control nontargeting sirna Search Results


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  • 99
    Millipore shrna nontarget control
    Transcriptional regulation of <t>SOCS2</t> by p53. a Inverse regulation of SOCS2 in the HCT116 p53(+/+) cells simultaneously treated with doxorubicin and <t>siRNA</t> against p53 (sip53) versus control (siC). b SOCS2 promoter activity in wild-type or mutant p53 HEK293 cells. Values represent the mean ± SD from three independent experiments. * P
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    Thermo Fisher control nontargeting shrna
    Transcriptional regulation of <t>SOCS2</t> by p53. a Inverse regulation of SOCS2 in the HCT116 p53(+/+) cells simultaneously treated with doxorubicin and <t>siRNA</t> against p53 (sip53) versus control (siC). b SOCS2 promoter activity in wild-type or mutant p53 HEK293 cells. Values represent the mean ± SD from three independent experiments. * P
    Control Nontargeting Shrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery control nontargeting shrna
    AMPK promotes Akt dephosphorylation in suspension via PHLPP. A and B, Representative immunoblots of MDA-MB-231 cells treated with DMSO or compound C (CC) and subjected to suspension (Sus) for 8 hours ( n = 5; A ), and cells grown in adherent (Att) or suspension (Sus) condition for 10 minutes and 8 hours ( n = 3; B ). C, Phosphatase assay performed with immunoprecipitated PHLPP2; IgG was used as control; n = 4. Error bars, mean ± SEM. D–G, Representative immunoblots of MDA-MB-231 cells harvested under conditions detailed below. D, Cells stably expressing <t>nontargeting</t> <t>shRNA</t> (NT) or shPHLPP2 (seq #5) and subjected to suspension (Sus) for 8 hours; n = 4. E, Cells treated with DMSO or compound C (CC) were subjected to suspension (Sus) for 8 hours; n = 3. F, Adherent cells stably expressing pTRIPZ empty vector (EV) or shAMPKα2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n = 3. G, Adherent cells pretreated with DMSO or AMPK inhibitor (CC) were treated with cycloheximide (CHX) for 20 minutes, followed by suspension culture (Sus) for indicated time points; n = 3. Graph represents quantification of PHLPP2. All values represent densitometric analyses of Western blots to quantify relative levels of specified proteins.
    Control Nontargeting Shrna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology control nontargeting sirna
    AMPK promotes Akt dephosphorylation in suspension via PHLPP. A and B, Representative immunoblots of MDA-MB-231 cells treated with DMSO or compound C (CC) and subjected to suspension (Sus) for 8 hours ( n = 5; A ), and cells grown in adherent (Att) or suspension (Sus) condition for 10 minutes and 8 hours ( n = 3; B ). C, Phosphatase assay performed with immunoprecipitated PHLPP2; IgG was used as control; n = 4. Error bars, mean ± SEM. D–G, Representative immunoblots of MDA-MB-231 cells harvested under conditions detailed below. D, Cells stably expressing <t>nontargeting</t> <t>shRNA</t> (NT) or shPHLPP2 (seq #5) and subjected to suspension (Sus) for 8 hours; n = 4. E, Cells treated with DMSO or compound C (CC) were subjected to suspension (Sus) for 8 hours; n = 3. F, Adherent cells stably expressing pTRIPZ empty vector (EV) or shAMPKα2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n = 3. G, Adherent cells pretreated with DMSO or AMPK inhibitor (CC) were treated with cycloheximide (CHX) for 20 minutes, followed by suspension culture (Sus) for indicated time points; n = 3. Graph represents quantification of PHLPP2. All values represent densitometric analyses of Western blots to quantify relative levels of specified proteins.
    Control Nontargeting Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery nontargeting control ntc sirna
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Nontargeting Control Ntc Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery control nontargeted sirna
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Control Nontargeted Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery control nontarget sirna
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Control Nontarget Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company control nontargeting shrna shnt
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Control Nontargeting Shrna Shnt, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology nontargeting control ntc
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Nontargeting Control Ntc, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company nontargeting sirna si control
    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with <t>nontargeting</t> control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the <t>WT/NTC/0-h</t> band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.
    Nontargeting Sirna Si Control, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology control nontargeted scramble sirna
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Control Nontargeted Scramble Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery sirna smartpool nontargeting control
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
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    Horizon Discovery control sigenome nontargeting sirna
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Control Sigenome Nontargeting Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nontargeted control shrnas
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Nontargeted Control Shrnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery accell control pool nontargeting sirna
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Accell Control Pool Nontargeting Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sigenome control pool nontargeting sirna
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Sigenome Control Pool Nontargeting Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore control nontargeting sirna
    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using <t>siRNA</t> technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and <t>nontargeted</t> siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p
    Control Nontargeting Sirna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Transcriptional regulation of SOCS2 by p53. a Inverse regulation of SOCS2 in the HCT116 p53(+/+) cells simultaneously treated with doxorubicin and siRNA against p53 (sip53) versus control (siC). b SOCS2 promoter activity in wild-type or mutant p53 HEK293 cells. Values represent the mean ± SD from three independent experiments. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Alterations in the p53-SOCS2 axis contribute to tumor growth in colon cancer

    doi: 10.1038/s12276-017-0001-1

    Figure Lengend Snippet: Transcriptional regulation of SOCS2 by p53. a Inverse regulation of SOCS2 in the HCT116 p53(+/+) cells simultaneously treated with doxorubicin and siRNA against p53 (sip53) versus control (siC). b SOCS2 promoter activity in wild-type or mutant p53 HEK293 cells. Values represent the mean ± SD from three independent experiments. * P

    Article Snippet: A lentivirus vector encoding shRNA targeting SOCS2 and shRNA nontarget control were used to transduce HCT116 (p53−/− ) and HCT116 (p53+/+ ) cells according to the manufacturer’s instructions (Sigma): 1.2 × 105 cells were seeded on 6-well plates overnight, then transduced with lentiviral particles at 10 MOI in the presence of 8 μg/ml hexadimethrine bromide (Sigma).

    Techniques: Activity Assay, Mutagenesis

    Inhibition of tumor growth by SOCS2 knockdown in p53-null cells. a Immunoblots from the extracts of colon cancer cells transduced with lentiviral particle encoding SOCS2 shRNA. b Colony formation after SOCS2 knockdown in HCT116 cells. Values represent the mean ± SD from three independent experiments. ** P

    Journal: Experimental & Molecular Medicine

    Article Title: Alterations in the p53-SOCS2 axis contribute to tumor growth in colon cancer

    doi: 10.1038/s12276-017-0001-1

    Figure Lengend Snippet: Inhibition of tumor growth by SOCS2 knockdown in p53-null cells. a Immunoblots from the extracts of colon cancer cells transduced with lentiviral particle encoding SOCS2 shRNA. b Colony formation after SOCS2 knockdown in HCT116 cells. Values represent the mean ± SD from three independent experiments. ** P

    Article Snippet: A lentivirus vector encoding shRNA targeting SOCS2 and shRNA nontarget control were used to transduce HCT116 (p53−/− ) and HCT116 (p53+/+ ) cells according to the manufacturer’s instructions (Sigma): 1.2 × 105 cells were seeded on 6-well plates overnight, then transduced with lentiviral particles at 10 MOI in the presence of 8 μg/ml hexadimethrine bromide (Sigma).

    Techniques: Inhibition, Western Blot, Transduction, shRNA

    In vitro phenotypic analysis of PAK1‐shRNA#1 mutants derived from different OS cell lines. (A) Representative images and quantification of colony formation assays showing the tumorigenic potential of PAK1‐silenced mutants and scramble controls. (B) Growth curves of PAK1‐shRNA mutants and scramble controls. Five wells per cell were measured; error bars represent standard deviations of the replicates. Experiments were replicated three times. (C) Results of ECM adhesion assays to measure the adhesion abilities of PAK1‐shRNA mutants and scramble controls. The amounts of adhered cells were measured by A 560 nm , and replicates were averaged per cell line for each of the indicated ECM components or the empty plate without precoated ECM component. Error bars represent standard deviations of the replicates, and asterisks denote statistical significance (Student's t ‐test; * P

    Journal: Molecular Oncology

    Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

    doi: 10.1002/1878-0261.12624

    Figure Lengend Snippet: In vitro phenotypic analysis of PAK1‐shRNA#1 mutants derived from different OS cell lines. (A) Representative images and quantification of colony formation assays showing the tumorigenic potential of PAK1‐silenced mutants and scramble controls. (B) Growth curves of PAK1‐shRNA mutants and scramble controls. Five wells per cell were measured; error bars represent standard deviations of the replicates. Experiments were replicated three times. (C) Results of ECM adhesion assays to measure the adhesion abilities of PAK1‐shRNA mutants and scramble controls. The amounts of adhered cells were measured by A 560 nm , and replicates were averaged per cell line for each of the indicated ECM components or the empty plate without precoated ECM component. Error bars represent standard deviations of the replicates, and asterisks denote statistical significance (Student's t ‐test; * P

    Article Snippet: 2.6 shRNA‐mediated silencing of PAK1 expression Two specific MISSION TRC Lentivirus shRNAs for PAK1 and an shRNA scramble nontarget control were used to generate PAK1‐silenced and control mutants in OS cell lines (MISSION® Lentiviral Transduction Particles, shRNA#1 clone ID: TRCN0000195500, shRNA#2 clone ID: TRCN0000195636, and MISSION® pLKO.1‐puro Non‐Mammalian shRNA Control Transduction Particles; Sigma‐Aldrich, St. Louis, MO, USA).

    Techniques: In Vitro, shRNA, Derivative Assay

    In vivo analysis of PAK1 silencing on OS metastasis. (A) Luminescence images of whole mice 4 weeks after tail‐vein injection of 143B PAK1‐shRNA#1 cells or scramble control cells. After an imaging analysis, mouse lungs were harvested and examined histologically to confirm the presence of pulmonary metastases. *One mouse in the scramble group developed micrometastasis in the lungs without detectable luminescence. Occurrence of metastasis was determined based on both luminescence and histopathological examination. The numbers of mice with or without metastases were compared between the shRNA and control groups (Fisher's exact test, P = 0.0286). (B) Luminescence images obtained early postinjection time points (0, 3, and 6 h) from mice injected with either the PAK1‐shRNA#1 or scramble control cells. The 24‐h image represents the luminescence image of the lung tissues harvested 24 h after tumor cell injection to illustrate tumor cell invasion of the lungs. (C) Representative fluorescence images of PAK1‐shRNA#1 and scramble cells at different time points (0, 3, and 6 h) after transduction with RFP‐actin in culture wells (20×). Experiments were replicated three times.

    Journal: Molecular Oncology

    Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

    doi: 10.1002/1878-0261.12624

    Figure Lengend Snippet: In vivo analysis of PAK1 silencing on OS metastasis. (A) Luminescence images of whole mice 4 weeks after tail‐vein injection of 143B PAK1‐shRNA#1 cells or scramble control cells. After an imaging analysis, mouse lungs were harvested and examined histologically to confirm the presence of pulmonary metastases. *One mouse in the scramble group developed micrometastasis in the lungs without detectable luminescence. Occurrence of metastasis was determined based on both luminescence and histopathological examination. The numbers of mice with or without metastases were compared between the shRNA and control groups (Fisher's exact test, P = 0.0286). (B) Luminescence images obtained early postinjection time points (0, 3, and 6 h) from mice injected with either the PAK1‐shRNA#1 or scramble control cells. The 24‐h image represents the luminescence image of the lung tissues harvested 24 h after tumor cell injection to illustrate tumor cell invasion of the lungs. (C) Representative fluorescence images of PAK1‐shRNA#1 and scramble cells at different time points (0, 3, and 6 h) after transduction with RFP‐actin in culture wells (20×). Experiments were replicated three times.

    Article Snippet: 2.6 shRNA‐mediated silencing of PAK1 expression Two specific MISSION TRC Lentivirus shRNAs for PAK1 and an shRNA scramble nontarget control were used to generate PAK1‐silenced and control mutants in OS cell lines (MISSION® Lentiviral Transduction Particles, shRNA#1 clone ID: TRCN0000195500, shRNA#2 clone ID: TRCN0000195636, and MISSION® pLKO.1‐puro Non‐Mammalian shRNA Control Transduction Particles; Sigma‐Aldrich, St. Louis, MO, USA).

    Techniques: In Vivo, Mouse Assay, Injection, shRNA, Imaging, Fluorescence, Transduction

    Migration inhibitory effect of PAK1 silencing on non‐OS cell lines with p27 mislocalization. (A) Western blotting with subcellular fractionation of three non‐OS cancer cell lines. GAPDH and HDAC were used as nuclear and cytoplasmic protein controls, respectively (C, cytoplasmic fraction; N, nuclear fractions). (B) Western blotting of PAK1‐shRNA mutants and parental controls in the three cancer cell lines showing high efficiency of PAK1 knockdown. (C) Representative images and quantification of transwell migration assays showing migrated cells in the three PAK1‐shRNA mutants relative to their parental cells. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). Error bars represent standard deviations of the replicates, and asterisks denote statistical significance (Student's t ‐test; * P

    Journal: Molecular Oncology

    Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

    doi: 10.1002/1878-0261.12624

    Figure Lengend Snippet: Migration inhibitory effect of PAK1 silencing on non‐OS cell lines with p27 mislocalization. (A) Western blotting with subcellular fractionation of three non‐OS cancer cell lines. GAPDH and HDAC were used as nuclear and cytoplasmic protein controls, respectively (C, cytoplasmic fraction; N, nuclear fractions). (B) Western blotting of PAK1‐shRNA mutants and parental controls in the three cancer cell lines showing high efficiency of PAK1 knockdown. (C) Representative images and quantification of transwell migration assays showing migrated cells in the three PAK1‐shRNA mutants relative to their parental cells. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). Error bars represent standard deviations of the replicates, and asterisks denote statistical significance (Student's t ‐test; * P

    Article Snippet: 2.6 shRNA‐mediated silencing of PAK1 expression Two specific MISSION TRC Lentivirus shRNAs for PAK1 and an shRNA scramble nontarget control were used to generate PAK1‐silenced and control mutants in OS cell lines (MISSION® Lentiviral Transduction Particles, shRNA#1 clone ID: TRCN0000195500, shRNA#2 clone ID: TRCN0000195636, and MISSION® pLKO.1‐puro Non‐Mammalian shRNA Control Transduction Particles; Sigma‐Aldrich, St. Louis, MO, USA).

    Techniques: Migration, Western Blot, Fractionation, shRNA, Staining, Software

    Effects of PAK1 silencing on tumor cell migration and actin stress fiber formation in three distinct OS cell lines with p27 mislocalization. (A) Western blots of total PAK1 in PAK1‐shRNA (shRNA#1 and #2) mutants and scramble controls from NES‐p27, 143B, and U2OS cell lines. (B) Representative images of the transwell migration assays of the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines. (C) Quantification of the transwell migration assays shown in B. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). The experiment was replicated three times. (D) Representative images and quantification of phalloidin staining showing the amount and distribution of actin stress fibers in the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines (20×). In the quantification analyses, error bars and asterisks represent standard deviations and statistical significance (Student's t ‐test; * P

    Journal: Molecular Oncology

    Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

    doi: 10.1002/1878-0261.12624

    Figure Lengend Snippet: Effects of PAK1 silencing on tumor cell migration and actin stress fiber formation in three distinct OS cell lines with p27 mislocalization. (A) Western blots of total PAK1 in PAK1‐shRNA (shRNA#1 and #2) mutants and scramble controls from NES‐p27, 143B, and U2OS cell lines. (B) Representative images of the transwell migration assays of the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines. (C) Quantification of the transwell migration assays shown in B. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). The experiment was replicated three times. (D) Representative images and quantification of phalloidin staining showing the amount and distribution of actin stress fibers in the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines (20×). In the quantification analyses, error bars and asterisks represent standard deviations and statistical significance (Student's t ‐test; * P

    Article Snippet: 2.6 shRNA‐mediated silencing of PAK1 expression Two specific MISSION TRC Lentivirus shRNAs for PAK1 and an shRNA scramble nontarget control were used to generate PAK1‐silenced and control mutants in OS cell lines (MISSION® Lentiviral Transduction Particles, shRNA#1 clone ID: TRCN0000195500, shRNA#2 clone ID: TRCN0000195636, and MISSION® pLKO.1‐puro Non‐Mammalian shRNA Control Transduction Particles; Sigma‐Aldrich, St. Louis, MO, USA).

    Techniques: Migration, Western Blot, shRNA, Staining, Software

    Inhibition of IGF1R alleviates collagen-induced arthritis and supports early neurogenesis. ( A ) IGF1R was inhibited with shRNA (shIGF1R, empty symbols) provided intraperitoneally on days 19, 26, and 33 (indicated by arrows). The control group received nontargeting scrambled RNA (shNT) (filled symbols). Inhibition of IGF1R alleviated severity of arthritis and weight loss ( Inset ) in mice. Inhibition of IGF1R ( B ) reduced IRS1 mRNA in spleen, ( C ) increased serum levels of IGF1, and reduced the cell density of IBA1 + cells in DG ( D ) and CA ( E ). ( F ) Change in density of pS 612 IRS1 + cells in DG. ( G and H ) Change in density of GFAP + cells in total DG and in DG sg . ( I ) Reduction in behavioral activity of shIGF1R-treated mice. ( J ) The brain sections show costaining for GFAP and DCX in DG. The dotted line indicates the borders of DG sg . The arrowheads indicate positive cells. ( K ) Different morphology of GFAP + cells in DG sg , distinct from vertical-oriented neuronal stem cells from astrocytes. ( L ) Heatmap shows transcriptional analysis of genes involved in neurogenesis and IGF1R signaling in the hippocampal tissue of shIGF1R-treated and shNT-treated RA mice, done by RT-PCR. Boxplots present median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test, between shIGF1R-treated mice (open boxes) vs. control, shNT (filled boxes). * P

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

    Article Title: Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis

    doi: 10.1073/pnas.1810553115

    Figure Lengend Snippet: Inhibition of IGF1R alleviates collagen-induced arthritis and supports early neurogenesis. ( A ) IGF1R was inhibited with shRNA (shIGF1R, empty symbols) provided intraperitoneally on days 19, 26, and 33 (indicated by arrows). The control group received nontargeting scrambled RNA (shNT) (filled symbols). Inhibition of IGF1R alleviated severity of arthritis and weight loss ( Inset ) in mice. Inhibition of IGF1R ( B ) reduced IRS1 mRNA in spleen, ( C ) increased serum levels of IGF1, and reduced the cell density of IBA1 + cells in DG ( D ) and CA ( E ). ( F ) Change in density of pS 612 IRS1 + cells in DG. ( G and H ) Change in density of GFAP + cells in total DG and in DG sg . ( I ) Reduction in behavioral activity of shIGF1R-treated mice. ( J ) The brain sections show costaining for GFAP and DCX in DG. The dotted line indicates the borders of DG sg . The arrowheads indicate positive cells. ( K ) Different morphology of GFAP + cells in DG sg , distinct from vertical-oriented neuronal stem cells from astrocytes. ( L ) Heatmap shows transcriptional analysis of genes involved in neurogenesis and IGF1R signaling in the hippocampal tissue of shIGF1R-treated and shNT-treated RA mice, done by RT-PCR. Boxplots present median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test, between shIGF1R-treated mice (open boxes) vs. control, shNT (filled boxes). * P

    Article Snippet: Lentiviral particles, MISSION TRCN0000023490 and TRCN0000023493 (Sigma-Aldrich), encoding shRNA targeting IGF1R (shIGF1R), or nontargeting shRNA controls, shNT (SHC002H; Sigma-Aldrich), were injected intraperitoneally (106 to 107 particles in 100 µL per mouse) on days 19, 26, and 33.

    Techniques: Inhibition, shRNA, Mouse Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

    AMPK promotes Akt dephosphorylation in suspension via PHLPP. A and B, Representative immunoblots of MDA-MB-231 cells treated with DMSO or compound C (CC) and subjected to suspension (Sus) for 8 hours ( n = 5; A ), and cells grown in adherent (Att) or suspension (Sus) condition for 10 minutes and 8 hours ( n = 3; B ). C, Phosphatase assay performed with immunoprecipitated PHLPP2; IgG was used as control; n = 4. Error bars, mean ± SEM. D–G, Representative immunoblots of MDA-MB-231 cells harvested under conditions detailed below. D, Cells stably expressing nontargeting shRNA (NT) or shPHLPP2 (seq #5) and subjected to suspension (Sus) for 8 hours; n = 4. E, Cells treated with DMSO or compound C (CC) were subjected to suspension (Sus) for 8 hours; n = 3. F, Adherent cells stably expressing pTRIPZ empty vector (EV) or shAMPKα2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n = 3. G, Adherent cells pretreated with DMSO or AMPK inhibitor (CC) were treated with cycloheximide (CHX) for 20 minutes, followed by suspension culture (Sus) for indicated time points; n = 3. Graph represents quantification of PHLPP2. All values represent densitometric analyses of Western blots to quantify relative levels of specified proteins.

    Journal: Cancer research

    Article Title: AMPK–Akt Double-Negative Feedback Loop in Breast Cancer Cells Regulates Their Adaptation to Matrix Deprivation

    doi: 10.1158/0008-5472.CAN-17-2090

    Figure Lengend Snippet: AMPK promotes Akt dephosphorylation in suspension via PHLPP. A and B, Representative immunoblots of MDA-MB-231 cells treated with DMSO or compound C (CC) and subjected to suspension (Sus) for 8 hours ( n = 5; A ), and cells grown in adherent (Att) or suspension (Sus) condition for 10 minutes and 8 hours ( n = 3; B ). C, Phosphatase assay performed with immunoprecipitated PHLPP2; IgG was used as control; n = 4. Error bars, mean ± SEM. D–G, Representative immunoblots of MDA-MB-231 cells harvested under conditions detailed below. D, Cells stably expressing nontargeting shRNA (NT) or shPHLPP2 (seq #5) and subjected to suspension (Sus) for 8 hours; n = 4. E, Cells treated with DMSO or compound C (CC) were subjected to suspension (Sus) for 8 hours; n = 3. F, Adherent cells stably expressing pTRIPZ empty vector (EV) or shAMPKα2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n = 3. G, Adherent cells pretreated with DMSO or AMPK inhibitor (CC) were treated with cycloheximide (CHX) for 20 minutes, followed by suspension culture (Sus) for indicated time points; n = 3. Graph represents quantification of PHLPP2. All values represent densitometric analyses of Western blots to quantify relative levels of specified proteins.

    Article Snippet: HA myr-Akt and GFP CA CaMKK were provided by Dr. Joseph Testa and Dr. Grahame D. Hardie, respectively, as kind gifts. shRNAs against PHLPP2 (RHS4531-EG23035) and the corresponding control nontargeting shRNA in pGIPZ vector (NT); and inducible shRNA against AMPKα2 (V2THS_57674) and the corresponding control empty pTRIPZ vector (EV) were procured from Dharmacon.

    Techniques: De-Phosphorylation Assay, Western Blot, Multiple Displacement Amplification, Phosphatase Assay, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Plasmid Preparation

    Focal adhesion characteristics and force measurements in REF52 cells depleted for tropomyosin. (A) F-actin stained with phalloidin and focal adhesions labeled with vinculin antibody in cells transfected with nontargeting siRNA (Ctrl), tropomyosin-3 siRNA (Tpm3), and siRNA against all tropmyosins (TpmT). Scale bar is 20 µm. (B) Distribution of focal adhesions size. (C) Distribution of focal adhesion aspect ratio. Images analyzed: n = 14 (Ctrl), n = 26 (KD Tpm3), and n = 20 (KD TpmT). The images were acquired on a W1 spinning-disk microscope. (D) Representative images of myosin regulator light chain (RLC-GFP) at time point of laser ablation are shown in green for Ctrl and TpmT KD cells. Scale bar is 20 µm. Associated kymographs of F-Tractin-tdTomato (magenta) and RLC-GFP (green) along a line drawn crossing the gap are displayed in the next row. Scale bar is 5 µm. Recoil velocity and maximum opening are characterized. The differences between groups are shown in the graph. The number of images quantified for opening: n = 26 (Ctrl), n = 10 (KD Tpm3), and n = 20 (KD TpmT). The number of movies quantified for recoil velocity: n = 15 (Ctrl), n = 8 (KD Tpm3), and n = 9 (KD TpmT). The images were obtained on a Nikon A1R confocal microscope. (E) Traction forces of cells. Left, merge representative images of fibronectin-Atto-647N (magenta), F-tractin-tdTomato (green), and traction force vectors for each pillar (yellow). Middle, merge and zoomed image (position indicated on left image) of RLC-GFP (green) and F-tractin-tdTomato (magenta) with a top-hat filter applied. Right, interpolated traction force map. Scale bar is 20 μm. (Graphs) Net contractile moment exerted by the cells on the substrate and total strain energy are plotted. The images were taken with an Olympus IX-81 inverted widefield fluorescence microscope.

    Journal: Molecular Biology of the Cell

    Article Title: Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

    doi: 10.1091/mbc.E19-02-0082

    Figure Lengend Snippet: Focal adhesion characteristics and force measurements in REF52 cells depleted for tropomyosin. (A) F-actin stained with phalloidin and focal adhesions labeled with vinculin antibody in cells transfected with nontargeting siRNA (Ctrl), tropomyosin-3 siRNA (Tpm3), and siRNA against all tropmyosins (TpmT). Scale bar is 20 µm. (B) Distribution of focal adhesions size. (C) Distribution of focal adhesion aspect ratio. Images analyzed: n = 14 (Ctrl), n = 26 (KD Tpm3), and n = 20 (KD TpmT). The images were acquired on a W1 spinning-disk microscope. (D) Representative images of myosin regulator light chain (RLC-GFP) at time point of laser ablation are shown in green for Ctrl and TpmT KD cells. Scale bar is 20 µm. Associated kymographs of F-Tractin-tdTomato (magenta) and RLC-GFP (green) along a line drawn crossing the gap are displayed in the next row. Scale bar is 5 µm. Recoil velocity and maximum opening are characterized. The differences between groups are shown in the graph. The number of images quantified for opening: n = 26 (Ctrl), n = 10 (KD Tpm3), and n = 20 (KD TpmT). The number of movies quantified for recoil velocity: n = 15 (Ctrl), n = 8 (KD Tpm3), and n = 9 (KD TpmT). The images were obtained on a Nikon A1R confocal microscope. (E) Traction forces of cells. Left, merge representative images of fibronectin-Atto-647N (magenta), F-tractin-tdTomato (green), and traction force vectors for each pillar (yellow). Middle, merge and zoomed image (position indicated on left image) of RLC-GFP (green) and F-tractin-tdTomato (magenta) with a top-hat filter applied. Right, interpolated traction force map. Scale bar is 20 μm. (Graphs) Net contractile moment exerted by the cells on the substrate and total strain energy are plotted. The images were taken with an Olympus IX-81 inverted widefield fluorescence microscope.

    Article Snippet: siRNA KD REF52 cells were transfected with ON-TARGETplus Rat siRNA SMARTpool library (Dharmacon) or a nontargeting control pool siRNA (D-001810-10-05; Dharmacon) at a concentration of 20 µM using electroporation (Neon; Invitrogen) following the manufacturer’s protocol.

    Techniques: Staining, Labeling, Transfection, Microscopy, HAT Assay, Fluorescence

    Organization of myosin II filaments in REF52 cells depleted for tropomyosin. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), siRNA against tropomyosin 1 (Tpm1), tropomyosin 2 (Tpm2), tropomyosin 3 (Tpm3), tropomyosin 4 (Tpm4), and a combination of tropmyosin 1, 2, 3, and 4 (TpmT), F-actin labeled with phalloidin and immunolabeled for myosin IIA. (B) Representative image of myosin IIA immunolabeled REF52 cells overexpressing tropomyosin 3.1 (Tpm3.1 OE). Images were taken with a SIM microscope. Scale bar is 10 µm.

    Journal: Molecular Biology of the Cell

    Article Title: Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

    doi: 10.1091/mbc.E19-02-0082

    Figure Lengend Snippet: Organization of myosin II filaments in REF52 cells depleted for tropomyosin. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), siRNA against tropomyosin 1 (Tpm1), tropomyosin 2 (Tpm2), tropomyosin 3 (Tpm3), tropomyosin 4 (Tpm4), and a combination of tropmyosin 1, 2, 3, and 4 (TpmT), F-actin labeled with phalloidin and immunolabeled for myosin IIA. (B) Representative image of myosin IIA immunolabeled REF52 cells overexpressing tropomyosin 3.1 (Tpm3.1 OE). Images were taken with a SIM microscope. Scale bar is 10 µm.

    Article Snippet: siRNA KD REF52 cells were transfected with ON-TARGETplus Rat siRNA SMARTpool library (Dharmacon) or a nontargeting control pool siRNA (D-001810-10-05; Dharmacon) at a concentration of 20 µM using electroporation (Neon; Invitrogen) following the manufacturer’s protocol.

    Techniques: Transfection, Labeling, Immunolabeling, Microscopy

    Depletion of alpha-actinin increases disorder of myosin II filaments along with increases in focal adhesion size, actomyosin tension, and traction forces. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), α-actinin-4 siRNA (KD Actn4), and the double KD of α-actinin-1 and -4 (KD Actn1 4), and immunolabeled for myosin IIA. Scale bar is 10 µm. (B) Quantification of myosin stack length and number of stacks longer than 500 nm identified. Images analyzed: n = 18 (Ctrl), n = 28 (KD Actn4), and n = 9 (KD Actn1 4). (C) Line scanning across myosin is shown in representative images immunolabeled for myosin IIA. Representative intensity profiles for Ctrl, KD Actn4, and KD Actn1 4 are presented. Number of line scans analyzed: n = 90 (Ctrl), n = 85 (KD Actn4), and n = 89 (KD Actn1 4). (D) Representative images of actin stained with phalloidin and immunolabeled vinculin shown for Ctrl, KD Actn4, and KD Actn1 4 cells. Scale bar is 20 µm. (E) Quantification of focal adhesions in Ctrl, KD Actn4, and KD Actn1 4 cells. Images analyzed: n = 14 (Ctrl), n = 22 (KD Actn4), and n = 8 (KD Actn1 4). The images were taken with a W1 spinning-disk microscope.

    Journal: Molecular Biology of the Cell

    Article Title: Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

    doi: 10.1091/mbc.E19-02-0082

    Figure Lengend Snippet: Depletion of alpha-actinin increases disorder of myosin II filaments along with increases in focal adhesion size, actomyosin tension, and traction forces. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), α-actinin-4 siRNA (KD Actn4), and the double KD of α-actinin-1 and -4 (KD Actn1 4), and immunolabeled for myosin IIA. Scale bar is 10 µm. (B) Quantification of myosin stack length and number of stacks longer than 500 nm identified. Images analyzed: n = 18 (Ctrl), n = 28 (KD Actn4), and n = 9 (KD Actn1 4). (C) Line scanning across myosin is shown in representative images immunolabeled for myosin IIA. Representative intensity profiles for Ctrl, KD Actn4, and KD Actn1 4 are presented. Number of line scans analyzed: n = 90 (Ctrl), n = 85 (KD Actn4), and n = 89 (KD Actn1 4). (D) Representative images of actin stained with phalloidin and immunolabeled vinculin shown for Ctrl, KD Actn4, and KD Actn1 4 cells. Scale bar is 20 µm. (E) Quantification of focal adhesions in Ctrl, KD Actn4, and KD Actn1 4 cells. Images analyzed: n = 14 (Ctrl), n = 22 (KD Actn4), and n = 8 (KD Actn1 4). The images were taken with a W1 spinning-disk microscope.

    Article Snippet: siRNA KD REF52 cells were transfected with ON-TARGETplus Rat siRNA SMARTpool library (Dharmacon) or a nontargeting control pool siRNA (D-001810-10-05; Dharmacon) at a concentration of 20 µM using electroporation (Neon; Invitrogen) following the manufacturer’s protocol.

    Techniques: Transfection, Immunolabeling, Staining, Microscopy

    Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with nontargeting control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the WT/NTC/0-h band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus Tropism Modulator UL148 Interacts with SEL1L, a Cellular Factor That Governs Endoplasmic Reticulum-Associated Degradation of the Viral Envelope Glycoprotein gO

    doi: 10.1128/JVI.00688-18

    Figure Lengend Snippet: Depletion of SEL1L or Hrd1 in HCMV-infected cells stabilizes gO during WT and UL148 -null infection. (A and B) Fibroblasts (HFFT cells) were reverse transfected with nontargeting control siRNAs (NTCs) or with siRNAs targeting SEL1L (A) or Hrd1 (B) NTCs. At 6 h posttransfection (hpt), the cells were infected at an MOI of 1 TCID 50 per cell with TB_gO-S (WT) or TB_148 STOP _gO-S (148 STOP ). At 96 hpi, the cells were pulsed for 20 min with 200 μCi/ml [ 35 S]Met-Cys and chased for the indicated times before lysis. Samples with equal TCA-precipitable counts per minute were subjected to S-AP, followed by PNGase F digestion to allow better resolution of gH and gO by SDS-PAGE. The dried gel was exposed to a phosphor screen to produce an autoradiograph. The densities of gO bands were calculated and expressed either as an absolute signal (“abs”) in relation to the WT/NTC/0-h band or as a normalized signal (“norm”) for each virus condition relative to the respective NTC/0-h band.

    Article Snippet: siGenome SMARTpool siRNAs targeting SEL1L and Hrd1 and nontargeting control (NTC) siRNA were purchased from Dharmacon.

    Techniques: Infection, Transfection, Lysis, SDS Page, Autoradiography

    Depletion of SEL1L in HCMV-infected cells increases steady-state gO levels. (A) Fibroblasts (HFFT cells) were reverse transfected with siRNA targeting SEL1L (SEL) or a nontargeting control (NTC). At 48 h posttransfection, the cells were infected with HCMV strain TB40/E (WT) or its UL148 -null derivative, TB_148 STOP (148 STOP ), at an MOI of 1 TCID 50 per cell. HCMV glycoprotein levels at 96 hpi were analyzed by Western blotting. (B) Treatment of lysates from panel A with EndoH (H) or mock digestion as a control (C). Extraneous lanes from this blot have been cropped from the image.

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus Tropism Modulator UL148 Interacts with SEL1L, a Cellular Factor That Governs Endoplasmic Reticulum-Associated Degradation of the Viral Envelope Glycoprotein gO

    doi: 10.1128/JVI.00688-18

    Figure Lengend Snippet: Depletion of SEL1L in HCMV-infected cells increases steady-state gO levels. (A) Fibroblasts (HFFT cells) were reverse transfected with siRNA targeting SEL1L (SEL) or a nontargeting control (NTC). At 48 h posttransfection, the cells were infected with HCMV strain TB40/E (WT) or its UL148 -null derivative, TB_148 STOP (148 STOP ), at an MOI of 1 TCID 50 per cell. HCMV glycoprotein levels at 96 hpi were analyzed by Western blotting. (B) Treatment of lysates from panel A with EndoH (H) or mock digestion as a control (C). Extraneous lanes from this blot have been cropped from the image.

    Article Snippet: siGenome SMARTpool siRNAs targeting SEL1L and Hrd1 and nontargeting control (NTC) siRNA were purchased from Dharmacon.

    Techniques: Infection, Transfection, Western Blot

    The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using siRNA technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and nontargeted siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p

    Journal: Antioxidants & Redox Signaling

    Article Title: Oral Monomethyl Fumarate Therapy Ameliorates Retinopathy in a Humanized Mouse Model of Sickle Cell Disease

    doi: 10.1089/ars.2016.6638

    Figure Lengend Snippet: The MMF-induced upregulation of γ-globin mRNA and suppression of Bcl11A mRNA in cultured RPE cells is dependent on Nrf2 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was knocked down in transformed human retinal pigment epithelial (ARPE-19) cells using siRNA technology as confirmed by (A) RT-qPCR analyses of Nrf2 mRNA expression and Western blotting and associated densitometric analyses (B) of Nrf2 protein. Treatment of control ARPE-19 (no siRNA) and nontargeted siRNA control ARPE-19 cells with MMF was associated with robust increases in Nrf2 mRNA and protein expression. However, the MMF-induced increases in Nrf2 mRNA and protein were abrogated significantly in ARPE-19 cells in which Nrf2 expression was knocked down using Nrf2-specific siRNA. Data in (A and B) are presented as mean ± SE, *p

    Article Snippet: Nrf2 small interfering RNA (siRNA) and control nontargeted (scramble) siRNA were obtained from Santa Cruz Biotechnology.

    Techniques: Cell Culture, Expressing, Derivative Assay, Transformation Assay, Quantitative RT-PCR, Western Blot