2×10 5 cells, given on day 2) and treated with vehicle or sorafenib. The experiment was performed twice and the results were pooled; n= 10, biologically independent animals per group. P -values were calculated using the two-sided Mantel-Cox test. (h) Quantification of IL-15 mRNA by qPCR in MOLM-13 (human FLT3-ITD + AML cell line) cells containing a non-silencing vector (MOLM-13 NS shRNA ) or an IRF7 knockdown vector (MOLM-13 IRF7 shRNA ). The MOLM-13 cells were exposed to the indicated concentrations of sorafenib. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n =6 biologically independent samples per group. The P -values were calculated using a two-sided Mann-Whitney U test. (i) Percentage survival of Rag2 −/− Il2rγ −/− recipient mice receiving MOLM-13 NS shRNA and MOLM-13 IRF7 shRNA as indicated. The experiment was performed twice and the results were pooled, n =12 biologically independent animals per group. P- values were calculated using a two-sided Mantel-Cox test. (j) Proposed mechanism as to how sorafenib leads to increased IL-15 transcription. Sorafenib inhibits FLT3 receptor tyrosine kinase signaling which normally leads to ATF4 production. Reduced ATF4 levels lead to less inhibition of IRF7 phosphorylation and activation. Active pIRF7 can translocate to the nucleus where it activates IL-15 transcription.

    Article Snippet: The lentiviral particles were generated by transfection of HEK293T-cells using Polyethylenimine (Polysciences) and the Trans-Lentiviral Packaging System (Dharmacon, Germany).

    Techniques: Western Blot, Mutagenesis, MANN-WHITNEY, Cell Culture, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, shRNA, Mouse Assay, Inhibition, Activation Assay

    Effects of NRF-1 on the viability and mRNA expression levels of HIF-1α, NRF-1 and NRF-2 of H9C2 cells. CoCl 2 -treated H9C2 cells were cultured in DMEM plus 10% FBS containing 200 or 400 µM CoCl 2 for 6 or 24 h. (A) GFP expression in lentiviral vector-transfected H9C2 cells, as observed by fluorescence microscopy (left); western blot analysis of NRF-1 expression levels in pLenti-H9C2 and NRF1-H9C2 cells (right). (B) CCK-8 assays were used to detect cell viability following treatment of pLenti-H9C2 and NRF1-H9C2 cells with 200 or 400 µM CoCl 2 for 6 or 24 h. (C) Reverse transcription-quantitative polymerase chain reaction was used to analyze HIF-1α, NRF-1 and NRF-2 mRNA expression levels in pLenti-H9C2 and NRF1-H9C2 cells. **P

    Journal: Molecular Medicine Reports

    Article Title: Effects of nuclear respiratory factor-1 on apoptosis and mitochondrial dysfunction induced by cobalt chloride in H9C2 cells

    doi: 10.3892/mmr.2019.9839

    Figure Lengend Snippet: Effects of NRF-1 on the viability and mRNA expression levels of HIF-1α, NRF-1 and NRF-2 of H9C2 cells. CoCl 2 -treated H9C2 cells were cultured in DMEM plus 10% FBS containing 200 or 400 µM CoCl 2 for 6 or 24 h. (A) GFP expression in lentiviral vector-transfected H9C2 cells, as observed by fluorescence microscopy (left); western blot analysis of NRF-1 expression levels in pLenti-H9C2 and NRF1-H9C2 cells (right). (B) CCK-8 assays were used to detect cell viability following treatment of pLenti-H9C2 and NRF1-H9C2 cells with 200 or 400 µM CoCl 2 for 6 or 24 h. (C) Reverse transcription-quantitative polymerase chain reaction was used to analyze HIF-1α, NRF-1 and NRF-2 mRNA expression levels in pLenti-H9C2 and NRF1-H9C2 cells. **P

    Article Snippet: Materials The lentiviral expression vector pLenti6.3-NRF1-IRES2-EGFP and lentiviral packaging plasmids (pLP1, pLP2 and pLP/VSVG) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

    Techniques: Expressing, Cell Culture, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Western Blot, CCK-8 Assay, Real-time Polymerase Chain Reaction