sirna oligonucleotides  (Thermo Fisher)


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    Structured Review

    Thermo Fisher sirna oligonucleotides
    RASSF1A regulation is dependent on FoxM1. <t>T84</t> and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with <t>siRNA</t> for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p
    Sirna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer"

    Article Title: Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer

    Journal: Cancers

    doi: 10.3390/cancers11020199

    RASSF1A regulation is dependent on FoxM1. T84 and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with siRNA for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p
    Figure Legend Snippet: RASSF1A regulation is dependent on FoxM1. T84 and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with siRNA for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p

    Techniques Used: Transfection, Plasmid Preparation, Over Expression, Expressing

    2) Product Images from "Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin"

    Article Title: Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.85

    Dilation of mitochondria and ER precedes DMC-induced cell death in breast cancer cells. ( a ) Cells were treated with 20 μ M DMC for 12 h and observed under a phase contrast microscope. Bars, 20 μ m. ( b ) Hematoxylin and eosin-stained sections of MDA-MB 435S xenografts treated with vehicle or 25 mg/kg DMC at intervals of 2 days for 20 days. Vacuoles are indicated by arrows. ( c ) MDA-MB 435S cells were pre-treated with the indicated specific inhibitors of autophagy (3-MA; bafiolmycin A1 (Bafilo. A1); CQ) and further treated with 20 μ M DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. ( d ) MDA-MB 435S or MDA-MB 231 cells were treated with 20 μ M DMC for the indicated time points and western blotting of autophagy-related proteins were performed. Western blotting of β-actin served as the loading control of protein samples. ( e ) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μ M DMC for 24 h. Knockdown of LC3 was confirmed by western blotting using anti-LC3 antibody. Western blotting of β -actin was served as a loading control (upper panel). Cellular viability was assessed using calcein-AM and EthD-1 (lower panel). ( f ) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μ M DMC for 16 h. Cellular morphologies were observed under a phase contrast microscope. Bar, 20 μm
    Figure Legend Snippet: Dilation of mitochondria and ER precedes DMC-induced cell death in breast cancer cells. ( a ) Cells were treated with 20 μ M DMC for 12 h and observed under a phase contrast microscope. Bars, 20 μ m. ( b ) Hematoxylin and eosin-stained sections of MDA-MB 435S xenografts treated with vehicle or 25 mg/kg DMC at intervals of 2 days for 20 days. Vacuoles are indicated by arrows. ( c ) MDA-MB 435S cells were pre-treated with the indicated specific inhibitors of autophagy (3-MA; bafiolmycin A1 (Bafilo. A1); CQ) and further treated with 20 μ M DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. ( d ) MDA-MB 435S or MDA-MB 231 cells were treated with 20 μ M DMC for the indicated time points and western blotting of autophagy-related proteins were performed. Western blotting of β-actin served as the loading control of protein samples. ( e ) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μ M DMC for 24 h. Knockdown of LC3 was confirmed by western blotting using anti-LC3 antibody. Western blotting of β -actin was served as a loading control (upper panel). Cellular viability was assessed using calcein-AM and EthD-1 (lower panel). ( f ) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μ M DMC for 16 h. Cellular morphologies were observed under a phase contrast microscope. Bar, 20 μm

    Techniques Used: Microscopy, Staining, Multiple Displacement Amplification, Ethidium Homodimer Assay, Western Blot, Transfection

    CHOP, but not Noxa, is critically involved in DMC-induced ER dilation and subsequent cell death . (a ) MDA-MB 435S cells transfected with CHOP or Noxa siRNA were further treated with 20 μ M DMC for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. ( b ) MDA-MB 435 cells were transfected with CHOP or Noxa siRNA and their knockdown was confirmed by western blotting of CHOP or Noxa. Effect of CHOP or Noxa knockdown on ubiquitinated proteins was examined by western blotting using anti-ubiquitin antibody. β -Actin expression was analyzed to confirm equal loading of the protein samples. ( c ) The sublines expressing the fluorescence selectively in ER (YFP-ER cells/435S) or mitochondria (YFP-Mito cells/435S) were transfected with CHOP siRNA and further treated with 20 μ M DMC for 16 h. Cells were observed under a fluorescence microscope. Bars, 20 μ m. ( d ) The changes in the widths of mitochondria-derived vacuoles and the ER-derived vacuoles by CHOP knockdown were quantitatively measured in YFP-Mito cells and YFP-ER cells treated with 20 μ M DMC for 16 h using AxioVision Rel. 4.8 software. CHOP knockdown significantly reduced the DMC-induced increase in the width of the ER. Results were repeated in three other experiments. In each experiment, 50 cells were scored as described in Materials and Methods section. ( e ) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) and then treated with 20 μ M DMC for 16 h. Treated cells were processed for immunocytochemistry of CHOP, PDI and COX IV. Bars, 20 μ m. ( f ) Tumors in nude mice treated with DMC (50 mg/kg) or curcumin (50 mg/kg) were collected after 25 days of treatments and tissue extracts were prepared for western blotting using anti-ubiquitin and anti-CHOP antibody. Western blotting of α -tubulin was examined to verify equal loading
    Figure Legend Snippet: CHOP, but not Noxa, is critically involved in DMC-induced ER dilation and subsequent cell death . (a ) MDA-MB 435S cells transfected with CHOP or Noxa siRNA were further treated with 20 μ M DMC for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. ( b ) MDA-MB 435 cells were transfected with CHOP or Noxa siRNA and their knockdown was confirmed by western blotting of CHOP or Noxa. Effect of CHOP or Noxa knockdown on ubiquitinated proteins was examined by western blotting using anti-ubiquitin antibody. β -Actin expression was analyzed to confirm equal loading of the protein samples. ( c ) The sublines expressing the fluorescence selectively in ER (YFP-ER cells/435S) or mitochondria (YFP-Mito cells/435S) were transfected with CHOP siRNA and further treated with 20 μ M DMC for 16 h. Cells were observed under a fluorescence microscope. Bars, 20 μ m. ( d ) The changes in the widths of mitochondria-derived vacuoles and the ER-derived vacuoles by CHOP knockdown were quantitatively measured in YFP-Mito cells and YFP-ER cells treated with 20 μ M DMC for 16 h using AxioVision Rel. 4.8 software. CHOP knockdown significantly reduced the DMC-induced increase in the width of the ER. Results were repeated in three other experiments. In each experiment, 50 cells were scored as described in Materials and Methods section. ( e ) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) and then treated with 20 μ M DMC for 16 h. Treated cells were processed for immunocytochemistry of CHOP, PDI and COX IV. Bars, 20 μ m. ( f ) Tumors in nude mice treated with DMC (50 mg/kg) or curcumin (50 mg/kg) were collected after 25 days of treatments and tissue extracts were prepared for western blotting using anti-ubiquitin and anti-CHOP antibody. Western blotting of α -tubulin was examined to verify equal loading

    Techniques Used: Multiple Displacement Amplification, Transfection, Ethidium Homodimer Assay, Western Blot, Expressing, Fluorescence, Microscopy, Derivative Assay, Software, Infection, shRNA, Immunocytochemistry, Mouse Assay

    3) Product Images from "The NIP7 protein is required for accurate pre-rRNA processing in human cells"

    Article Title: The NIP7 protein is required for accurate pre-rRNA processing in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq758

    Northern blot analysis of pre-rRNAs detected by using probes complementary to the 5′-ETS and ITS1. ( A ) Northern blot using probe P1 complementary to the 5′-ETS upstream site A0. ( B ) Northern blot using probe P2 complementary to the 5′-ETS downstream site A0. ( C ) Northern blot using probe P3 complementary to ITS1 upstream site 2c. ( D ) and ( E ) Northern blots using probes P1 and P3 of longer electrophoresis runs. ( F ) Northern blot of cells treated with leptomycin B using probe P3 complementary to ITS1 upstream site 2c. ( G ) Structure of the 47S pre-rRNA and pre-rRNA intermediates that are most affected in NIP7-depleted cells. The positions of the probes P1, P2 and P3 used in the northern blots shown in A–D are indicated. HEK293, parental cells; SC, cells transfected with the scrambled shRNA; CP4, cells transfected with shRNA against the NIP7 mRNA.
    Figure Legend Snippet: Northern blot analysis of pre-rRNAs detected by using probes complementary to the 5′-ETS and ITS1. ( A ) Northern blot using probe P1 complementary to the 5′-ETS upstream site A0. ( B ) Northern blot using probe P2 complementary to the 5′-ETS downstream site A0. ( C ) Northern blot using probe P3 complementary to ITS1 upstream site 2c. ( D ) and ( E ) Northern blots using probes P1 and P3 of longer electrophoresis runs. ( F ) Northern blot of cells treated with leptomycin B using probe P3 complementary to ITS1 upstream site 2c. ( G ) Structure of the 47S pre-rRNA and pre-rRNA intermediates that are most affected in NIP7-depleted cells. The positions of the probes P1, P2 and P3 used in the northern blots shown in A–D are indicated. HEK293, parental cells; SC, cells transfected with the scrambled shRNA; CP4, cells transfected with shRNA against the NIP7 mRNA.

    Techniques Used: Northern Blot, Electrophoresis, Transfection, shRNA

    Northern blot analysis of pre-rRNAs detected by using probes complementary to the 5.8S rRNA, ITS2 and the 28S rRNA. ( A ) Northern blot using probe P4 complementary to the 5.8S rRNA. ( B ) Northern blot using probe P5 complementary to ITS2 upstream site 4b. ( C ) Northern blot using probe P6 complementary to ITS2 downstream site 4b. ( D ) Northern blot using probe P7 complementary to the 28S rRNA. ( E ) Ethidium bromide staining of an RNA gel showing the amounts of RNA loaded in each lane. ( F ) Enlarged figures showing the gel region of the high molecular weight pre-rRNAs detected with probes P5, P6 and P7. HEK293, parental cells; SC, cells transfected with the scrambled shRNA; CP4, cells transfected with shRNA against the NIP7 mRNA. ( G ) Structure of the 47S pre-rRNA. The positions of the probes P4, P5, P6 and P7 used in the northern blots shown in A-D are indicated. Asterisk indicates an unidentified band.
    Figure Legend Snippet: Northern blot analysis of pre-rRNAs detected by using probes complementary to the 5.8S rRNA, ITS2 and the 28S rRNA. ( A ) Northern blot using probe P4 complementary to the 5.8S rRNA. ( B ) Northern blot using probe P5 complementary to ITS2 upstream site 4b. ( C ) Northern blot using probe P6 complementary to ITS2 downstream site 4b. ( D ) Northern blot using probe P7 complementary to the 28S rRNA. ( E ) Ethidium bromide staining of an RNA gel showing the amounts of RNA loaded in each lane. ( F ) Enlarged figures showing the gel region of the high molecular weight pre-rRNAs detected with probes P5, P6 and P7. HEK293, parental cells; SC, cells transfected with the scrambled shRNA; CP4, cells transfected with shRNA against the NIP7 mRNA. ( G ) Structure of the 47S pre-rRNA. The positions of the probes P4, P5, P6 and P7 used in the northern blots shown in A-D are indicated. Asterisk indicates an unidentified band.

    Techniques Used: Northern Blot, Staining, Molecular Weight, Transfection, shRNA

    Analysis of polysomes by sucrose density gradient fractionation. ( A ) Polysome profile of HEK293 cells. ( B ) Polysome profile of control cells expressing the scrambled shRNA. ( C ) Polysome profiles of clone CP4 transfected with shRNA targeting the NIP7 mRNA. ( D ) Quantitation of the 60S/40S subunit ratio of HEK293 cells, control scrambled shRNA cells (SC) and of CP4 cells in the polysome profiles shown in A, B and C, respectively. CP4 cells contain a significant lower amount of 40S ribosomal subunits. P -value was obtained by using a one-sided Student’s t -test ( P
    Figure Legend Snippet: Analysis of polysomes by sucrose density gradient fractionation. ( A ) Polysome profile of HEK293 cells. ( B ) Polysome profile of control cells expressing the scrambled shRNA. ( C ) Polysome profiles of clone CP4 transfected with shRNA targeting the NIP7 mRNA. ( D ) Quantitation of the 60S/40S subunit ratio of HEK293 cells, control scrambled shRNA cells (SC) and of CP4 cells in the polysome profiles shown in A, B and C, respectively. CP4 cells contain a significant lower amount of 40S ribosomal subunits. P -value was obtained by using a one-sided Student’s t -test ( P

    Techniques Used: Fractionation, Expressing, shRNA, Transfection, Quantitation Assay

    Analysis of NIP7 knockdown. ( A ) NIP7 mRNA levels as determined by quantitative RT–qPCR in HEK293 derivative cells. HEK293, parental cells; SC, cells transfected with a scrambled shRNA; CP4 and CP6, cells transfected with shRNA against the NIP7 mRNA. NIP7 mRNA quantitation was performed using three different RNA extractions from clones CP4 and CP6. ( B ) NIP7 mRNA levels in transiently transfected MCF10A and HeLa cells. SC, control cells transfected with scrambled RNA; SiRNA, cells transfected with siRNA against NIP7. The histogram corresponds to one of the three independent transfections of MFC10A and HeLa cells using three replicates for each cell treatment. The amount of NIP7 mRNA in cells treated with the scrambled shRNA was considered as 100% to calculate its relative levels in the parental cells (HEK293) and in cells expressing the RNAi against the NIP7 mRNA (CP4) or in cells transfected with siRNA against the NIP7 mRNA. ( C ) Immunoblot showing the levels of the NIP7 protein in HEK293 derivative cells β-actin was used as an internal control. ( D ) Immunoblot using antiserum for the NIP7 protein in transiently transfected MCF10A and HeLa cells. ( E ) Proliferation rate of HEK293 derivative cells expressing the scrambled shRNA (SC) and the RNAi against the NIP7 mRNA (CP4) over a 7-day period. ( F ) Proliferation rate of transiently transfected MCF10A and HeLa cells. The graphs correspond to one of the two independent proliferation assays performed using three replicates for each cell treatment.
    Figure Legend Snippet: Analysis of NIP7 knockdown. ( A ) NIP7 mRNA levels as determined by quantitative RT–qPCR in HEK293 derivative cells. HEK293, parental cells; SC, cells transfected with a scrambled shRNA; CP4 and CP6, cells transfected with shRNA against the NIP7 mRNA. NIP7 mRNA quantitation was performed using three different RNA extractions from clones CP4 and CP6. ( B ) NIP7 mRNA levels in transiently transfected MCF10A and HeLa cells. SC, control cells transfected with scrambled RNA; SiRNA, cells transfected with siRNA against NIP7. The histogram corresponds to one of the three independent transfections of MFC10A and HeLa cells using three replicates for each cell treatment. The amount of NIP7 mRNA in cells treated with the scrambled shRNA was considered as 100% to calculate its relative levels in the parental cells (HEK293) and in cells expressing the RNAi against the NIP7 mRNA (CP4) or in cells transfected with siRNA against the NIP7 mRNA. ( C ) Immunoblot showing the levels of the NIP7 protein in HEK293 derivative cells β-actin was used as an internal control. ( D ) Immunoblot using antiserum for the NIP7 protein in transiently transfected MCF10A and HeLa cells. ( E ) Proliferation rate of HEK293 derivative cells expressing the scrambled shRNA (SC) and the RNAi against the NIP7 mRNA (CP4) over a 7-day period. ( F ) Proliferation rate of transiently transfected MCF10A and HeLa cells. The graphs correspond to one of the two independent proliferation assays performed using three replicates for each cell treatment.

    Techniques Used: Quantitative RT-PCR, Transfection, shRNA, Quantitation Assay, Clone Assay, Expressing

    Analysis of primer extension products generated by reverse transcriptase. ( A ) Primer extension products using primer PE1 that is complementary to the 18S rRNA downstream site 1. ( B ) Primer extension products using primer PE2 that is complementary to ITS1 downstream site 2b. ( C ) Primer extension products using primer PE3 that is complementary to the 5.8S rRNA downstream site 3. U m 14 indicates the position of the 2- O -methyluridine at position 14 of the 5.8S rRNA. U7 and A17 indicate two unexpected primer extension stops. 1, 2b, 3 and 3L indicate the primer extension products extended to the respective processing sties. Arrowheads indicate the bands corresponding to the primers which were not extended: PE1, 30 nt; PE2, 26 nt and PE3, 28 nt, respectively. HEK293, parental cells; SC, cells transfected with scRNA; CP4, cells transfected with shRNA against the NIP7 mRNA. ( D ) Structure of the 47S pre-rRNA with the indication of the position of the oligonucleotides PE1, PE2 and PE3 used in the primer extension assays shown in A, B and C.
    Figure Legend Snippet: Analysis of primer extension products generated by reverse transcriptase. ( A ) Primer extension products using primer PE1 that is complementary to the 18S rRNA downstream site 1. ( B ) Primer extension products using primer PE2 that is complementary to ITS1 downstream site 2b. ( C ) Primer extension products using primer PE3 that is complementary to the 5.8S rRNA downstream site 3. U m 14 indicates the position of the 2- O -methyluridine at position 14 of the 5.8S rRNA. U7 and A17 indicate two unexpected primer extension stops. 1, 2b, 3 and 3L indicate the primer extension products extended to the respective processing sties. Arrowheads indicate the bands corresponding to the primers which were not extended: PE1, 30 nt; PE2, 26 nt and PE3, 28 nt, respectively. HEK293, parental cells; SC, cells transfected with scRNA; CP4, cells transfected with shRNA against the NIP7 mRNA. ( D ) Structure of the 47S pre-rRNA with the indication of the position of the oligonucleotides PE1, PE2 and PE3 used in the primer extension assays shown in A, B and C.

    Techniques Used: Generated, Transfection, shRNA

    4) Product Images from "HCV 3a Core Protein Increases Lipid Droplet Cholesteryl Ester Content via a Mechanism Dependent on Sphingolipid Biosynthesis"

    Article Title: HCV 3a Core Protein Increases Lipid Droplet Cholesteryl Ester Content via a Mechanism Dependent on Sphingolipid Biosynthesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115309

    Effect of HCV core protein expression on mRNA levels of key enzymes of sphingolipid biosynthesis, cell treatment with inhibitors of sphingolipid biosynthesis and silencing of SPT. ( A ) The expression of key enzymes involved in sphingolipid biosynthesis (SGMS1: sphingomyelin synthase; SPTLC2: serine palmitoyl transferase; UGCG: glucosylceramide synthase; CerS 2, 5 and 6: ceramide synthase 2, 5 and 6) was assessed after transduction of Huh-7 cells with HCV core protein of genotype 3a, 2a or GFP at three days post-transduction by RT-qPCR. Results are represented as mean ±SEM. ( B, a–c ) Huh-7 cells were pre-treated with 200 nM myriocin for 24 hours before transduction with HCV 3a core-expressing lentivector and then treated with 200 nM myriocin for an additional 48 hours. Control cells (a), untreated core 3a transduced Huh-7 cells (b) and core 3a transduced Huh-7 cells treated with myriocin (c). ( B , d–f) Cells were transfected with either scrambled siRNA (SCR) or siRNA targeting SPT (siSPT) 24 hours prior transduction. Control cells (d), core 3a transduced Huh-7 cells transfected with SCR (e) and core 3a transduced Huh-7 cells transfected with siSPT (f). Images show representative single optical confocal immuno-fluorescence sections of ORO (red) and anti-core 3a (green) staining. Overlay images are shown. ( C ) CE levels were measured in myriocin-treated or SPT-silenced cell conditions using the cholesterol/cholesteryl ester quantitation kit. Values were normalized to the amount of protein. Results are represented as mean ±SEM of at least 3 independent experiments.
    Figure Legend Snippet: Effect of HCV core protein expression on mRNA levels of key enzymes of sphingolipid biosynthesis, cell treatment with inhibitors of sphingolipid biosynthesis and silencing of SPT. ( A ) The expression of key enzymes involved in sphingolipid biosynthesis (SGMS1: sphingomyelin synthase; SPTLC2: serine palmitoyl transferase; UGCG: glucosylceramide synthase; CerS 2, 5 and 6: ceramide synthase 2, 5 and 6) was assessed after transduction of Huh-7 cells with HCV core protein of genotype 3a, 2a or GFP at three days post-transduction by RT-qPCR. Results are represented as mean ±SEM. ( B, a–c ) Huh-7 cells were pre-treated with 200 nM myriocin for 24 hours before transduction with HCV 3a core-expressing lentivector and then treated with 200 nM myriocin for an additional 48 hours. Control cells (a), untreated core 3a transduced Huh-7 cells (b) and core 3a transduced Huh-7 cells treated with myriocin (c). ( B , d–f) Cells were transfected with either scrambled siRNA (SCR) or siRNA targeting SPT (siSPT) 24 hours prior transduction. Control cells (d), core 3a transduced Huh-7 cells transfected with SCR (e) and core 3a transduced Huh-7 cells transfected with siSPT (f). Images show representative single optical confocal immuno-fluorescence sections of ORO (red) and anti-core 3a (green) staining. Overlay images are shown. ( C ) CE levels were measured in myriocin-treated or SPT-silenced cell conditions using the cholesterol/cholesteryl ester quantitation kit. Values were normalized to the amount of protein. Results are represented as mean ±SEM of at least 3 independent experiments.

    Techniques Used: Expressing, Single-particle Tracking, Transduction, Quantitative RT-PCR, Transfection, Fluorescence, Staining, Quantitation Assay

    5) Product Images from "Salmonella Suppresses the TRIF-Dependent Type I Interferon Response in Macrophages"

    Article Title: Salmonella Suppresses the TRIF-Dependent Type I Interferon Response in Macrophages

    Journal: mBio

    doi: 10.1128/mBio.02051-15

    IFN-β production in response to Salmonella infection requires TRIF. (A and B) Immortalized macrophages (iMacs) deficient in both MyD88 and TRIF (MyD88 −/− /TRIF −/− ) were pretreated with rapamycin (1 nM) for 1 h (A) or were depleted of FAK by siRNA before incubation with Δ invG Salmonella (B) (MOI, 100) for 5 h. Lysates were immunoblotted with the indicated antibodies; n = 3. (C) Macrophages were treated as described for panels A and B before assessment of IFN-β mRNA levels. IFN-β amounts in infected cells were calculated relative to those seen with uninfected controls. n.s., not significant; n = 3. (D) WT iMacs were depleted of TRIF or MyD88 before pretreatment with rapamycin (1 nM) for 1 h prior to infection with Δ InvG Salmonella for 5 h. Lysates were immunoblotted with the indicated antibodies; n = 3. (E) Macrophages were treated as described for panel D before assessment of IFN-β mRNA levels. IFN-β amounts in infected cells were calculated relative to those seen with uninfected controls. *, P
    Figure Legend Snippet: IFN-β production in response to Salmonella infection requires TRIF. (A and B) Immortalized macrophages (iMacs) deficient in both MyD88 and TRIF (MyD88 −/− /TRIF −/− ) were pretreated with rapamycin (1 nM) for 1 h (A) or were depleted of FAK by siRNA before incubation with Δ invG Salmonella (B) (MOI, 100) for 5 h. Lysates were immunoblotted with the indicated antibodies; n = 3. (C) Macrophages were treated as described for panels A and B before assessment of IFN-β mRNA levels. IFN-β amounts in infected cells were calculated relative to those seen with uninfected controls. n.s., not significant; n = 3. (D) WT iMacs were depleted of TRIF or MyD88 before pretreatment with rapamycin (1 nM) for 1 h prior to infection with Δ InvG Salmonella for 5 h. Lysates were immunoblotted with the indicated antibodies; n = 3. (E) Macrophages were treated as described for panel D before assessment of IFN-β mRNA levels. IFN-β amounts in infected cells were calculated relative to those seen with uninfected controls. *, P

    Techniques Used: Infection, Incubation

    6) Product Images from "NME7 is a functional component of the γ-tubulin ring complex"

    Article Title: NME7 is a functional component of the γ-tubulin ring complex

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E13-06-0339

    NME7 is a component of the γTuRC. (A) Proteins were immunoprecipitated from HEK293T extracts by using anti-NME7, anti-GCP5, and anti–immunoglobulin G (control) antibodies, and the immunoprecipitates were probed by means of immunoblotting. (B) HEK293T extracts were fractionated by centrifuging them over a continuous sucrose gradient, and the gradient fractions obtained were immunoblotted. (C) Gradient fractions containing γ-tubulin of the low–molecular weight species (fractions 2–5) and the γTuRC (fractions 8–11) were pooled and used for anti-NME7 immunoprecipitation. The immunoprecipitates were probed by means of immunoblotting. (D) Sucrose-gradient fractions of extracts of HEK293T cells transfected with an nme7 -targeting siRNA (NME7 RNAi) were probed on immunoblots. (E) Extracts of HEK293T cells expressing FLAG-tagged NME7 fragments were used for immunoprecipitation with an anti-FLAG antibody. The precipitated proteins were immunoblotted for γ-tubulin and NME7 fragments (anti-FLAG). A schematic representation of the NME7 constructs is shown below. (F) Extracts of HEK293T cells expressing FLAG-tagged wild-type (WT) and mutant NME7 proteins were immunoprecipitated with anti-FLAG antibodies. The precipitates were probed using anti–γ-tubulin, anti-FLAG, and anti-GCP6 antibodies.
    Figure Legend Snippet: NME7 is a component of the γTuRC. (A) Proteins were immunoprecipitated from HEK293T extracts by using anti-NME7, anti-GCP5, and anti–immunoglobulin G (control) antibodies, and the immunoprecipitates were probed by means of immunoblotting. (B) HEK293T extracts were fractionated by centrifuging them over a continuous sucrose gradient, and the gradient fractions obtained were immunoblotted. (C) Gradient fractions containing γ-tubulin of the low–molecular weight species (fractions 2–5) and the γTuRC (fractions 8–11) were pooled and used for anti-NME7 immunoprecipitation. The immunoprecipitates were probed by means of immunoblotting. (D) Sucrose-gradient fractions of extracts of HEK293T cells transfected with an nme7 -targeting siRNA (NME7 RNAi) were probed on immunoblots. (E) Extracts of HEK293T cells expressing FLAG-tagged NME7 fragments were used for immunoprecipitation with an anti-FLAG antibody. The precipitated proteins were immunoblotted for γ-tubulin and NME7 fragments (anti-FLAG). A schematic representation of the NME7 constructs is shown below. (F) Extracts of HEK293T cells expressing FLAG-tagged wild-type (WT) and mutant NME7 proteins were immunoprecipitated with anti-FLAG antibodies. The precipitates were probed using anti–γ-tubulin, anti-FLAG, and anti-GCP6 antibodies.

    Techniques Used: Immunoprecipitation, Molecular Weight, Transfection, Western Blot, Expressing, Construct, Mutagenesis

    NME7 is detected in cultured cells and depleted after RNAi. (A) Schematic representation of NME7 domains. (B) Detection of NME7 isoforms. Lysates of HEK293T cells were probed using anti-NME7 and anti–γ-tubulin antibodies. Cells transfected with NME7a or NME7b were also analyzed by means of anti-NME7 immunoblotting. (C) HEK293T cells transfected with siRNA oligonucleotides were immunoblotted with anti-NME7 and anti–β-actin antibodies. The intensity of NME7 bands was quantified and normalized relative to the corresponding β-actin bands to estimate RNAi efficiency.
    Figure Legend Snippet: NME7 is detected in cultured cells and depleted after RNAi. (A) Schematic representation of NME7 domains. (B) Detection of NME7 isoforms. Lysates of HEK293T cells were probed using anti-NME7 and anti–γ-tubulin antibodies. Cells transfected with NME7a or NME7b were also analyzed by means of anti-NME7 immunoblotting. (C) HEK293T cells transfected with siRNA oligonucleotides were immunoblotted with anti-NME7 and anti–β-actin antibodies. The intensity of NME7 bands was quantified and normalized relative to the corresponding β-actin bands to estimate RNAi efficiency.

    Techniques Used: Cell Culture, Transfection

    NME7 localizes at centrosomes through assembly into the γTuRC. (A) HeLa cells were transfected with a control siRNA or with siRNAs against NME7, γ-tubulin, or GCP4. Protein expression was analyzed by means of immunoblotting. (B) siRNA-transfected cells were immunostained and examined using fluorescence microscopy. Boxed areas show magnified centrosomes. Scale bar, 5 μm. (C) Centrosomal intensities of NME7 and γ-tubulin were measured in cells transfected with siRNAs targeting γ-tubulin or GCP4. Data are presented after background subtraction; the data shown are representative of three independent experiments.
    Figure Legend Snippet: NME7 localizes at centrosomes through assembly into the γTuRC. (A) HeLa cells were transfected with a control siRNA or with siRNAs against NME7, γ-tubulin, or GCP4. Protein expression was analyzed by means of immunoblotting. (B) siRNA-transfected cells were immunostained and examined using fluorescence microscopy. Boxed areas show magnified centrosomes. Scale bar, 5 μm. (C) Centrosomal intensities of NME7 and γ-tubulin were measured in cells transfected with siRNAs targeting γ-tubulin or GCP4. Data are presented after background subtraction; the data shown are representative of three independent experiments.

    Techniques Used: Transfection, Expressing, Fluorescence, Microscopy

    7) Product Images from "Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cells"

    Article Title: Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cells

    Journal: Data in Brief

    doi: 10.1016/j.dib.2019.104943

    Levels of normal or nonsense-mutated human β-globin transcripts are not affected by depletion of DIS3L2 in HeLa cells. (A) Western blot analysis of HeLa cells lysates transfected (+) with control Luciferase siRNA (LUC siRNA), or with siRNA targeting the human DIS3L2. Following to the siRNA-mediated silencing, cells were transfected with the plasmids expressing wild type (βWT), NMD-resistant (β15), or NMD-sensitive (β26, β39) human β-globin mRNAs. Primary antibodies against DIS3L2 and α-tubulin (loading control) were used in this blotting to monitor DIS3L2 knockdown. Identification of each band is indicated on the left side of the image. (B) Representative Northern blot analysis of total RNA obtained from HeLa cells with (+) or without (−) depletion of DIS3L2. Fifteen micrograms of total RNA were separated under denaturing conditions on an MOPS/formaldehyde agarose gel (1%) and human β-globin variants βWT, β15, β26 and β39 were detected with a specific probe for exon 1. Loading was controlled by detection of 28S rRNA. (C) Bar plot shows fold-change of each sample mRNA level relative to the control [βWT at Luciferase (LUC) siRNA-treated cells], arbitrarily set to 1. RT-qPCR was used to determine mRNA levels by using primers specific for human β-globin gene, and for glyceraldehyde-3-phosphate dehydrogenase gene. Mean and standard deviation (SD) of four independent biological replicates are shown in the bar plot.
    Figure Legend Snippet: Levels of normal or nonsense-mutated human β-globin transcripts are not affected by depletion of DIS3L2 in HeLa cells. (A) Western blot analysis of HeLa cells lysates transfected (+) with control Luciferase siRNA (LUC siRNA), or with siRNA targeting the human DIS3L2. Following to the siRNA-mediated silencing, cells were transfected with the plasmids expressing wild type (βWT), NMD-resistant (β15), or NMD-sensitive (β26, β39) human β-globin mRNAs. Primary antibodies against DIS3L2 and α-tubulin (loading control) were used in this blotting to monitor DIS3L2 knockdown. Identification of each band is indicated on the left side of the image. (B) Representative Northern blot analysis of total RNA obtained from HeLa cells with (+) or without (−) depletion of DIS3L2. Fifteen micrograms of total RNA were separated under denaturing conditions on an MOPS/formaldehyde agarose gel (1%) and human β-globin variants βWT, β15, β26 and β39 were detected with a specific probe for exon 1. Loading was controlled by detection of 28S rRNA. (C) Bar plot shows fold-change of each sample mRNA level relative to the control [βWT at Luciferase (LUC) siRNA-treated cells], arbitrarily set to 1. RT-qPCR was used to determine mRNA levels by using primers specific for human β-globin gene, and for glyceraldehyde-3-phosphate dehydrogenase gene. Mean and standard deviation (SD) of four independent biological replicates are shown in the bar plot.

    Techniques Used: Western Blot, Transfection, Luciferase, Expressing, Northern Blot, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    8) Product Images from "Antitumor effects of the antiparasitic agent ivermectin via inhibition of Yes-associated protein 1 expression in gastric cancer"

    Article Title: Antitumor effects of the antiparasitic agent ivermectin via inhibition of Yes-associated protein 1 expression in gastric cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22587

    The antiproliferative effects of ivermectin were dependent on YAP1 expression ( A ) Immunoblotting for total protein expression of YAP1 in YAP1 siRNA-transfected MKN1 and MKN7 cells and control siRNA-transfected cells. ( B ) MTT proliferation assays. The proliferation rates of YAP1 siRNA-transfected MKN1 and MKN7 cells were compared with that of control siRNA-transfected cells. * P
    Figure Legend Snippet: The antiproliferative effects of ivermectin were dependent on YAP1 expression ( A ) Immunoblotting for total protein expression of YAP1 in YAP1 siRNA-transfected MKN1 and MKN7 cells and control siRNA-transfected cells. ( B ) MTT proliferation assays. The proliferation rates of YAP1 siRNA-transfected MKN1 and MKN7 cells were compared with that of control siRNA-transfected cells. * P

    Techniques Used: Expressing, Transfection, MTT Assay

    9) Product Images from "SNHG5 promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization"

    Article Title: SNHG5 promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization

    Journal: Nature Communications

    doi: 10.1038/ncomms13875

    SNHG5 targets mRNAs in the cytoplasm. ( a ) Schematic representation of biotinylated antisense DNA probes complementary to the SNHG5 transcript Even (green) and Odd (red) used for the RNA pool-down. ( b ) qRT–PCR was performed to assess the % of SNHG5 RNA retrieved from HCT116 cell lysates following pull-down with streptavidin beads. Data was normalized to input (1:100) and GAPDH mRNA included as negative control. Error bars indicate±s.d. for the mean of three independent experiments. ( c ) Venn diagram representing the number of SNHG5 pull-down peaks commonly enriched with both the Even and Odd probe sets. ( d ) Validation of the RIA-seq results by qRT–PCR. Data for each target transcript is normalized on input (1:100). Error bars indicate±s.d. for the mean of three independent experiments. ( e ) Left, qRT–PCR. SNHG5 expression levels were measured 48 h after transfection of HCT116 cells. Data was normalized to the GAPDH housekeeping gene and plotted relative to the siRNA control. Error bars indicate the mean±s.e. of three independent experiments. Right, western blot. HCT116 cells 48 h after transfection with the indicated siRNAs. The optical density (OD) of protein bands is indicated relative to corresponding loading control GAPDH and normalized relative the non-targeting siControl. ( f ) Transcript stability of SNHG5 targets were measured by qRT–PCR in HCT116 cells 36 h after transfection with the indicated siRNAs. The cells were treated with Triptolide at a final concentration of 10 μM for the last 5 h. The mRNA levels are plotted relative to the corresponding expression levels in the untreated cells. Error bars indicate the mean±s.e. of three independent experiments (Student's t -test paired).
    Figure Legend Snippet: SNHG5 targets mRNAs in the cytoplasm. ( a ) Schematic representation of biotinylated antisense DNA probes complementary to the SNHG5 transcript Even (green) and Odd (red) used for the RNA pool-down. ( b ) qRT–PCR was performed to assess the % of SNHG5 RNA retrieved from HCT116 cell lysates following pull-down with streptavidin beads. Data was normalized to input (1:100) and GAPDH mRNA included as negative control. Error bars indicate±s.d. for the mean of three independent experiments. ( c ) Venn diagram representing the number of SNHG5 pull-down peaks commonly enriched with both the Even and Odd probe sets. ( d ) Validation of the RIA-seq results by qRT–PCR. Data for each target transcript is normalized on input (1:100). Error bars indicate±s.d. for the mean of three independent experiments. ( e ) Left, qRT–PCR. SNHG5 expression levels were measured 48 h after transfection of HCT116 cells. Data was normalized to the GAPDH housekeeping gene and plotted relative to the siRNA control. Error bars indicate the mean±s.e. of three independent experiments. Right, western blot. HCT116 cells 48 h after transfection with the indicated siRNAs. The optical density (OD) of protein bands is indicated relative to corresponding loading control GAPDH and normalized relative the non-targeting siControl. ( f ) Transcript stability of SNHG5 targets were measured by qRT–PCR in HCT116 cells 36 h after transfection with the indicated siRNAs. The cells were treated with Triptolide at a final concentration of 10 μM for the last 5 h. The mRNA levels are plotted relative to the corresponding expression levels in the untreated cells. Error bars indicate the mean±s.e. of three independent experiments (Student's t -test paired).

    Techniques Used: Quantitative RT-PCR, Negative Control, Expressing, Transfection, Western Blot, Concentration Assay

    SNHG5 impairs the association between STAU1 protein and the SPATS2 mRNA. ( a ) Left, western blot for STAU1 and SPATS2 in HCT116 cells transfected with indicated siRNAs for 72 h. OD of protein bands is indicated relative to corresponding loading control GAPDH and normalized relative the non-targeting siControl. Right, qRT–PCR. HCT116 cells were transfected as described above. SPATS2 expression data are shown normalized to the GAPDH housekeeping gene and normalized to the non-targeting control siRNA. ( b ) qRT–PCR. HCT116 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection the cells were treated with Triptolide at a final concentration of 10 μM for 5 h and the RNA subsequently extracted. The percentage retrieved SPATS2 mRNA was obtained by normalizing to the corresponding expression levels in the untreated cells. ( c ) RNA immunoprecipitation. HCT116 were transfected with the indicated siRNAs for 48 h. The cytosolic fraction was isolated from the ultraviolet-cross-linked cells and the IP performed with a STAU1-specific antibody. Rabbit IgG were included as negative control for the immunoprecipitation. The RNA was extracted, retro-transcribed and the SPATS2 mRNA levels evaluated. ARF1 was included as positive control and H3 mRNA as negative control. Error bars indicate±s.d. relative for the mean of three independent experiments (* P value
    Figure Legend Snippet: SNHG5 impairs the association between STAU1 protein and the SPATS2 mRNA. ( a ) Left, western blot for STAU1 and SPATS2 in HCT116 cells transfected with indicated siRNAs for 72 h. OD of protein bands is indicated relative to corresponding loading control GAPDH and normalized relative the non-targeting siControl. Right, qRT–PCR. HCT116 cells were transfected as described above. SPATS2 expression data are shown normalized to the GAPDH housekeeping gene and normalized to the non-targeting control siRNA. ( b ) qRT–PCR. HCT116 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection the cells were treated with Triptolide at a final concentration of 10 μM for 5 h and the RNA subsequently extracted. The percentage retrieved SPATS2 mRNA was obtained by normalizing to the corresponding expression levels in the untreated cells. ( c ) RNA immunoprecipitation. HCT116 were transfected with the indicated siRNAs for 48 h. The cytosolic fraction was isolated from the ultraviolet-cross-linked cells and the IP performed with a STAU1-specific antibody. Rabbit IgG were included as negative control for the immunoprecipitation. The RNA was extracted, retro-transcribed and the SPATS2 mRNA levels evaluated. ARF1 was included as positive control and H3 mRNA as negative control. Error bars indicate±s.d. relative for the mean of three independent experiments (* P value

    Techniques Used: Western Blot, Transfection, Quantitative RT-PCR, Expressing, Concentration Assay, Immunoprecipitation, Isolation, Negative Control, Positive Control

    10) Product Images from "Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase"

    Article Title: Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase

    Journal: Diabetes

    doi: 10.2337/db16-0023

    TAT inhibits TH in neurons. CATH.a cells were transfected with TAT plasmid or TAT siRNA for 24 h, and proteins were extracted from treated cells for Western blotting evaluation of TH level. A : Representative Western blot bands of TH in TAT knockdown (si-TAT) and overexpressing (OE)-TAT cells. GAPDH is a loading control. B : Densitometric analyses of TH level in the si-TAT neuronal cells. The bar graph shows relative upregulation of TH after knockdown of TAT. C : Densitometric analyses of TH level in the OE-TAT cells. The bar graph shows relative downregulation of TH after TAT OE. Values are mean ± SEM ( n = 3).
    Figure Legend Snippet: TAT inhibits TH in neurons. CATH.a cells were transfected with TAT plasmid or TAT siRNA for 24 h, and proteins were extracted from treated cells for Western blotting evaluation of TH level. A : Representative Western blot bands of TH in TAT knockdown (si-TAT) and overexpressing (OE)-TAT cells. GAPDH is a loading control. B : Densitometric analyses of TH level in the si-TAT neuronal cells. The bar graph shows relative upregulation of TH after knockdown of TAT. C : Densitometric analyses of TH level in the OE-TAT cells. The bar graph shows relative downregulation of TH after TAT OE. Values are mean ± SEM ( n = 3).

    Techniques Used: Transfection, Plasmid Preparation, Western Blot

    11) Product Images from "LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage"

    Article Title: LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage

    Journal: The FASEB Journal

    doi: 10.1096/fj.12-216671

    siRNA-mediated knockdown of LRP-1 impairs ADAMTS-5 endocytosis in human chondrocytes. Human chondrocytes transfected with nontargeting siRNA (siCtrl) or LRP-1 targeting siRNA (siLRP-1) were cultured for 2 d in DMEM containing 10% FCS. A ) Results of TaqMan
    Figure Legend Snippet: siRNA-mediated knockdown of LRP-1 impairs ADAMTS-5 endocytosis in human chondrocytes. Human chondrocytes transfected with nontargeting siRNA (siCtrl) or LRP-1 targeting siRNA (siLRP-1) were cultured for 2 d in DMEM containing 10% FCS. A ) Results of TaqMan

    Techniques Used: Transfection, Cell Culture

    12) Product Images from "Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase II?"

    Article Title: Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase II?

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201202155

    KIF4 and SMC2 act in parallel to shape mitotic chromosomes and are opposed by Topo IIα. (A) SMC2 ON/OFF cells treated as shown were washed in either PBS or 75 mM KCl and fixed in methanol/acetic acid. (B) SMC2 ON/OFF cells were transfected with either control or KIF4 or Topo IIα siRNA as in Fig. 6, B–D . Simultaneous transfection of the SMC2 ON/OFF cells with KIF4 and Topo IIα siRNA oligos gave the triple knockout. Cells were washed in PBS or 75 mM KCl (hypotonic treatment), fixed in methanol acetic acid, and stained with DAPI. Bars, 5 µm.
    Figure Legend Snippet: KIF4 and SMC2 act in parallel to shape mitotic chromosomes and are opposed by Topo IIα. (A) SMC2 ON/OFF cells treated as shown were washed in either PBS or 75 mM KCl and fixed in methanol/acetic acid. (B) SMC2 ON/OFF cells were transfected with either control or KIF4 or Topo IIα siRNA as in Fig. 6, B–D . Simultaneous transfection of the SMC2 ON/OFF cells with KIF4 and Topo IIα siRNA oligos gave the triple knockout. Cells were washed in PBS or 75 mM KCl (hypotonic treatment), fixed in methanol acetic acid, and stained with DAPI. Bars, 5 µm.

    Techniques Used: Activated Clotting Time Assay, Transfection, Triple Knockout, Staining

    KIF4 partially colocalizes with SMC2 and topo IIα and affects condensin localization on the chromosome axis. (A) SMC2 OFF cells expressing SMC2-TrAP were stained with guinea pig anti-topo IIα, mouse anti-SBP, and rabbit anti-KIF4 plus DAPI for the DNA. Bar, 5 µm. (B and C) Asynchronously growing SMC2 OFF :SMC2-GFP-STS cells were transfected with either control or KIF4 siRNA oligos. After 20–24 h, these cells were fixed in PFA/PBS and stained by anti–CENP-T antibody and DAPI. Bars, 2 µm. (D) Quantification of B and C. Intensity of SMC2-GFP signal was measured at 5 kinetochores (Kt), 5 chromosome arms (Arm), and 5 areas in the cytosol in each of 20 cells. (E–G) Asynchronously growing SMC2 OFF :SMC2-GFP-STS cells (E), CAP-H OFF :CAP-H-GFP-TrAP cells (F), or CAP-D3 OFF :CAP-D3-GFP-TrAP cells (G) were treated as in B and C and stained with DAPI. Bars, 2 µm.
    Figure Legend Snippet: KIF4 partially colocalizes with SMC2 and topo IIα and affects condensin localization on the chromosome axis. (A) SMC2 OFF cells expressing SMC2-TrAP were stained with guinea pig anti-topo IIα, mouse anti-SBP, and rabbit anti-KIF4 plus DAPI for the DNA. Bar, 5 µm. (B and C) Asynchronously growing SMC2 OFF :SMC2-GFP-STS cells were transfected with either control or KIF4 siRNA oligos. After 20–24 h, these cells were fixed in PFA/PBS and stained by anti–CENP-T antibody and DAPI. Bars, 2 µm. (D) Quantification of B and C. Intensity of SMC2-GFP signal was measured at 5 kinetochores (Kt), 5 chromosome arms (Arm), and 5 areas in the cytosol in each of 20 cells. (E–G) Asynchronously growing SMC2 OFF :SMC2-GFP-STS cells (E), CAP-H OFF :CAP-H-GFP-TrAP cells (F), or CAP-D3 OFF :CAP-D3-GFP-TrAP cells (G) were treated as in B and C and stained with DAPI. Bars, 2 µm.

    Techniques Used: Expressing, Staining, Transfection

    13) Product Images from "Vitamin D Analogs Differentially Control Antimicrobial Peptide/"Alarmin"Expression in Psoriasis"

    Article Title: Vitamin D Analogs Differentially Control Antimicrobial Peptide/"Alarmin"Expression in Psoriasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006340

    VDR and the MEK/ERK signaling pathway are involved in cathelicidin induction by vitamin D analogs. To characterize the role of the VDR in increased cathelicidin expression after treatment with vitamin D analogs, NHEK were transfected with siRNA to decrease VDR expression before stimulation with vitamin D analogs ZK191784, ZK203278 and calcipotriol (all at 10 −8 M). Silencing of VDR was confirmed by qPCR (A; left panel) and Western blot (B; middle panel). siRNA suppression of VDR significantly reduced the induction of cathelicidin mRNA by all vitamin D analogs after 24 hours (A; right panel) (* P
    Figure Legend Snippet: VDR and the MEK/ERK signaling pathway are involved in cathelicidin induction by vitamin D analogs. To characterize the role of the VDR in increased cathelicidin expression after treatment with vitamin D analogs, NHEK were transfected with siRNA to decrease VDR expression before stimulation with vitamin D analogs ZK191784, ZK203278 and calcipotriol (all at 10 −8 M). Silencing of VDR was confirmed by qPCR (A; left panel) and Western blot (B; middle panel). siRNA suppression of VDR significantly reduced the induction of cathelicidin mRNA by all vitamin D analogs after 24 hours (A; right panel) (* P

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    14) Product Images from "New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways"

    Article Title: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021039

    NTH1-catalyzed DNA glycosylase activity towards pyrimidine-derived hydantoins in HeLa cell extracts. 5′-[ 32 P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. ( C ) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods .
    Figure Legend Snippet: NTH1-catalyzed DNA glycosylase activity towards pyrimidine-derived hydantoins in HeLa cell extracts. 5′-[ 32 P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. ( C ) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods .

    Techniques Used: Activity Assay, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Purification, Recombinant, Generated

    APE1-catalyzed nucleotide incision activity towards pyrimidine-derived hydantoins in HeLa cells extracts. 3′-[α- 32 P]-ddATP-labelled oligonucleotide duplexes were incubated with either 0.5 µg of HeLa cells extract or a purified protein either under “NIR+Zn 2+ ” (lanes 2–3, 6 and 9–10, 13) or under “BER+EDTA” conditions (lanes 4–5, 7 and 11–12, 14) for 1 h at 37°C. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lanes 2 and 4, 5OH-Hyd•G incubated with extracts from HeLa cells treated with the non-specific siRNA (100 nM); lanes 3 and 5, 5OH-Hyd•G incubated with extract from HeLa cells treated with the APE1-specific siRNA (100 nM); lane 6, 5OH-Hyd•G treated with 1 nM APE1; lane 7, 5OH-Hyd•G treated with 5 nM NEIL1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of siRNA-induced down-regulation of the APE1 expression in HeLa cells. ( C ) Graphic representation of the mean values of DNA glycosylase (BER) and AP endonuclease (NIR) activities in extracts, representing amounts of the 13-mer and 14-mer products, respectively. The cleavage activities in each cell free extract were normalized to the relative densitometry values of actin bands on western blot in panel B. For details see Materials and Methods .
    Figure Legend Snippet: APE1-catalyzed nucleotide incision activity towards pyrimidine-derived hydantoins in HeLa cells extracts. 3′-[α- 32 P]-ddATP-labelled oligonucleotide duplexes were incubated with either 0.5 µg of HeLa cells extract or a purified protein either under “NIR+Zn 2+ ” (lanes 2–3, 6 and 9–10, 13) or under “BER+EDTA” conditions (lanes 4–5, 7 and 11–12, 14) for 1 h at 37°C. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lanes 2 and 4, 5OH-Hyd•G incubated with extracts from HeLa cells treated with the non-specific siRNA (100 nM); lanes 3 and 5, 5OH-Hyd•G incubated with extract from HeLa cells treated with the APE1-specific siRNA (100 nM); lane 6, 5OH-Hyd•G treated with 1 nM APE1; lane 7, 5OH-Hyd•G treated with 5 nM NEIL1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of siRNA-induced down-regulation of the APE1 expression in HeLa cells. ( C ) Graphic representation of the mean values of DNA glycosylase (BER) and AP endonuclease (NIR) activities in extracts, representing amounts of the 13-mer and 14-mer products, respectively. The cleavage activities in each cell free extract were normalized to the relative densitometry values of actin bands on western blot in panel B. For details see Materials and Methods .

    Techniques Used: Activity Assay, Derivative Assay, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing

    15) Product Images from "Integrin ?9?1 promotes malignant tumor growth and metastasis by potentiating epithelial-mesenchymal transition"

    Article Title: Integrin ?9?1 promotes malignant tumor growth and metastasis by potentiating epithelial-mesenchymal transition

    Journal: Oncogene

    doi: 10.1038/onc.2012.41

    α9β1-induced malignant tumor growth and metastasis is inhibited followingα9 knockdown A, Flow cytometry for expression of α9β1 in LLC-1 cells transduced with non-targeted (shRNA-Cont) or α9β1-targeted (shRNA-α9) shRNA. B, Cell adhesion C, Haptotaxis (using α9β1-specific ligand, TnfnRAA) and chemotaxis (using 10% FBS) and D, Cell invasion assays in shRNA-Cont (grey bars) or shRNA-α9 (black bars) LLC-1 grown on plastic (no matrix) or TnfnRAA or pFN as indicated. E, Immunoblots to detect EMT and signaling proteins as indicated in lysates from LLC-1 cells expressing shRNA-cont (grey) or shRNA-α9, and grown with (+) or without (−) TnfnRAA. F, Volume of mouse flank tumors from subcutaneous injection of LLC-1 cells expressing shRNA-Cont (grey) or shRNA-α9 (black) ** p
    Figure Legend Snippet: α9β1-induced malignant tumor growth and metastasis is inhibited followingα9 knockdown A, Flow cytometry for expression of α9β1 in LLC-1 cells transduced with non-targeted (shRNA-Cont) or α9β1-targeted (shRNA-α9) shRNA. B, Cell adhesion C, Haptotaxis (using α9β1-specific ligand, TnfnRAA) and chemotaxis (using 10% FBS) and D, Cell invasion assays in shRNA-Cont (grey bars) or shRNA-α9 (black bars) LLC-1 grown on plastic (no matrix) or TnfnRAA or pFN as indicated. E, Immunoblots to detect EMT and signaling proteins as indicated in lysates from LLC-1 cells expressing shRNA-cont (grey) or shRNA-α9, and grown with (+) or without (−) TnfnRAA. F, Volume of mouse flank tumors from subcutaneous injection of LLC-1 cells expressing shRNA-Cont (grey) or shRNA-α9 (black) ** p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Transduction, shRNA, Chemotaxis Assay, Western Blot, Injection

    α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signaling A, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.
    Figure Legend Snippet: α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signaling A, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.

    Techniques Used: Western Blot, Cell Culture, Staining, Transfection, Migration

    16) Product Images from "Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores"

    Article Title: Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201304197

    Partial loss of Knl1, RZZ, and Mad1 at Zwint1-depleted kinetochores. (A) Immunoblot of endogenous Zwint1 and α-tubulin (loading control) from HeLa cells treated with control or Zwint1 siRNA for 48 h. (B, E, F, H, and I) Nocodazole-treated HeLa cells treated with either control luciferase or Zwint1 siRNA were immunostained using anti-Hec1 9G3 (B, E, F, and H) or anti-Zwint1 (I) and either anti-Zwint1 (B), anti-Knl1 (E), anti-Rod (F), anti-Zwilch (H), or anti-Mad1 (I) antibodies. (C, D, G, and J) Kinetochore fluorescence was normalized relative to Hec1 (C, D, and G) or ACA (J) in control and Zwint1-depleted cells. n = 100 kinetochores; P
    Figure Legend Snippet: Partial loss of Knl1, RZZ, and Mad1 at Zwint1-depleted kinetochores. (A) Immunoblot of endogenous Zwint1 and α-tubulin (loading control) from HeLa cells treated with control or Zwint1 siRNA for 48 h. (B, E, F, H, and I) Nocodazole-treated HeLa cells treated with either control luciferase or Zwint1 siRNA were immunostained using anti-Hec1 9G3 (B, E, F, and H) or anti-Zwint1 (I) and either anti-Zwint1 (B), anti-Knl1 (E), anti-Rod (F), anti-Zwilch (H), or anti-Mad1 (I) antibodies. (C, D, G, and J) Kinetochore fluorescence was normalized relative to Hec1 (C, D, and G) or ACA (J) in control and Zwint1-depleted cells. n = 100 kinetochores; P

    Techniques Used: Luciferase, Fluorescence

    Complete loss of Zwint1, RZZ, and Mad1 at Knl1-depleted kinetochores. (A–I) Nocodazole-treated HeLa cells treated with either control luciferase or Knl1 siRNA were immunostained using anti-Hec1 9G3 (A, C, E, and G) or anti-Knl1 (H) and either anti-Knl1 (A), anti-Zwint1 (B), anti-Rod (E), anti-Zwilch (G), or anti-Mad1 (H) antibodies. Kinetochore fluorescence was normalized relative to Hec1 (B, D, and F) or ACA (I) in control and Knl1-depleted cells. n = 100 kinetochores; P
    Figure Legend Snippet: Complete loss of Zwint1, RZZ, and Mad1 at Knl1-depleted kinetochores. (A–I) Nocodazole-treated HeLa cells treated with either control luciferase or Knl1 siRNA were immunostained using anti-Hec1 9G3 (A, C, E, and G) or anti-Knl1 (H) and either anti-Knl1 (A), anti-Zwint1 (B), anti-Rod (E), anti-Zwilch (G), or anti-Mad1 (H) antibodies. Kinetochore fluorescence was normalized relative to Hec1 (B, D, and F) or ACA (I) in control and Knl1-depleted cells. n = 100 kinetochores; P

    Techniques Used: Luciferase, Fluorescence

    17) Product Images from "The FIP3-Rab11 Protein Complex Regulates Recycling Endosome Targeting to the Cleavage Furrow during Late Cytokinesis D⃞V⃞"

    Article Title: The FIP3-Rab11 Protein Complex Regulates Recycling Endosome Targeting to the Cleavage Furrow during Late Cytokinesis D⃞V⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E04-10-0927

    FIP3 and Rab11 regulate the late stage of cytokinesis. (A) HeLa cells were transfected with FIP3 siRNA and incubated for 48 h. Cells were then harvested and levels of FIP3 protein determined by immunoblotting. (B) HeLa cells were transfected with FIP3
    Figure Legend Snippet: FIP3 and Rab11 regulate the late stage of cytokinesis. (A) HeLa cells were transfected with FIP3 siRNA and incubated for 48 h. Cells were then harvested and levels of FIP3 protein determined by immunoblotting. (B) HeLa cells were transfected with FIP3

    Techniques Used: Transfection, Incubation

    18) Product Images from "SOCS3 Drives Proteasomal Degradation of TBK1 and Negatively Regulates Antiviral Innate Immunity"

    Article Title: SOCS3 Drives Proteasomal Degradation of TBK1 and Negatively Regulates Antiviral Innate Immunity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00090-15

    Knockdown of SOCS3 activates the IFN-β signaling pathway. (A) qRT-PCR (left) and RT-PCR (right) analysis of SOCS3 knockdown efficiency at the mRNA level in HeLa cells. (B) qRT-PCR analysis of SOCS3 knockdown efficiency at the mRNA level in 293T cells. (C) Immunoblot analysis of the knockdown of exogenous SOCS3 in 293T cells transfected with Flag-tagged SOCS3 plasmid and siRNA control or SOCS3-specific siRNAs. (D) Real-time PCR analysis of IFN-β mRNA in 293T cells transfected with control siRNA or siRNA against SOCS3. (E) 293T cells were transfected with siRNA control (si-control) or si-1 for 36 h and further transfected with plasmids harboring genes for the IFN-β promoter and TK reporter genes, with or without a TBK1 expression vector. The activity of the IFN-β promoter was analyzed in dual-luciferase assays. (F) HeLa cells were transfected with si-control or si-1 and further transfected with or without TBK1 plasmid. The transcription of IFN-β mRNA was evaluated by real-time PCR. (G) Thirty-six hours after transfection with siRNAs, 293T cells were transfected with the indicated plasmids. Luciferase assays were performed to evaluate the activity of the ISRE reporter. (H) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with IRF3 plasmid (50 ng/ml) and si-control or si-1. (I) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with the indicated siRNA or SOCS3 rescue plasmid. Overexpressed and endogenous SOCS3 is indicated with an arrow. (J) Primary MEF cells were transfected with si-control or si-1. The expression levels of TBK1 and SOCS3 were evaluated by IB analysis. Error bars indicate standard deviations. *, P
    Figure Legend Snippet: Knockdown of SOCS3 activates the IFN-β signaling pathway. (A) qRT-PCR (left) and RT-PCR (right) analysis of SOCS3 knockdown efficiency at the mRNA level in HeLa cells. (B) qRT-PCR analysis of SOCS3 knockdown efficiency at the mRNA level in 293T cells. (C) Immunoblot analysis of the knockdown of exogenous SOCS3 in 293T cells transfected with Flag-tagged SOCS3 plasmid and siRNA control or SOCS3-specific siRNAs. (D) Real-time PCR analysis of IFN-β mRNA in 293T cells transfected with control siRNA or siRNA against SOCS3. (E) 293T cells were transfected with siRNA control (si-control) or si-1 for 36 h and further transfected with plasmids harboring genes for the IFN-β promoter and TK reporter genes, with or without a TBK1 expression vector. The activity of the IFN-β promoter was analyzed in dual-luciferase assays. (F) HeLa cells were transfected with si-control or si-1 and further transfected with or without TBK1 plasmid. The transcription of IFN-β mRNA was evaluated by real-time PCR. (G) Thirty-six hours after transfection with siRNAs, 293T cells were transfected with the indicated plasmids. Luciferase assays were performed to evaluate the activity of the ISRE reporter. (H) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with IRF3 plasmid (50 ng/ml) and si-control or si-1. (I) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with the indicated siRNA or SOCS3 rescue plasmid. Overexpressed and endogenous SOCS3 is indicated with an arrow. (J) Primary MEF cells were transfected with si-control or si-1. The expression levels of TBK1 and SOCS3 were evaluated by IB analysis. Error bars indicate standard deviations. *, P

    Techniques Used: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Luciferase

    SOCS3 targets TBK1 for degradation. (A) Plasmids carrying genes for adaptors of the RIG-I signal were separately transfected into 293T cells with or without Myc-SOCS3 cotransfection. IB was performed to identify the abundance of these adaptors (Flag-tagged MAVS, TRAF3, TBK1, TANK, IKKε, and IRF3). (B and C) 293T or HeLa cells were transfected with the indicated plasmids. The expression levels of TBK1 and p-TBK1 (S172) were evaluated by Western blotting. (D and E) The time courses of SOCS3-mediated degradation of TBK1 and inhibition of IFN-β activity were evaluated by Western blotting and a dual-luciferase assay in 293T cells, respectively. (F) 293T cells were transfected with control siRNA (si-control) or si-1 (SOCS3-specific siRNA) by using Lipofectamine 2000 for 36 h, followed by the transfection with Flag-TBK1. IB analysis showed the expression of TBK1 and SOCS3 proteins. (G) Constructs of Myc-tagged wild-type SOCS3 and its truncations (left). Flag-tagged TBK1 and wild-type or mutated Myc-SOCS3 were transfected into 293T cells for 24 h, and then IB for TBK1 expression levels (right). (H) 293T cells were transfected with IRF3 (50 ng/ml), Flag-TBK1, and Myc-SOCS3 wild type or truncations for 24 h, and IRF3 phosphorylation and total IRF3 were analyzed by IB. (I) A dual-luciferase assay was performed to identify the activation of the IFN-β reporter in 293T cells transfected with the indicated plasmids. (J) 293T cells were transfected with the indicated plasmids for 24 h. Cells lysates were incubated with protein G beads plus anti-Flag Ab at 4°C overnight, followed by immunoblot analysis with anti-p-TBK1 Ab. (K) 293T cells were transfected with increasing amounts of SOCS3 plasmid and Flag-TBK1-S172A mutant or Flag-TBK1-K38A mutant for 24 h. The expression of the two mutants was evaluated by Western blotting. Error bars indicate standard deviations. **, P
    Figure Legend Snippet: SOCS3 targets TBK1 for degradation. (A) Plasmids carrying genes for adaptors of the RIG-I signal were separately transfected into 293T cells with or without Myc-SOCS3 cotransfection. IB was performed to identify the abundance of these adaptors (Flag-tagged MAVS, TRAF3, TBK1, TANK, IKKε, and IRF3). (B and C) 293T or HeLa cells were transfected with the indicated plasmids. The expression levels of TBK1 and p-TBK1 (S172) were evaluated by Western blotting. (D and E) The time courses of SOCS3-mediated degradation of TBK1 and inhibition of IFN-β activity were evaluated by Western blotting and a dual-luciferase assay in 293T cells, respectively. (F) 293T cells were transfected with control siRNA (si-control) or si-1 (SOCS3-specific siRNA) by using Lipofectamine 2000 for 36 h, followed by the transfection with Flag-TBK1. IB analysis showed the expression of TBK1 and SOCS3 proteins. (G) Constructs of Myc-tagged wild-type SOCS3 and its truncations (left). Flag-tagged TBK1 and wild-type or mutated Myc-SOCS3 were transfected into 293T cells for 24 h, and then IB for TBK1 expression levels (right). (H) 293T cells were transfected with IRF3 (50 ng/ml), Flag-TBK1, and Myc-SOCS3 wild type or truncations for 24 h, and IRF3 phosphorylation and total IRF3 were analyzed by IB. (I) A dual-luciferase assay was performed to identify the activation of the IFN-β reporter in 293T cells transfected with the indicated plasmids. (J) 293T cells were transfected with the indicated plasmids for 24 h. Cells lysates were incubated with protein G beads plus anti-Flag Ab at 4°C overnight, followed by immunoblot analysis with anti-p-TBK1 Ab. (K) 293T cells were transfected with increasing amounts of SOCS3 plasmid and Flag-TBK1-S172A mutant or Flag-TBK1-K38A mutant for 24 h. The expression of the two mutants was evaluated by Western blotting. Error bars indicate standard deviations. **, P

    Techniques Used: Transfection, Cotransfection, Expressing, Western Blot, Inhibition, Activity Assay, Luciferase, Construct, Activation Assay, Incubation, Plasmid Preparation, Mutagenesis

    SOCS3 increases K48-linked ubiquitination of TBK1. (A) Immunoblot analysis of TBK1 expression in 293T cells with DMSO or MG132 treatment for 6 h after transfection for 24 h. (B) 293T cells were transfected with plasmids harboring genes for wild-type or mutated HA-Ub with or without Myc-SOCS3 cotransfection for 24 h. Cell lysates were incubated with protein G beads plus anti-TBK1 Ab at 4°C overnight, followed by immunoblot analysis with anti-HA Ab. (C) 293T cells were transfected with control siRNA (si-control) or si-1 for 24 h, followed by further transfection with HA-Ub plasmids for 24 h. Cell lysates were used to perform ubiquitination assays. (D) Ubiquitination assays of endogenous TBK1. 293T cells were transfected with HA-Ub and SOCS3 plasmid or poly(I·C), and then the cell lysates were incubated with protein G beads plus anti-TBK1 Ab at 4°C overnight, followed by immunoblot analysis with anti-HA Ab. (E) 293T cells were transfected with plasmids harboring genes for Flag-TBK1, His-Ub, and Myc-tagged wild-type or truncated SOCS3 for 24 h, followed by immunoprecipitation with protein G beads plus anti-Flag Ab and immunoblot analysis with anti-His Ab. (F) 293T cells were transfected with Flag-TBK1, HA-Ub(K63), or Myc-SOCS3 wild type or ΔBOX for 24 h, and then lysates were analyzed in ubiquitination assays.
    Figure Legend Snippet: SOCS3 increases K48-linked ubiquitination of TBK1. (A) Immunoblot analysis of TBK1 expression in 293T cells with DMSO or MG132 treatment for 6 h after transfection for 24 h. (B) 293T cells were transfected with plasmids harboring genes for wild-type or mutated HA-Ub with or without Myc-SOCS3 cotransfection for 24 h. Cell lysates were incubated with protein G beads plus anti-TBK1 Ab at 4°C overnight, followed by immunoblot analysis with anti-HA Ab. (C) 293T cells were transfected with control siRNA (si-control) or si-1 for 24 h, followed by further transfection with HA-Ub plasmids for 24 h. Cell lysates were used to perform ubiquitination assays. (D) Ubiquitination assays of endogenous TBK1. 293T cells were transfected with HA-Ub and SOCS3 plasmid or poly(I·C), and then the cell lysates were incubated with protein G beads plus anti-TBK1 Ab at 4°C overnight, followed by immunoblot analysis with anti-HA Ab. (E) 293T cells were transfected with plasmids harboring genes for Flag-TBK1, His-Ub, and Myc-tagged wild-type or truncated SOCS3 for 24 h, followed by immunoprecipitation with protein G beads plus anti-Flag Ab and immunoblot analysis with anti-His Ab. (F) 293T cells were transfected with Flag-TBK1, HA-Ub(K63), or Myc-SOCS3 wild type or ΔBOX for 24 h, and then lysates were analyzed in ubiquitination assays.

    Techniques Used: Expressing, Transfection, Cotransfection, Incubation, Plasmid Preparation, Immunoprecipitation

    SOCS3 facilitates viral replication. (A) 293T cells were transfected with or without TBK1 for 24 h, followed by WSN infection for 0 h, 12 h, or 16 h. The transcription of vRNAs ( NP , NA , PA , and M1 ) was evaluated by qRT-PCR. (B) 293T cells were transfected with or without SOCS3 for 24 h, followed by WSN infection for 0 h, 12 h, or 16 h. The transcription of vRNAs ( NP , NA , PA , and M1 ) was evaluated by qRT-PCR. (C) Control siRNA or SOCS3-specific siRNA (si-1) was transfected into A549 cells for 48 h, followed by WSN infection for 0 h, 12 h, or 16 h. qRT-PCR was performed to detect the expression of vRNAs ( NP , NA , PA , and M1 ). (D) IRF3 phosphorylation was evaluated in 293T cells transfected with IRF3 (50 ng/ml) and Flag-3.0 or Flag-SOCS3 for 24 h followed by VSV infection for the indicated times. (E) 293T cells were transfected with the indicated plasmids for 24 h, and then cells were infected with WSN for an additional 0 h, 12 h, 16 h, or 24 h. IB was used for analysis of the phosphorylation of IRF3 and total IRF3. (F) Immunoblot analysis results showing the expression of TBK1 and SOCS3 in primary MEF cells with WSN virus infection. (G and H) ELISA results for IFN-β production at the protein level in RAW 264.7 cells or primary MEF cells with 10 μg/ml or 1 μg/ml poly(I·C) stimulation, respectively. Error bars indicate standard deviations.
    Figure Legend Snippet: SOCS3 facilitates viral replication. (A) 293T cells were transfected with or without TBK1 for 24 h, followed by WSN infection for 0 h, 12 h, or 16 h. The transcription of vRNAs ( NP , NA , PA , and M1 ) was evaluated by qRT-PCR. (B) 293T cells were transfected with or without SOCS3 for 24 h, followed by WSN infection for 0 h, 12 h, or 16 h. The transcription of vRNAs ( NP , NA , PA , and M1 ) was evaluated by qRT-PCR. (C) Control siRNA or SOCS3-specific siRNA (si-1) was transfected into A549 cells for 48 h, followed by WSN infection for 0 h, 12 h, or 16 h. qRT-PCR was performed to detect the expression of vRNAs ( NP , NA , PA , and M1 ). (D) IRF3 phosphorylation was evaluated in 293T cells transfected with IRF3 (50 ng/ml) and Flag-3.0 or Flag-SOCS3 for 24 h followed by VSV infection for the indicated times. (E) 293T cells were transfected with the indicated plasmids for 24 h, and then cells were infected with WSN for an additional 0 h, 12 h, 16 h, or 24 h. IB was used for analysis of the phosphorylation of IRF3 and total IRF3. (F) Immunoblot analysis results showing the expression of TBK1 and SOCS3 in primary MEF cells with WSN virus infection. (G and H) ELISA results for IFN-β production at the protein level in RAW 264.7 cells or primary MEF cells with 10 μg/ml or 1 μg/ml poly(I·C) stimulation, respectively. Error bars indicate standard deviations.

    Techniques Used: Transfection, Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

    19) Product Images from "Mitochondrial glycerol 3‐phosphate dehydrogenase promotes skeletal muscle regeneration"

    Article Title: Mitochondrial glycerol 3‐phosphate dehydrogenase promotes skeletal muscle regeneration

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201809390

    mGPDH effect occurs via the CaMKKβ/AMPK control of mitochondrial biogenesis Mitochondrial DNA (A), nuclear‐encoded OXPHOS genes (B), respirometry analysis (C), and immunoblots of mGPDH, phospho‐Thr172 AMPK (p‐AMPK), total AMPK (AMPK), phospho‐Ser79‐ACC (p‐ACC), total ACC and PGC1α, and corresponding quantifications represent mGPDH, p‐AMPK, p‐ACC, and PGC1α protein levels (D–F) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. Immunoblots of p‐AMPK, p‐ACC, and PGC1α and corresponding quantifications represent p‐AMPK, p‐ACC, and PGC1α protein levels (G), mitochondrial DNA (H), and nuclear‐encoded OXPHOS genes combined by NDUFS8 , SDHb , Uqcrc1 , COX5b , and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation. NAD + /NADH ratio (J) and immunoprecipitation analysis for PGC1α acetyl‐lysine (Ac‐Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. Immunoblot of c‐myc and myogenin (L) and corresponding quantifications represent c‐myc and myogenin protein levels (M), representative images of MyHC immunofluorescence (N), fusion index (O), and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L, M) or 72 h (N–P) after differentiation. Immunoblots of p‐AMPK, p‐ACC, PGC1α, and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO‐609 at 24 h after differentiation. Quantifications represent p‐AMPK, p‐ACC, PGC1α, and myogenin protein levels. Immunoblots of p‐AMPK and p‐ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca 2+ chelator BAPTA‐AM at 24 h after differentiation. Quantifications represent p‐AMPK and p‐ACC protein levels. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (N). In panels (A, B, D–M, Q, and R), n = 3; in panel (C), n = 10; in panels (N–P), n = 15. * P
    Figure Legend Snippet: mGPDH effect occurs via the CaMKKβ/AMPK control of mitochondrial biogenesis Mitochondrial DNA (A), nuclear‐encoded OXPHOS genes (B), respirometry analysis (C), and immunoblots of mGPDH, phospho‐Thr172 AMPK (p‐AMPK), total AMPK (AMPK), phospho‐Ser79‐ACC (p‐ACC), total ACC and PGC1α, and corresponding quantifications represent mGPDH, p‐AMPK, p‐ACC, and PGC1α protein levels (D–F) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. Immunoblots of p‐AMPK, p‐ACC, and PGC1α and corresponding quantifications represent p‐AMPK, p‐ACC, and PGC1α protein levels (G), mitochondrial DNA (H), and nuclear‐encoded OXPHOS genes combined by NDUFS8 , SDHb , Uqcrc1 , COX5b , and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation. NAD + /NADH ratio (J) and immunoprecipitation analysis for PGC1α acetyl‐lysine (Ac‐Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. Immunoblot of c‐myc and myogenin (L) and corresponding quantifications represent c‐myc and myogenin protein levels (M), representative images of MyHC immunofluorescence (N), fusion index (O), and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L, M) or 72 h (N–P) after differentiation. Immunoblots of p‐AMPK, p‐ACC, PGC1α, and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO‐609 at 24 h after differentiation. Quantifications represent p‐AMPK, p‐ACC, PGC1α, and myogenin protein levels. Immunoblots of p‐AMPK and p‐ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca 2+ chelator BAPTA‐AM at 24 h after differentiation. Quantifications represent p‐AMPK and p‐ACC protein levels. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (N). In panels (A, B, D–M, Q, and R), n = 3; in panel (C), n = 10; in panels (N–P), n = 15. * P

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Immunofluorescence

    mGPDH regulates myoblast differentiation qRT–PCR (A) and immunoblot (B) of mGPDH, myogenin, and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to β‐actin. Immunoblot of mGPDH, voltage‐dependent anion channel (VDAC), and cytochrome c (Cyt C) levels in mitochondrial lysate during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to COX IV. Activity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiation. Representative images of MyHC immunofluorescence (E) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH; the fusion index (F) and the distribution of nuclei per myotube (G) were calculated at day 5 after differentiation. Immunoblot of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantification (I–K) represents the levels of the indicated protein normalized to β‐actin at the indicated day after differentiation. qRT–PCR analysis of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with the siRNA or the overexpression plasmid for mGPDH at day 4 after differentiation. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (E). In panels (A–D) and (H–M), n = 3; in panels (E–G), n = 15. * P
    Figure Legend Snippet: mGPDH regulates myoblast differentiation qRT–PCR (A) and immunoblot (B) of mGPDH, myogenin, and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to β‐actin. Immunoblot of mGPDH, voltage‐dependent anion channel (VDAC), and cytochrome c (Cyt C) levels in mitochondrial lysate during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to COX IV. Activity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiation. Representative images of MyHC immunofluorescence (E) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH; the fusion index (F) and the distribution of nuclei per myotube (G) were calculated at day 5 after differentiation. Immunoblot of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantification (I–K) represents the levels of the indicated protein normalized to β‐actin at the indicated day after differentiation. qRT–PCR analysis of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with the siRNA or the overexpression plasmid for mGPDH at day 4 after differentiation. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (E). In panels (A–D) and (H–M), n = 3; in panels (E–G), n = 15. * P

    Techniques Used: Quantitative RT-PCR, Activity Assay, Immunofluorescence, Transfection, Over Expression, Plasmid Preparation

    Effect of cGPDH on myoblast differentiation cGPDH expression during C2C12 myocyte differentiation. Representative images of MyHC immunofluorescence (B) of C2C12 myocytes transfected with siRNA targeting cGPDH; the fusion index (C) and the distribution of nuclei per myotube (D) were calculated. qRT–PCR (E) and Western blot analysis (F) of myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting cGPDH. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (B). In panels (A–F), n = 3. * P
    Figure Legend Snippet: Effect of cGPDH on myoblast differentiation cGPDH expression during C2C12 myocyte differentiation. Representative images of MyHC immunofluorescence (B) of C2C12 myocytes transfected with siRNA targeting cGPDH; the fusion index (C) and the distribution of nuclei per myotube (D) were calculated. qRT–PCR (E) and Western blot analysis (F) of myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting cGPDH. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (B). In panels (A–F), n = 3. * P

    Techniques Used: Expressing, Immunofluorescence, Transfection, Quantitative RT-PCR, Western Blot

    20) Product Images from "Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition"

    Article Title: Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.001379

    Acute induction of CDC25C or suppression of WEE1 partially reverses the effect of AMPK activation on the G 2 /M-phase transition. A , model showing that the G 2 /M-phase transition is regulated by CDC25C and WEE1. B , schematic view of cell synchronization and the indicated treatments. Synchronized HeLa cells that stably express reverse tetracycline-controlled transactivator and doxycycline-inducible CDC25C were treated with doxycycline when cells were released from the second thymidine block (G 1 /S boundary). Synchronized HeLa cells were transfected with WEE1 siRNA at the G 1 /S boundary or treated with WEE1 inhibitor at G 2 phase (7 h after cells were released from the second thymidine block), respectively. AMPK activators and nocodazole were added when cells are in G 2 phase. C , the percentage of M-phase cells in HeLa cells transfected with scramble siRNA or WEE1 siRNA was determined by phosphorylation of histone H3 ( pH3 ) staining. The percentage of M-phase cells in vehicle-treated cells was set to 100, and the value of AMPK activator–treated cells was normalized to that of vehicle-treated cells. Error bars , S.D. of triplicate samples ( left ). Cell lysates were blotted with the indicated antibodies ( right ). D , the percentage of M-phase cells in HeLa cells that stably express reverse tetracycline transcriptional activator ( Ctrl ) or CDC25C-S216A following doxycycline treatment was determined by phosphorylation of histone H3 ( pH3 ) staining. The percentage of M-phase cells in vehicle-treated cells were set to 100, and the value of AMPK activator–treated cells was normalized to that of vehicle-treated cells. Error bars , S.D. of triplicate samples ( left ). Cell lysates were blotted with the indicated antibodies ( right ). *, p
    Figure Legend Snippet: Acute induction of CDC25C or suppression of WEE1 partially reverses the effect of AMPK activation on the G 2 /M-phase transition. A , model showing that the G 2 /M-phase transition is regulated by CDC25C and WEE1. B , schematic view of cell synchronization and the indicated treatments. Synchronized HeLa cells that stably express reverse tetracycline-controlled transactivator and doxycycline-inducible CDC25C were treated with doxycycline when cells were released from the second thymidine block (G 1 /S boundary). Synchronized HeLa cells were transfected with WEE1 siRNA at the G 1 /S boundary or treated with WEE1 inhibitor at G 2 phase (7 h after cells were released from the second thymidine block), respectively. AMPK activators and nocodazole were added when cells are in G 2 phase. C , the percentage of M-phase cells in HeLa cells transfected with scramble siRNA or WEE1 siRNA was determined by phosphorylation of histone H3 ( pH3 ) staining. The percentage of M-phase cells in vehicle-treated cells was set to 100, and the value of AMPK activator–treated cells was normalized to that of vehicle-treated cells. Error bars , S.D. of triplicate samples ( left ). Cell lysates were blotted with the indicated antibodies ( right ). D , the percentage of M-phase cells in HeLa cells that stably express reverse tetracycline transcriptional activator ( Ctrl ) or CDC25C-S216A following doxycycline treatment was determined by phosphorylation of histone H3 ( pH3 ) staining. The percentage of M-phase cells in vehicle-treated cells were set to 100, and the value of AMPK activator–treated cells was normalized to that of vehicle-treated cells. Error bars , S.D. of triplicate samples ( left ). Cell lysates were blotted with the indicated antibodies ( right ). *, p

    Techniques Used: Activation Assay, Sublimation, Stable Transfection, Blocking Assay, Transfection, Staining

    21) Product Images from "The Replication Protein Cdc6 Suppresses Centrosome Over-Duplication in a Manner Independent of Its ATPase Activity"

    Article Title: The Replication Protein Cdc6 Suppresses Centrosome Over-Duplication in a Manner Independent of Its ATPase Activity

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2017.0191

    Depletion of Cdc6 induces centrosome over-duplication in a manner independent of cell cycle arrest (A) FACS analysis was performed with propidium iodide ( upper panel ) and BrdU ( lower panel ) staining of U2OS cells transfected with siRNA for the indicated time. Replicating S-phase cells are indicated in the dashed boxes. The proportion of replicating S-phase cells is shown below the FACS profiles. (B–D) Cells transfected with siRNA for 72 h were immunostained with anti-cyclin E (B), ant-cyclin A (C), or anti-cyclin B (D) and anti-γ-tubulin antibodies. The ratios of cells containing the indicated number of γ-tubulin spots ( bottom panel ) were determined as described in Figs. 1C and 1D . Values represent means ± standard deviations from at least 100 cells in each of the three independent experiments. siGL3, control siRNA GL3; siCdc6, Cdc6-specific siRNA; 1C, one γ-tubulin spot; 2C-L, two linked γ-tubulin spots; 2C-S, two separated γ-tubulin spots; 3 ≥ C, more than two γ-tubulin spots.
    Figure Legend Snippet: Depletion of Cdc6 induces centrosome over-duplication in a manner independent of cell cycle arrest (A) FACS analysis was performed with propidium iodide ( upper panel ) and BrdU ( lower panel ) staining of U2OS cells transfected with siRNA for the indicated time. Replicating S-phase cells are indicated in the dashed boxes. The proportion of replicating S-phase cells is shown below the FACS profiles. (B–D) Cells transfected with siRNA for 72 h were immunostained with anti-cyclin E (B), ant-cyclin A (C), or anti-cyclin B (D) and anti-γ-tubulin antibodies. The ratios of cells containing the indicated number of γ-tubulin spots ( bottom panel ) were determined as described in Figs. 1C and 1D . Values represent means ± standard deviations from at least 100 cells in each of the three independent experiments. siGL3, control siRNA GL3; siCdc6, Cdc6-specific siRNA; 1C, one γ-tubulin spot; 2C-L, two linked γ-tubulin spots; 2C-S, two separated γ-tubulin spots; 3 ≥ C, more than two γ-tubulin spots.

    Techniques Used: FACS, Staining, Transfection

    Centrosome over-duplication and premature centrosome separation in Cdc6-depleted cells (A) U2OS cells transected with the Cdc6-specific siRNA (siCdc6) or control GL3-siRNA (siGL3) were analyzed by immunoblotting with indicated antibodies at indicated times after transfection. (B) U2OS cells transfected with siGL3 ( upper panel ) or siCdc6 ( lower panel ) for 72 h were co-immunostained with antibodies specific to γ-tubulin (green) and CP110 (red); representative images are displayed. DNA was stained with DAPI. Fields containing centrosomes are shown at higher magnification in insets. Scale bar = 10 μm. (C) Number of γ-tubulin spots per cell was determined at the indicated time points after siRNA transfection and described in terms of the ratios over total cell number. 1C, one γ-tubulin spot; 2C-L, two linked γ-tubulin spots; 2C-S, two separated γ-tubulin spots; 3 ≥ C, more than two γ-tubulin spots. (D) After Cdc6-depletion for 72 h, the cells were co-immunostained with antibodies specific to CP110 and γ-tubulin. Centriole amplification was determined by observing CP110 spots, while centrosome number was determined by observing γ-tubulin spots. The number of γ-tubulin spots per cell is presented on the x -axis. The ratios of cells containing the indicated number of CP110 spots (top of the figure) and the average number of CP110 spots to the number of γ-tubulin spots are presented on the left and right of the y -axis, respectively. (E) Cdc6-siRNA-resistant FLAG-Cdc6-inducible U2OS Tet-On cells were transfected with the indicated siRNA for 72 h and doxycycline (2 mg/ml) was added to the culture medium for 48 h, prior to collecting the cells. The indicated proteins were detected by immunoblotting. Ctrl, U2OS Tet-On control cells; WT, FLAG-Cdc6-inducible U2OS Tet-On cells. (F) Cells from (E) were immunostained with anti-γ-tubulin antibody, and the number of γ-tubulin spots per cell was counted, as described in (C). Dox, doxycycline. Values represent mean ± standard deviation. At least 100 cells/experimental group were analyzed in three independent experiments.
    Figure Legend Snippet: Centrosome over-duplication and premature centrosome separation in Cdc6-depleted cells (A) U2OS cells transected with the Cdc6-specific siRNA (siCdc6) or control GL3-siRNA (siGL3) were analyzed by immunoblotting with indicated antibodies at indicated times after transfection. (B) U2OS cells transfected with siGL3 ( upper panel ) or siCdc6 ( lower panel ) for 72 h were co-immunostained with antibodies specific to γ-tubulin (green) and CP110 (red); representative images are displayed. DNA was stained with DAPI. Fields containing centrosomes are shown at higher magnification in insets. Scale bar = 10 μm. (C) Number of γ-tubulin spots per cell was determined at the indicated time points after siRNA transfection and described in terms of the ratios over total cell number. 1C, one γ-tubulin spot; 2C-L, two linked γ-tubulin spots; 2C-S, two separated γ-tubulin spots; 3 ≥ C, more than two γ-tubulin spots. (D) After Cdc6-depletion for 72 h, the cells were co-immunostained with antibodies specific to CP110 and γ-tubulin. Centriole amplification was determined by observing CP110 spots, while centrosome number was determined by observing γ-tubulin spots. The number of γ-tubulin spots per cell is presented on the x -axis. The ratios of cells containing the indicated number of CP110 spots (top of the figure) and the average number of CP110 spots to the number of γ-tubulin spots are presented on the left and right of the y -axis, respectively. (E) Cdc6-siRNA-resistant FLAG-Cdc6-inducible U2OS Tet-On cells were transfected with the indicated siRNA for 72 h and doxycycline (2 mg/ml) was added to the culture medium for 48 h, prior to collecting the cells. The indicated proteins were detected by immunoblotting. Ctrl, U2OS Tet-On control cells; WT, FLAG-Cdc6-inducible U2OS Tet-On cells. (F) Cells from (E) were immunostained with anti-γ-tubulin antibody, and the number of γ-tubulin spots per cell was counted, as described in (C). Dox, doxycycline. Values represent mean ± standard deviation. At least 100 cells/experimental group were analyzed in three independent experiments.

    Techniques Used: Transfection, Staining, Amplification, Standard Deviation

    22) Product Images from "BIX-01294 sensitizes renal cancer Caki cells to TRAIL-induced apoptosis through downregulation of survivin expression and upregulation of DR5 expression"

    Article Title: BIX-01294 sensitizes renal cancer Caki cells to TRAIL-induced apoptosis through downregulation of survivin expression and upregulation of DR5 expression

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0035-8

    Knockdown of G9a sensitizes Caki cells to TRAIL-mediated apoptosis. a Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA. Twenty-four hours after transfection, cells were treated with 50 ng/ml TRAIL for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry (upper panel). The protein levels of PARP, G9a, and actin were determined by western blotting. (lower panel). b Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA or treated with 10 μM BIX for 24 h. The protein levels of G9a, XIAP, survivin, DR5, and actin were determined by western blotting. c Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA. Twenty-four hours after transfection, cells were treated 50 ng/ml TRAIL in the presence or absence of 10 μM BIX for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry. The protein levels of PARP, G9a, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a represent the mean ± SD from three independent samples; * p
    Figure Legend Snippet: Knockdown of G9a sensitizes Caki cells to TRAIL-mediated apoptosis. a Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA. Twenty-four hours after transfection, cells were treated with 50 ng/ml TRAIL for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry (upper panel). The protein levels of PARP, G9a, and actin were determined by western blotting. (lower panel). b Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA or treated with 10 μM BIX for 24 h. The protein levels of G9a, XIAP, survivin, DR5, and actin were determined by western blotting. c Caki cells were transiently transfected control siRNA (Cont siRNA) or G9a siRNA. Twenty-four hours after transfection, cells were treated 50 ng/ml TRAIL in the presence or absence of 10 μM BIX for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry. The protein levels of PARP, G9a, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a represent the mean ± SD from three independent samples; * p

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Western Blot

    Upregulation of DR5 expression is associated with BIX-01294 plus TRAIL-induced apoptosis. a , b Caki cells were treated with the indicated concentrations of BIX for 24 h. The mRNA levels of DR5 and actin were determined by RT-PCR a and qPCR b . c Caki cells were treated with 10 μM BIX for 24 h. The cell surface expression level of DR5 was measured by flow cytometry analysis. d , e Caki cells were transiently transfected control siRNA (Cont siRNA) or DR5 siRNA. Twenty-four hours after transfection, cells were treated with 50 ng/ml TRAIL in the presence or absence of 10 μM BIX for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry d . The protein levels of PARP, DR5 and actin were determined by western blotting. The level of actin was used as a loading control e . The values in b , c , and d represent the mean ± SD from three independent samples; * p
    Figure Legend Snippet: Upregulation of DR5 expression is associated with BIX-01294 plus TRAIL-induced apoptosis. a , b Caki cells were treated with the indicated concentrations of BIX for 24 h. The mRNA levels of DR5 and actin were determined by RT-PCR a and qPCR b . c Caki cells were treated with 10 μM BIX for 24 h. The cell surface expression level of DR5 was measured by flow cytometry analysis. d , e Caki cells were transiently transfected control siRNA (Cont siRNA) or DR5 siRNA. Twenty-four hours after transfection, cells were treated with 50 ng/ml TRAIL in the presence or absence of 10 μM BIX for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry d . The protein levels of PARP, DR5 and actin were determined by western blotting. The level of actin was used as a loading control e . The values in b , c , and d represent the mean ± SD from three independent samples; * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Transfection, Western Blot

    23) Product Images from "TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content"

    Article Title: TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

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

    doi: 10.1073/pnas.1323785111

    TM6SF2 siRNA inhibition decreases secretion of TRLs by human hepatoma Huh7 and HepG2 cells. ( A and C ) Western blot analysis of human hepatoma Huh7 ( A ) and HepG2 ( C ) cells treated for 48 h with TM6SF2 -specific or control siRNA probes. ( B and D ) Quantitation
    Figure Legend Snippet: TM6SF2 siRNA inhibition decreases secretion of TRLs by human hepatoma Huh7 and HepG2 cells. ( A and C ) Western blot analysis of human hepatoma Huh7 ( A ) and HepG2 ( C ) cells treated for 48 h with TM6SF2 -specific or control siRNA probes. ( B and D ) Quantitation

    Techniques Used: Inhibition, Western Blot, Quantitation Assay

    TM6SF2 siRNA inhibition increases lipid droplet content of human hepatoma Huh7 and HepG2 cells. Human hepatoma Huh7 ( A − D ) and HepG2 ( E − H ) cells were analyzed 48 h after siRNA inhibition with TM6SF2 -specific or control siRNA probes. ( A
    Figure Legend Snippet: TM6SF2 siRNA inhibition increases lipid droplet content of human hepatoma Huh7 and HepG2 cells. Human hepatoma Huh7 ( A − D ) and HepG2 ( E − H ) cells were analyzed 48 h after siRNA inhibition with TM6SF2 -specific or control siRNA probes. ( A

    Techniques Used: Inhibition

    24) Product Images from "Low Density Lipoprotein Receptor-related Protein 1 (LRP1)-mediated Endocytic Clearance of a Disintegrin and Metalloproteinase with Thrombospondin Motifs-4 (ADAMTS-4)"

    Article Title: Low Density Lipoprotein Receptor-related Protein 1 (LRP1)-mediated Endocytic Clearance of a Disintegrin and Metalloproteinase with Thrombospondin Motifs-4 (ADAMTS-4)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.545376

    siRNA-mediated knockdown of LRP1 impairs ADAMTS-4 endocytosis in human chondrocytes. Human chondrocytes ( n = 3) transfected with non-targeting siRNA ( siCtrl ) or LRP1 targeting siRNA ( siLRP1 ) were cultured for 2 days in DMEM containing 10% FCS. A , results
    Figure Legend Snippet: siRNA-mediated knockdown of LRP1 impairs ADAMTS-4 endocytosis in human chondrocytes. Human chondrocytes ( n = 3) transfected with non-targeting siRNA ( siCtrl ) or LRP1 targeting siRNA ( siLRP1 ) were cultured for 2 days in DMEM containing 10% FCS. A , results

    Techniques Used: Transfection, Cell Culture

    25) Product Images from "A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion ▿ Invasion ▿ †"

    Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion ▿ Invasion ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00362-10

    DvKPI limits rickettsial colonization of the tick midgut. Forty-eight hours after unfed female ticks were injected with DvKPI siRNA or a control siRNA, R. montanensis was delivered per os . Midguts were dissected 24 h after delivery of rickettsiae for measurement of burden. (A) Protein levels for the DvKPI siRNA treatment group were reduced compared to those for the siRNA control group. (B) Transcript levels in the DvKPI siRNA-treated ticks ( n = 9) were reduced 52% from levels observed in the control siRNA-treated ticks ( n = 7). (C) Accordingly, we observed a 90% increase in rickettsial abundance for the DvKPI siRNA-treated ticks ( n = 12) compared to the control siRNA-treated ticks ( n = 8). Transcript and rickettsial abundance averages represent individual tick replicates. Each experiment was run at least twice. P values were derived using one-tailed Student's t test. Horizontal bars represents the means.
    Figure Legend Snippet: DvKPI limits rickettsial colonization of the tick midgut. Forty-eight hours after unfed female ticks were injected with DvKPI siRNA or a control siRNA, R. montanensis was delivered per os . Midguts were dissected 24 h after delivery of rickettsiae for measurement of burden. (A) Protein levels for the DvKPI siRNA treatment group were reduced compared to those for the siRNA control group. (B) Transcript levels in the DvKPI siRNA-treated ticks ( n = 9) were reduced 52% from levels observed in the control siRNA-treated ticks ( n = 7). (C) Accordingly, we observed a 90% increase in rickettsial abundance for the DvKPI siRNA-treated ticks ( n = 12) compared to the control siRNA-treated ticks ( n = 8). Transcript and rickettsial abundance averages represent individual tick replicates. Each experiment was run at least twice. P values were derived using one-tailed Student's t test. Horizontal bars represents the means.

    Techniques Used: Injection, Derivative Assay, One-tailed Test

    26) Product Images from "Claudin 11 regulates bone homeostasis via bidirectional EphB4-EphrinB2 signaling"

    Article Title: Claudin 11 regulates bone homeostasis via bidirectional EphB4-EphrinB2 signaling

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0076-3

    Cldn11 negatively modulates osteoclastogenesis via interaction with the EphrinB2 ligand. a The expression of Cldn11, EphrinB2, and EphB4 and the co-localization of these proteins in osteoclast precursor cells were observed with a confocal imaging assay. b The mRNA expression of EphrinB2 and EphB4 was evaluated during preOC differentiation into mature OCs by quantitative RT-PCR. c BMMs were cultured in the presence of M-CSF (10 ng/mL) for 1 day prior to cell lysis. Lysates were immunoprecipitated with EphrinB2 or EphB4 antibody and subsequently immunoblotted with Cldn11 antibody. d BMMs transfected with siControl or siCldn11 were cultured and lysed in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 24 and 48 h. Lysates were immunoprecipitated with EphrinB2 antibody and subsequently immunoblotted with Cldn11 antibody. c , d Co-immunoprecipitated samples (top) or total cell extracts (bottom) were subjected to western blotting to detect Cldn11, EphrinB2, EphB4, and β-actin. e BMMs were transfected with control siRNA or siCldn11, and one of the siCldn11 groups was treated with EphB4-Fc. Subsequently, the cells were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, cells were fixed and stained (left), and the number of TRAP-positive MNCs was counted (right). ( f ) Mature OCs transfected with siControl or siCldn11 from the co-culture system were seeded on dentin slices, and one of the siCldn11 groups was treated with EphB4-Fc, and then mature OCs were cultured for 48 h. Attached cells on the plates were removed and photographed under a light microscope (left). Pit areas were quantified using Image-Pro Plus (Ver 4.5) software (right). * P
    Figure Legend Snippet: Cldn11 negatively modulates osteoclastogenesis via interaction with the EphrinB2 ligand. a The expression of Cldn11, EphrinB2, and EphB4 and the co-localization of these proteins in osteoclast precursor cells were observed with a confocal imaging assay. b The mRNA expression of EphrinB2 and EphB4 was evaluated during preOC differentiation into mature OCs by quantitative RT-PCR. c BMMs were cultured in the presence of M-CSF (10 ng/mL) for 1 day prior to cell lysis. Lysates were immunoprecipitated with EphrinB2 or EphB4 antibody and subsequently immunoblotted with Cldn11 antibody. d BMMs transfected with siControl or siCldn11 were cultured and lysed in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 24 and 48 h. Lysates were immunoprecipitated with EphrinB2 antibody and subsequently immunoblotted with Cldn11 antibody. c , d Co-immunoprecipitated samples (top) or total cell extracts (bottom) were subjected to western blotting to detect Cldn11, EphrinB2, EphB4, and β-actin. e BMMs were transfected with control siRNA or siCldn11, and one of the siCldn11 groups was treated with EphB4-Fc. Subsequently, the cells were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, cells were fixed and stained (left), and the number of TRAP-positive MNCs was counted (right). ( f ) Mature OCs transfected with siControl or siCldn11 from the co-culture system were seeded on dentin slices, and one of the siCldn11 groups was treated with EphB4-Fc, and then mature OCs were cultured for 48 h. Attached cells on the plates were removed and photographed under a light microscope (left). Pit areas were quantified using Image-Pro Plus (Ver 4.5) software (right). * P

    Techniques Used: Expressing, Imaging, Quantitative RT-PCR, Cell Culture, Lysis, Immunoprecipitation, Transfection, Western Blot, Staining, Co-Culture Assay, Light Microscopy, Software

    Knockdown of Cldn11 promotes OC differentiation. a BMMs were transfected with non-target control siRNA (siControl) or siRNA targeting Cldn11 (siCldn11), and the level of Cldn11 protein expression was analyzed by western blotting to confirm the knockdown efficiency of siCldn11. b , c BMMs transduced with siControl or siCldn11 were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. b Cultured cells were stained for TRAP. c The number of TRAP-positive MNCs (nuclei > 5) was counted. d , e OBs and BMCs transfected with siControl or siCldn11 were co-cultured for 7 days in a medium containing IL-1 (10 ng/mL) or PGE 2 (10 −6 M) and VitD 3 (10 −8 M). d After culturing, cells were fixed, and stained for TRAP. e The number of TRAP-positive MNCs (nuclei > 5) was determined. f Mature OCs transfected with siControl or siCldn11 from the co-culture system were seeded in a 48-well plate for 48 h, in a hydroxyapatite-coated plate for 24 h, or in dentin slices for 48 h. The cells attached to the 48-well plate were stained with TRAP solution (left), and those attached to the hydroxyapatite-coated plate (middle) and dentin slices (right) were removed. Then, the plates were photographed under a light microscope. g The number of TRAP-positive MNCs (nuclei > 5) was counted (left), and relative resorption areas in the hydroxyapatite-coated plate and dentin slices were quantified using Image-Pro Plus (Ver 4.5) software (right). h , i BMMs were transduced with siControl or siCldn11 and h then cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for the indicated time. i Infected BMMs were serum starved for 6 h and stimulated with RANKL (100 ng/mL) for the indicated time points. Total cell lysates were analyzed by performing western blotting with the indicated antibodies. β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. * P
    Figure Legend Snippet: Knockdown of Cldn11 promotes OC differentiation. a BMMs were transfected with non-target control siRNA (siControl) or siRNA targeting Cldn11 (siCldn11), and the level of Cldn11 protein expression was analyzed by western blotting to confirm the knockdown efficiency of siCldn11. b , c BMMs transduced with siControl or siCldn11 were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. b Cultured cells were stained for TRAP. c The number of TRAP-positive MNCs (nuclei > 5) was counted. d , e OBs and BMCs transfected with siControl or siCldn11 were co-cultured for 7 days in a medium containing IL-1 (10 ng/mL) or PGE 2 (10 −6 M) and VitD 3 (10 −8 M). d After culturing, cells were fixed, and stained for TRAP. e The number of TRAP-positive MNCs (nuclei > 5) was determined. f Mature OCs transfected with siControl or siCldn11 from the co-culture system were seeded in a 48-well plate for 48 h, in a hydroxyapatite-coated plate for 24 h, or in dentin slices for 48 h. The cells attached to the 48-well plate were stained with TRAP solution (left), and those attached to the hydroxyapatite-coated plate (middle) and dentin slices (right) were removed. Then, the plates were photographed under a light microscope. g The number of TRAP-positive MNCs (nuclei > 5) was counted (left), and relative resorption areas in the hydroxyapatite-coated plate and dentin slices were quantified using Image-Pro Plus (Ver 4.5) software (right). h , i BMMs were transduced with siControl or siCldn11 and h then cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for the indicated time. i Infected BMMs were serum starved for 6 h and stimulated with RANKL (100 ng/mL) for the indicated time points. Total cell lysates were analyzed by performing western blotting with the indicated antibodies. β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. * P

    Techniques Used: Transfection, Expressing, Western Blot, Transduction, Cell Culture, Staining, Co-Culture Assay, Light Microscopy, Software, Infection

    27) Product Images from "STAT3 Activity Promotes Programmed-Death Ligand 1 Expression and Suppresses Immune Responses in Breast Cancer"

    Article Title: STAT3 Activity Promotes Programmed-Death Ligand 1 Expression and Suppresses Immune Responses in Breast Cancer

    Journal: Cancers

    doi: 10.3390/cancers11101479

    Expression patterns of STAT3 and PD-L1 in human breast cancer subtypes and regulation of PD-L1 by STAT3 in breast cancer cell lines. ( A ) Expression levels of PD-L1 transcript, ( B ) pSTAT3-GS score and ( C ) STAT3 transcript in triple-negative versus non-triple negative breast cancer patients. ( D ) Inhibition of STAT3 activity by using STAT3 inhibitor C-188-9 (XIII) resulted in decreased levels of PD-L1 expression in immunoblot 48 h following treatment in MDA-MB-231 breast cancer cell line. ( E ) Knocking down STAT3 using specific siRNA construct led to decreased levels of PD-L1 in the transiently transfected BT549 cell line. Stable transfection of SKBR3 breast cancer cell line with a STAT3 overexpressing plasmid (STAT3c) resulted in increased levels of PD-L1 ( F ) protein and ( G ) transcript expression. qPCR data are illustrated as the fold change relative to control and normalized to β-actin. They represent one out of three independent experiments and are depicted as the mean (±standard error of the mean, SEM; ** p
    Figure Legend Snippet: Expression patterns of STAT3 and PD-L1 in human breast cancer subtypes and regulation of PD-L1 by STAT3 in breast cancer cell lines. ( A ) Expression levels of PD-L1 transcript, ( B ) pSTAT3-GS score and ( C ) STAT3 transcript in triple-negative versus non-triple negative breast cancer patients. ( D ) Inhibition of STAT3 activity by using STAT3 inhibitor C-188-9 (XIII) resulted in decreased levels of PD-L1 expression in immunoblot 48 h following treatment in MDA-MB-231 breast cancer cell line. ( E ) Knocking down STAT3 using specific siRNA construct led to decreased levels of PD-L1 in the transiently transfected BT549 cell line. Stable transfection of SKBR3 breast cancer cell line with a STAT3 overexpressing plasmid (STAT3c) resulted in increased levels of PD-L1 ( F ) protein and ( G ) transcript expression. qPCR data are illustrated as the fold change relative to control and normalized to β-actin. They represent one out of three independent experiments and are depicted as the mean (±standard error of the mean, SEM; ** p

    Techniques Used: Expressing, Inhibition, Activity Assay, Multiple Displacement Amplification, Construct, Transfection, Stable Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    28) Product Images from "Protein Inhibitor of Activated STAT, PIASy Regulates ?-Smooth Muscle Actin Expression by Interacting with E12 in Mesangial Cells"

    Article Title: Protein Inhibitor of Activated STAT, PIASy Regulates ?-Smooth Muscle Actin Expression by Interacting with E12 in Mesangial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041186

    Effect of siRNA specific for PIASy on TGF-β mediated α-SMA expression in mesangial cells. (A) Mouse MCs were transfected with siRNA against PIASy or control siRNA. 12 hours after the transfection, MCs were serum-starved in starving medium (0.5% bovine serum albumin/DMEM) for 36 h. MCs were then stimulated with TGF-β (1 ng/ml) for 24 hours. Total cell lysates were examined by Western blot analysis using anti-PIASy and anti-α-SMA antibodies. Representative data from three independent experiments is shown. (B and C ) Optical densitometry of PIASy and α-SMA in Western blotting. The values of PIASy, E12 and α-SMA were normalized for that of GAPDH and compared with the values of cells transfected with control siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P
    Figure Legend Snippet: Effect of siRNA specific for PIASy on TGF-β mediated α-SMA expression in mesangial cells. (A) Mouse MCs were transfected with siRNA against PIASy or control siRNA. 12 hours after the transfection, MCs were serum-starved in starving medium (0.5% bovine serum albumin/DMEM) for 36 h. MCs were then stimulated with TGF-β (1 ng/ml) for 24 hours. Total cell lysates were examined by Western blot analysis using anti-PIASy and anti-α-SMA antibodies. Representative data from three independent experiments is shown. (B and C ) Optical densitometry of PIASy and α-SMA in Western blotting. The values of PIASy, E12 and α-SMA were normalized for that of GAPDH and compared with the values of cells transfected with control siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P

    Techniques Used: Expressing, Transfection, Western Blot

    Effect of siRNA specific for PIASy and E12 in cell proliferation in MCs. siRNA specific for E12 did not affect the BrdU incorporation, but siRNA specific for PIASy obviously enhanced the cell proliferation 48 hours later after the transfection. The enhancing effect was significantly attenuated in combination with the knockdown of E12 by the RNA interference. Values were expressed as means ± SD (n = 3).
    Figure Legend Snippet: Effect of siRNA specific for PIASy and E12 in cell proliferation in MCs. siRNA specific for E12 did not affect the BrdU incorporation, but siRNA specific for PIASy obviously enhanced the cell proliferation 48 hours later after the transfection. The enhancing effect was significantly attenuated in combination with the knockdown of E12 by the RNA interference. Values were expressed as means ± SD (n = 3).

    Techniques Used: BrdU Incorporation Assay, Transfection

    Effect of siRNA specific for PIASy and E12 in α-SMA regulation in MCs. (A) PIASy mRNA and protein were reduced by transfection of siRNA against PIASy in mouse MCs. Gene expression of PIASy was examined by quantitative RT-PCR using mRNA of MCs transfected with siRNA against PIASy or control siRNA (20 nM). (B) α-SMA mRNA expression was increased by transfection of siRNA against PIASy. (C) Western blots of MCs transfected with siRNA against PIASy or control siRNA. (D and E) Optical densitometry of PIASy and α-SMA in immunoblotting. (F) E12 mRNA and protein were reduced by transfection of siRNA against E12 in mouse MCs. Gene expression of E12 was examined by quantitative RT-PCR using mRNA of MCs transfected with siRNA against E12 or control siRNA (10 nM). (G) α-SMA mRNA expression was suppressed by transfection of siRNA against E12. (H ) Western blot analysis of MCs transfected with siRNA against E12 or control siRNA. (I and J) Optical densitometry of E12 and α-SMA in immunoblotting. Means of four independent experiments are shown. The results were presented as the fold-increase or decrease compared with the values of cells transfected with control siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P
    Figure Legend Snippet: Effect of siRNA specific for PIASy and E12 in α-SMA regulation in MCs. (A) PIASy mRNA and protein were reduced by transfection of siRNA against PIASy in mouse MCs. Gene expression of PIASy was examined by quantitative RT-PCR using mRNA of MCs transfected with siRNA against PIASy or control siRNA (20 nM). (B) α-SMA mRNA expression was increased by transfection of siRNA against PIASy. (C) Western blots of MCs transfected with siRNA against PIASy or control siRNA. (D and E) Optical densitometry of PIASy and α-SMA in immunoblotting. (F) E12 mRNA and protein were reduced by transfection of siRNA against E12 in mouse MCs. Gene expression of E12 was examined by quantitative RT-PCR using mRNA of MCs transfected with siRNA against E12 or control siRNA (10 nM). (G) α-SMA mRNA expression was suppressed by transfection of siRNA against E12. (H ) Western blot analysis of MCs transfected with siRNA against E12 or control siRNA. (I and J) Optical densitometry of E12 and α-SMA in immunoblotting. Means of four independent experiments are shown. The results were presented as the fold-increase or decrease compared with the values of cells transfected with control siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot

    29) Product Images from "Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase"

    Article Title: Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase

    Journal: Diabetes

    doi: 10.2337/db16-0023

    TAT inhibits TH in neurons. CATH.a cells were transfected with TAT plasmid or TAT siRNA for 24 h, and proteins were extracted from treated cells for Western blotting evaluation of TH level. A : Representative Western blot bands of TH in TAT knockdown (si-TAT) and overexpressing (OE)-TAT cells. GAPDH is a loading control. B : Densitometric analyses of TH level in the si-TAT neuronal cells. The bar graph shows relative upregulation of TH after knockdown of TAT. C : Densitometric analyses of TH level in the OE-TAT cells. The bar graph shows relative downregulation of TH after TAT OE. Values are mean ± SEM ( n = 3).
    Figure Legend Snippet: TAT inhibits TH in neurons. CATH.a cells were transfected with TAT plasmid or TAT siRNA for 24 h, and proteins were extracted from treated cells for Western blotting evaluation of TH level. A : Representative Western blot bands of TH in TAT knockdown (si-TAT) and overexpressing (OE)-TAT cells. GAPDH is a loading control. B : Densitometric analyses of TH level in the si-TAT neuronal cells. The bar graph shows relative upregulation of TH after knockdown of TAT. C : Densitometric analyses of TH level in the OE-TAT cells. The bar graph shows relative downregulation of TH after TAT OE. Values are mean ± SEM ( n = 3).

    Techniques Used: Transfection, Plasmid Preparation, Western Blot

    30) Product Images from "JMJD6 promotes melanoma carcinogenesis through regulation of the alternative splicing of PAK1, a key MAPK signaling component"

    Article Title: JMJD6 promotes melanoma carcinogenesis through regulation of the alternative splicing of PAK1, a key MAPK signaling component

    Journal: Molecular Cancer

    doi: 10.1186/s12943-017-0744-2

    JMJD6 is Transcriptionally Activated by c-Jun. a A375 cells were transfected with control siRNA or BRAF siRNAs. Total RNAs and proteins were extracted and analyzed for the expressions of BRAF and JMJD6 by qRT-PCR and Western blotting, respectively. b c-Jun recognized consensus site was identified in the promoter region of JMJD6 using a bioinformatics website ( http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ). The number represents the nucleotide position relative to the transcription start site (+1). The mutated nucleotides are underlined. c Luciferase reporter assays in A 375 cells transfected with JMJD6-Luc or mut-JMJD6-Luc together with c-Jun and renilla as indicated. d qChIP assays (upper) and ChIP assays (lower) of the occupancy of c-Jun in the promoter region of JMJD6 in A375 cells. FOXK1 and Igr5 intron 3 serve as positive and negative control, respectively. e A375 cells were transfected with vector, c-Jun, or treated with control siRNA or c-Jun siRNAs. Total RNAs and proteins were extracted and analyzed for the expression of JMJD6 by qRT-PCR and Western blotting, respectively. f Proposed model of the JMJD6 in melanoma carcinogenesis. In melanoma cancer cells, JMJD6, through regulating PAK1 alternative splicing and positively influencing the MAPK signaling, promotes melanogenesis, cell proliferation, invasion and angiogenesis. Hyperactive MAPK signaling leads to the phosphorylation of c-Jun, which, in turn, transcriptionally activates JMJD6 expression. Such a self-enhancing molecular system favors the development and progression of melanoma
    Figure Legend Snippet: JMJD6 is Transcriptionally Activated by c-Jun. a A375 cells were transfected with control siRNA or BRAF siRNAs. Total RNAs and proteins were extracted and analyzed for the expressions of BRAF and JMJD6 by qRT-PCR and Western blotting, respectively. b c-Jun recognized consensus site was identified in the promoter region of JMJD6 using a bioinformatics website ( http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ). The number represents the nucleotide position relative to the transcription start site (+1). The mutated nucleotides are underlined. c Luciferase reporter assays in A 375 cells transfected with JMJD6-Luc or mut-JMJD6-Luc together with c-Jun and renilla as indicated. d qChIP assays (upper) and ChIP assays (lower) of the occupancy of c-Jun in the promoter region of JMJD6 in A375 cells. FOXK1 and Igr5 intron 3 serve as positive and negative control, respectively. e A375 cells were transfected with vector, c-Jun, or treated with control siRNA or c-Jun siRNAs. Total RNAs and proteins were extracted and analyzed for the expression of JMJD6 by qRT-PCR and Western blotting, respectively. f Proposed model of the JMJD6 in melanoma carcinogenesis. In melanoma cancer cells, JMJD6, through regulating PAK1 alternative splicing and positively influencing the MAPK signaling, promotes melanogenesis, cell proliferation, invasion and angiogenesis. Hyperactive MAPK signaling leads to the phosphorylation of c-Jun, which, in turn, transcriptionally activates JMJD6 expression. Such a self-enhancing molecular system favors the development and progression of melanoma

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Chromatin Immunoprecipitation, Negative Control, Plasmid Preparation, Expressing

    JMJD6 Enhances MAPK Signaling in Melanoma Cells through Regulation of Alternative Splicing. a Bioinformatics analysis of the different AS events that were identified in JMJD6-depleted A375 cells using the DAVID Functional Annotation Tool (DAVID, https://david.ncifcrf.gov /). b RNA-IP assay in A375 cells was performed with IgG or JMJD6 antibody followed by qRT-PCR with primer pairs for the intron-exon junction in PAK1 pre-mRNA. c The transcripts of full length (PAK1) and exon 15-skipped PAK1 (PAK1Δ15) were analyzed by RT-PCR using RNA extracted from control siRNA, siJMJD6#1 or siJMJD6#2-treated A375 cells. Arrows indicate the location of primers for RT-PCR analyses. Quantitation was done by densitometry and expressed as signals of PAK1/PAK1Δ15 ratios. d Schemes illustrating primer and probe design to detect PAK1 and PAK1Δ15 are shown. A375 cells were transfected with control siRNA/vector, siJMJD6#1 or siJMJD6#2, JMJD6, or JMJD6m, and TaqMan assays were performed to determine the ratio of PAK1 to PAK1Δ15 expression. e, f A375 and 451Lu cells were treated with control siRNA, siJMJD6#1, siJMJD6#2, vector, JMJD6 or JMJD6m, and Western blotting analysis was performed with antibodies as indicated. g A schematic representation of the structure of PAK1 and PAK1Δ15. The PBD (p21-binding domain), AID (auto inhibitory domain), and kinase domain are shown. The PAK1Δ15 is lack of 15th exon (517-533 aa) thus has an incomplete kinase domain. h A375 and 451Lu cells were transfected with vector, FLAG-PAK1 or FLAG-PAK1Δ15 expression plasmids, and Western blotting analysis was performed with antibodies as indicated
    Figure Legend Snippet: JMJD6 Enhances MAPK Signaling in Melanoma Cells through Regulation of Alternative Splicing. a Bioinformatics analysis of the different AS events that were identified in JMJD6-depleted A375 cells using the DAVID Functional Annotation Tool (DAVID, https://david.ncifcrf.gov /). b RNA-IP assay in A375 cells was performed with IgG or JMJD6 antibody followed by qRT-PCR with primer pairs for the intron-exon junction in PAK1 pre-mRNA. c The transcripts of full length (PAK1) and exon 15-skipped PAK1 (PAK1Δ15) were analyzed by RT-PCR using RNA extracted from control siRNA, siJMJD6#1 or siJMJD6#2-treated A375 cells. Arrows indicate the location of primers for RT-PCR analyses. Quantitation was done by densitometry and expressed as signals of PAK1/PAK1Δ15 ratios. d Schemes illustrating primer and probe design to detect PAK1 and PAK1Δ15 are shown. A375 cells were transfected with control siRNA/vector, siJMJD6#1 or siJMJD6#2, JMJD6, or JMJD6m, and TaqMan assays were performed to determine the ratio of PAK1 to PAK1Δ15 expression. e, f A375 and 451Lu cells were treated with control siRNA, siJMJD6#1, siJMJD6#2, vector, JMJD6 or JMJD6m, and Western blotting analysis was performed with antibodies as indicated. g A schematic representation of the structure of PAK1 and PAK1Δ15. The PBD (p21-binding domain), AID (auto inhibitory domain), and kinase domain are shown. The PAK1Δ15 is lack of 15th exon (517-533 aa) thus has an incomplete kinase domain. h A375 and 451Lu cells were transfected with vector, FLAG-PAK1 or FLAG-PAK1Δ15 expression plasmids, and Western blotting analysis was performed with antibodies as indicated

    Techniques Used: Functional Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Binding Assay

    JMJD6 Promotes Melanogenesis in Melanoma Cells. a A375 and 451Lu cells were transfected with control siRNA/vector, siJMJD6#1, siJMJD6#2, JMJD6, or JMJD6m. Total RNAs were extracted and analyzed for the mRNA expression of MITF by qRT-PCR. Results were presented as the mean ± SD. b A375 and 451Lu cells were transfected with control siRNA/vector, siJMJD6#1, siJMJD6#2, JMJD6, or JMJD6m. Western blotting analysis was performed with antibodies against indicated proteins. c B16F10 cells were infected with lentiviruses carrying control shRNA or Jmjd6 shRNA, and/or infected with retroviruses carrying vector or expression plasmids for PAK1, PAK1Δ15, JMJD6, or JMJD6m, and treated with α-MSH (100 nM). The culture dishes of B16F10 cells were photographed. d The B16F10 cells infected with viruses carrying indicated constructs were incubated with α-MSH (100 nM) for 24 h. Melanin content was measured at 475 nm and normalized to the protein concentration. Each bar represents the mean ± SD. Each independent experiment was performed at least three times
    Figure Legend Snippet: JMJD6 Promotes Melanogenesis in Melanoma Cells. a A375 and 451Lu cells were transfected with control siRNA/vector, siJMJD6#1, siJMJD6#2, JMJD6, or JMJD6m. Total RNAs were extracted and analyzed for the mRNA expression of MITF by qRT-PCR. Results were presented as the mean ± SD. b A375 and 451Lu cells were transfected with control siRNA/vector, siJMJD6#1, siJMJD6#2, JMJD6, or JMJD6m. Western blotting analysis was performed with antibodies against indicated proteins. c B16F10 cells were infected with lentiviruses carrying control shRNA or Jmjd6 shRNA, and/or infected with retroviruses carrying vector or expression plasmids for PAK1, PAK1Δ15, JMJD6, or JMJD6m, and treated with α-MSH (100 nM). The culture dishes of B16F10 cells were photographed. d The B16F10 cells infected with viruses carrying indicated constructs were incubated with α-MSH (100 nM) for 24 h. Melanin content was measured at 475 nm and normalized to the protein concentration. Each bar represents the mean ± SD. Each independent experiment was performed at least three times

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Infection, shRNA, Construct, Incubation, Protein Concentration

    31) Product Images from "A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds"

    Article Title: A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141083

    HMOX2 siRNA-mediated knockdown and hemin or Tin-PPIX treatment have similar effects on β-K562 hemoglobinization levels. A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HMOX2. Two siRNAs (see also S4A Fig ) were tested, with two technical replicates. C) Cells were treated with 50μM of either hemin or Tin-PPIX. A, C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1 . E) RTqPCR on α-, γ- and β-globins from cells treated with hemin or Tin-PPIX. Histograms show levels of globins expression relative to GAPDH (n = 3).
    Figure Legend Snippet: HMOX2 siRNA-mediated knockdown and hemin or Tin-PPIX treatment have similar effects on β-K562 hemoglobinization levels. A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HMOX2. Two siRNAs (see also S4A Fig ) were tested, with two technical replicates. C) Cells were treated with 50μM of either hemin or Tin-PPIX. A, C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1 . E) RTqPCR on α-, γ- and β-globins from cells treated with hemin or Tin-PPIX. Histograms show levels of globins expression relative to GAPDH (n = 3).

    Techniques Used: Transfection, Negative Control, Immunofluorescence, Expressing

    High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation. A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and dacinostat (see also S3 Fig ). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1 .
    Figure Legend Snippet: High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation. A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and dacinostat (see also S3 Fig ). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1 .

    Techniques Used: Activation Assay, Transfection, Negative Control, Immunofluorescence

    32) Product Images from "VPS72/YL1-Mediated H2A.Z Deposition Is Required for Nuclear Reassembly after Mitosis"

    Article Title: VPS72/YL1-Mediated H2A.Z Deposition Is Required for Nuclear Reassembly after Mitosis

    Journal: Cells

    doi: 10.3390/cells9071702

    VPS72 downregulation extends telophase: ( A ) HeLa cells expressing mCherry-H2B and EGFP-IBB (importin β binding domain of importin α as a nuclear import substrate) were transfected with 20 nM siRNA oligos against pontin, reptin or both proteins or with control oligos; 30–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. Length of anaphase (blue) and telophase (yellow) is indicated. The median time spent in late anaphase and telophase is shown for mitotic events from 2 independent experiments. Bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn´s multiple comparisons test. ( B ) Western blot and quantitation showing the downregulation of pontin and reptin at 55 h post-transfection with 20 nM siRNA oligos in HeLa cells expressing mCherry-H2B and EGFP-IBB. ( C ) HeLa cells expressing mCherry-H2B and EGFP-IBB were transfected with 20 nM siRNA oligos against the indicated pontin/reptin interactors or control oligos; 30–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. The median time spent in late anaphase and telophase is shown for mitotic events from 2 independent experiments. Colored bars indicate medians, and error bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn´s multiple comparisons test. The significance values for the candidates are NUFIP1 ( p = 0.0029), YEAST4 ( p > 0.9), HDAC3 ( p = 0.03), VPS72 ( p
    Figure Legend Snippet: VPS72 downregulation extends telophase: ( A ) HeLa cells expressing mCherry-H2B and EGFP-IBB (importin β binding domain of importin α as a nuclear import substrate) were transfected with 20 nM siRNA oligos against pontin, reptin or both proteins or with control oligos; 30–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. Length of anaphase (blue) and telophase (yellow) is indicated. The median time spent in late anaphase and telophase is shown for mitotic events from 2 independent experiments. Bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn´s multiple comparisons test. ( B ) Western blot and quantitation showing the downregulation of pontin and reptin at 55 h post-transfection with 20 nM siRNA oligos in HeLa cells expressing mCherry-H2B and EGFP-IBB. ( C ) HeLa cells expressing mCherry-H2B and EGFP-IBB were transfected with 20 nM siRNA oligos against the indicated pontin/reptin interactors or control oligos; 30–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. The median time spent in late anaphase and telophase is shown for mitotic events from 2 independent experiments. Colored bars indicate medians, and error bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn´s multiple comparisons test. The significance values for the candidates are NUFIP1 ( p = 0.0029), YEAST4 ( p > 0.9), HDAC3 ( p = 0.03), VPS72 ( p

    Techniques Used: Expressing, Binding Assay, Transfection, Imaging, Western Blot, Quantitation Assay

    H2A.Z depletion mimics the nuclear assembly defects observed in VPS72 depletion. ( A ) Egg extracts were treated with either control or VPS72 aa 1-97 beads to deplete H2A.Z (ΔH2A.Z) and were supplemented where indicated with recombinant H2A.Z-H2B. The extracts were incubated with sperm chromatin for 120 min, fixed with 4% paraformaldehyde and 0.5% glutaraldehyde, stained with DAPI and analyzed by confocal microscopy. Scale bars, 10 µm. ( B ) Nuclei were assembled as in A, were fixed in 4% paraformaldehyde, were stained for H2A.Z and DAPI, and were analyzed by confocal microscopy. Scale bar, 10 µm. ( C ) Nuclear size and H2A.Z signal were quantified as in Figure 6 E. n = 3 experiments, n > 20 structures per experiment and condition. Statistical significance was determined using two-tailed student’s test. ( D ) VPS72 was depleted from egg extracts and supplemented, where indicated, with 1 µM recombinant H2A.Z-H2B. Extracts were incubated with sperm chromatin for 120 min, were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde, were stained with DAPI and were analyzed by confocal microscopy. Nuclear size was quantified as in C. ( E ) HeLa cells expressing mCherry-H2B were transfected with 20 nM siRNA oligos against H2A.Z; 24–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. Colored bars indicate medians and error bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( F ) Western blot and quantitation show the downregulation of H2A.Z at 48 h post-transfection (siRNA 20 nM) in HeLa cells stably expressing mCherry-H2B.
    Figure Legend Snippet: H2A.Z depletion mimics the nuclear assembly defects observed in VPS72 depletion. ( A ) Egg extracts were treated with either control or VPS72 aa 1-97 beads to deplete H2A.Z (ΔH2A.Z) and were supplemented where indicated with recombinant H2A.Z-H2B. The extracts were incubated with sperm chromatin for 120 min, fixed with 4% paraformaldehyde and 0.5% glutaraldehyde, stained with DAPI and analyzed by confocal microscopy. Scale bars, 10 µm. ( B ) Nuclei were assembled as in A, were fixed in 4% paraformaldehyde, were stained for H2A.Z and DAPI, and were analyzed by confocal microscopy. Scale bar, 10 µm. ( C ) Nuclear size and H2A.Z signal were quantified as in Figure 6 E. n = 3 experiments, n > 20 structures per experiment and condition. Statistical significance was determined using two-tailed student’s test. ( D ) VPS72 was depleted from egg extracts and supplemented, where indicated, with 1 µM recombinant H2A.Z-H2B. Extracts were incubated with sperm chromatin for 120 min, were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde, were stained with DAPI and were analyzed by confocal microscopy. Nuclear size was quantified as in C. ( E ) HeLa cells expressing mCherry-H2B were transfected with 20 nM siRNA oligos against H2A.Z; 24–72 h after transfection, cells were analyzed by life cell imaging and the length of mitotic exit (time from metaphase to anaphase transition until end of telophase) was determined. Colored bars indicate medians and error bars represent interquartile range. Statistical significance at alpha = 0.001 was determined using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( F ) Western blot and quantitation show the downregulation of H2A.Z at 48 h post-transfection (siRNA 20 nM) in HeLa cells stably expressing mCherry-H2B.

    Techniques Used: Recombinant, Incubation, Staining, Confocal Microscopy, Two Tailed Test, Expressing, Transfection, Imaging, Western Blot, Quantitation Assay, Stable Transfection

    33) Product Images from "Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression"

    Article Title: Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.472

    Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. ( a ) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. ( b ) Caki cells were treated with 30 μ M cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. ( c ) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. ( d and e ) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA ( c ) and Bim ( d ) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μ M cafestol (Caf) and 0.1 μ M ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry ( d and e , upper panel). Equal amounts of cell lysates (60 μ g) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels ( d and e ) represent the mean±S.D. from three independent samples. * P
    Figure Legend Snippet: Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. ( a ) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. ( b ) Caki cells were treated with 30 μ M cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. ( c ) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. ( d and e ) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA ( c ) and Bim ( d ) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μ M cafestol (Caf) and 0.1 μ M ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry ( d and e , upper panel). Equal amounts of cell lysates (60 μ g) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels ( d and e ) represent the mean±S.D. from three independent samples. * P

    Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Transfection, Flow Cytometry, Cytometry, Nucleic Acid Electrophoresis

    34) Product Images from "Rottlerin enhances IL-1?-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells"

    Article Title: Rottlerin enhances IL-1?-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2011.43.12.077

    Rottlerin and IL-1β-induced COX-2 expression is independent of PKC δ inhibition. (A) MDA-MB-231 cells were transfected with PKC δ siRNA or control siRNA. After transfection, cells were treated with or without the indicated concentrations
    Figure Legend Snippet: Rottlerin and IL-1β-induced COX-2 expression is independent of PKC δ inhibition. (A) MDA-MB-231 cells were transfected with PKC δ siRNA or control siRNA. After transfection, cells were treated with or without the indicated concentrations

    Techniques Used: Expressing, Inhibition, Multiple Displacement Amplification, Transfection

    35) Product Images from "Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids"

    Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2013.82

    Inhibition of MFG-E8 expression by RNA interference or genetic knockout results in reduced phagocytosis promotion by dexamethasone treatment. ( a ) THP-1 monocytes were electroporated with an MFG-E8-specific siRNA oligonucleotide or the corresponding scrambled
    Figure Legend Snippet: Inhibition of MFG-E8 expression by RNA interference or genetic knockout results in reduced phagocytosis promotion by dexamethasone treatment. ( a ) THP-1 monocytes were electroporated with an MFG-E8-specific siRNA oligonucleotide or the corresponding scrambled

    Techniques Used: Inhibition, Expressing, Knock-Out

    36) Product Images from "GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism"

    Article Title: GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism

    Journal: Tissue Engineering and Regenerative Medicine

    doi: 10.1007/s13770-018-0163-5

    Effect of CXCR4 siRNA on the G-CSF or GM-CSF-induced BM MSCs migration in vitro . BM MSCs from SD rats were treated at passage 4 with 100 ng/mL of G-CSF or GM-CSF with or without prior transfection of CXCR4 siRNA. A Migration of BM MSCs was examined in the untreated, G-CSF, or GM-CSF groups. Data are presented as the mean values with standard deviations (n = 3/group). ** p  
    Figure Legend Snippet: Effect of CXCR4 siRNA on the G-CSF or GM-CSF-induced BM MSCs migration in vitro . BM MSCs from SD rats were treated at passage 4 with 100 ng/mL of G-CSF or GM-CSF with or without prior transfection of CXCR4 siRNA. A Migration of BM MSCs was examined in the untreated, G-CSF, or GM-CSF groups. Data are presented as the mean values with standard deviations (n = 3/group). ** p  

    Techniques Used: Migration, In Vitro, Transfection

    37) Product Images from "Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion *"

    Article Title: Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion *

    Journal:

    doi: 10.1074/jbc.M109.072421

    Depletion of RhoGDI enhances glucose-induced Cdc42 activation. A, MIN6 cells were transfected with three different commercially available RhoGDIα siRNA ( siA , siB , and siC ) or negative control ( siCon ) oligonucleotides using Lipofectamine 2000 as
    Figure Legend Snippet: Depletion of RhoGDI enhances glucose-induced Cdc42 activation. A, MIN6 cells were transfected with three different commercially available RhoGDIα siRNA ( siA , siB , and siC ) or negative control ( siCon ) oligonucleotides using Lipofectamine 2000 as

    Techniques Used: Activation Assay, Transfection, Negative Control

    38) Product Images from "CIIA prevents SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling"

    Article Title: CIIA prevents SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00179

    CIIA inhibits SOD1(G93A)-induced activation of ASK1 . NSC34 cells cells were stably transfected with plasmid vectors encoding Flag-tagged SOD1(WT) or SOD1(G93A) along with vectors for GFP or CIIA siRNA. (A,B) Cell lysates of the stable transfectants were examined for ASK1 and p38 kinase activities by immune complex kinase assay (A) or for intracellular ROS production by DCF fluorescence (B) . The data are means ± s.e.m. from three independent experiments. * P
    Figure Legend Snippet: CIIA inhibits SOD1(G93A)-induced activation of ASK1 . NSC34 cells cells were stably transfected with plasmid vectors encoding Flag-tagged SOD1(WT) or SOD1(G93A) along with vectors for GFP or CIIA siRNA. (A,B) Cell lysates of the stable transfectants were examined for ASK1 and p38 kinase activities by immune complex kinase assay (A) or for intracellular ROS production by DCF fluorescence (B) . The data are means ± s.e.m. from three independent experiments. * P

    Techniques Used: Activation Assay, Stable Transfection, Transfection, Plasmid Preparation, Immune Complex Kinase Assay, Fluorescence

    Depletion of CIIA by RNAi potentiates SOD1(G93A)-induced cytotoxicity. (A) Neuro2a (N2a) cells expressing Flag-tagged human SOD1(WT) or SOD1(G93A) were transfected for 48 h with si RNAs specific for GFP or CIIA mRNAs. The cells were stained with annexin V and PI, and the percentages of apoptotic cells were determined by flow cytometry. (B) NSC34 cells expressing Flag-tagged SOD1(WT) or SOD1(G93A) were transfected for 48 h with plasmids encoding RFP along with GFP (control) or CIIA siRNA. The cells were then examined for apoptosis by TUNEL assay. Left, representative images of the assay (scale bar, 100 μm). Arrows indicate TUNEL-positive cells. Right, the data are means ± s.e.m. from three independent experiments.
    Figure Legend Snippet: Depletion of CIIA by RNAi potentiates SOD1(G93A)-induced cytotoxicity. (A) Neuro2a (N2a) cells expressing Flag-tagged human SOD1(WT) or SOD1(G93A) were transfected for 48 h with si RNAs specific for GFP or CIIA mRNAs. The cells were stained with annexin V and PI, and the percentages of apoptotic cells were determined by flow cytometry. (B) NSC34 cells expressing Flag-tagged SOD1(WT) or SOD1(G93A) were transfected for 48 h with plasmids encoding RFP along with GFP (control) or CIIA siRNA. The cells were then examined for apoptosis by TUNEL assay. Left, representative images of the assay (scale bar, 100 μm). Arrows indicate TUNEL-positive cells. Right, the data are means ± s.e.m. from three independent experiments.

    Techniques Used: Expressing, Transfection, Staining, Flow Cytometry, Cytometry, TUNEL Assay

    CIIA inhibits SOD1(G93A)-induced reduction of mitochondria membrane potential (Δψ m ) and the release of cytochrome c . NSC34 cells were stably transfected with plasmid vectors encoding Flag-tagged SOD1(WT) or SOD1(G93A) along with vectors for GFP or CIIA siRNA. (A) mitochondria membrane potential (Δψ m ) was analyzed by measuring the fluorescence intensity of tetramethyl rhodamine methyl ester (TMRM, a mitochondria potential sensor). Quantitative data are mean ± s.e.m. from three independent experiments. * P
    Figure Legend Snippet: CIIA inhibits SOD1(G93A)-induced reduction of mitochondria membrane potential (Δψ m ) and the release of cytochrome c . NSC34 cells were stably transfected with plasmid vectors encoding Flag-tagged SOD1(WT) or SOD1(G93A) along with vectors for GFP or CIIA siRNA. (A) mitochondria membrane potential (Δψ m ) was analyzed by measuring the fluorescence intensity of tetramethyl rhodamine methyl ester (TMRM, a mitochondria potential sensor). Quantitative data are mean ± s.e.m. from three independent experiments. * P

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Fluorescence

    39) Product Images from "Distinct and separable activities of the endocytic clathrin coat components Fcho1/2 and AP-2 in developmental patterning"

    Article Title: Distinct and separable activities of the endocytic clathrin coat components Fcho1/2 and AP-2 in developmental patterning

    Journal: Nature Cell Biology

    doi: 10.1038/ncb2473

    AP-2–clathrin coats persist in FCHO1 and FCHO2 siRNA-treated HeLa and BS-C-1 cells. ( a ) Biochemical validation of FCHO1 transcript silencing in GFP-FCHO1-expressing HeLa cells by immunoblot analysis of lysates from mock or FCHO1 siRNA transfected cells. An ON-TARGET plus SMART pool (siRNA p ; Dharmacon) was used. Replicate immunoblot with an anti-β tubulin mAb to verify equivalent loading of lysates. ( b ) HeLa cells subject to mock, FCHO1 or FCHO2 siRNA p silencing and also ectopically expressing GFP-FCHO1 (along with RFP to mark co-transfected cells) were fixed and stained with Hoechst 33258. Representative, color-separated or merged, confocal optical sections are show. Note that both the FCHO1 and FCHO2 siRNA p sets efficiently quench the GFP-FCHO1 fluorescence. Scale bar: 10 μm. ( c ) Biochemical verification of FCHO2 siRNA p - or siRNA s -mediated silencing in HeLa cells by immunoblotting. SDS-PAGE resolved lysates from cells subjected to the indicated treatments were immunoblotted with antibodies against either GFP or FCHO2 and β tubulin as a loading control. Notice that the FCHO2 siRNA p suppresses both the transfected GFP-FCHO2 as well as the endogenous protein in these cell populations highly efficiently. ( d-e ) Representative single confocal optical sections of HeLa cells subjected to mock (d) or combined FCHO1+FCHO2 siRNA p (e) followed by fixation and immunodetection of the endogenous AP-2 α subunit using mAb AP.6. Note that although the roughly regular patterning and surface density of AP-2–clathrin structures is diminished upon knockdown of FCHO1/2, AP-2-positive puncta still persist and, generally appear to increase in size and are more irregularly deposited (arrowheads). ( f ) Biochemical verification of FCHO1+FCHO2 siRNA p - or siRNA s -mediated silencing in BS-C-1 cells by immunoblotting. Notice, again, that the Stealth (Invitrogen) FCHO2 siRNA s very effectively extinguishes the endogenous FCHO2 in these populations of cells. Mock transfected HeLa cells included for comparison. ( g-j ) Representative single confocal optical sections of clathrin LCa-GFP expressing BS-C-1 cells subjected to mock (g) or combined FCHO1 siRNA p +FCHO2 siRNA s (h) followed by fixation and immunodetection of the endogenous AP-2 α subunit using mAb AP.6. The insets (i, j) show a color-separated enlarged region corresponding to the boxed area in h. Surface AP-2-positive puncta clearly persist in these FCHO1+2-silenced BS-C-1 cells as well (arrowheads). Note that only a subset of clathrin-labeled structures is AP-2 positive in both the mock and FCHO1+2-silenced cells, as clathrin normally assembles on other intracellular structures as well. Scale bar: 10 μm.
    Figure Legend Snippet: AP-2–clathrin coats persist in FCHO1 and FCHO2 siRNA-treated HeLa and BS-C-1 cells. ( a ) Biochemical validation of FCHO1 transcript silencing in GFP-FCHO1-expressing HeLa cells by immunoblot analysis of lysates from mock or FCHO1 siRNA transfected cells. An ON-TARGET plus SMART pool (siRNA p ; Dharmacon) was used. Replicate immunoblot with an anti-β tubulin mAb to verify equivalent loading of lysates. ( b ) HeLa cells subject to mock, FCHO1 or FCHO2 siRNA p silencing and also ectopically expressing GFP-FCHO1 (along with RFP to mark co-transfected cells) were fixed and stained with Hoechst 33258. Representative, color-separated or merged, confocal optical sections are show. Note that both the FCHO1 and FCHO2 siRNA p sets efficiently quench the GFP-FCHO1 fluorescence. Scale bar: 10 μm. ( c ) Biochemical verification of FCHO2 siRNA p - or siRNA s -mediated silencing in HeLa cells by immunoblotting. SDS-PAGE resolved lysates from cells subjected to the indicated treatments were immunoblotted with antibodies against either GFP or FCHO2 and β tubulin as a loading control. Notice that the FCHO2 siRNA p suppresses both the transfected GFP-FCHO2 as well as the endogenous protein in these cell populations highly efficiently. ( d-e ) Representative single confocal optical sections of HeLa cells subjected to mock (d) or combined FCHO1+FCHO2 siRNA p (e) followed by fixation and immunodetection of the endogenous AP-2 α subunit using mAb AP.6. Note that although the roughly regular patterning and surface density of AP-2–clathrin structures is diminished upon knockdown of FCHO1/2, AP-2-positive puncta still persist and, generally appear to increase in size and are more irregularly deposited (arrowheads). ( f ) Biochemical verification of FCHO1+FCHO2 siRNA p - or siRNA s -mediated silencing in BS-C-1 cells by immunoblotting. Notice, again, that the Stealth (Invitrogen) FCHO2 siRNA s very effectively extinguishes the endogenous FCHO2 in these populations of cells. Mock transfected HeLa cells included for comparison. ( g-j ) Representative single confocal optical sections of clathrin LCa-GFP expressing BS-C-1 cells subjected to mock (g) or combined FCHO1 siRNA p +FCHO2 siRNA s (h) followed by fixation and immunodetection of the endogenous AP-2 α subunit using mAb AP.6. The insets (i, j) show a color-separated enlarged region corresponding to the boxed area in h. Surface AP-2-positive puncta clearly persist in these FCHO1+2-silenced BS-C-1 cells as well (arrowheads). Note that only a subset of clathrin-labeled structures is AP-2 positive in both the mock and FCHO1+2-silenced cells, as clathrin normally assembles on other intracellular structures as well. Scale bar: 10 μm.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence, SDS Page, Immunodetection, Labeling

    Binding properties of FCHO1 ( a ) Cartoon of FCHO1 with the location and relative binding properties of the various truncations tested. ( b ) Coomassie-stained gel and blot of supernatant (S) and pellet (P) fractions of a GST pull-down assay with brain cytosol and immobilized GST or the indicated GST-FCHO1 or GST-ARH fragments. Immunoblotted with anti-clathrin heavy chain (HC) and AP-1/2 β1/2-subunit antibodies. Molecular mass standards (kDa) are shown, and large adaptor subunits (arrowheads) indicated. ( c ) Pull-down assay with FCHO1 overexpressing HeLa cell lysate and immobilized GST or the indicated GST-fusion proteins. Two independent anti-FCHO1 antibodies used for detection. Non-specific bands (asterisks) are indicated. ( d ) Pull-down assay with FCHO1 overexpressing HeLa lysates and immobilized GST, GST-α C appendage or the indicated α C appendage point mutants. ( e - l ) Mock or AP-2 α-subunit siRNA-treated HeLa cells transfected with either GFP-FCHO1 or GFP-FCHO2 as indicated were incubated with Alexa568 transferrin on ice before fixation. Representative confocal images show AP-2 silencing leads to loss of transferrin clusters on the surface. Insets (f,h,i,l) provide color-separated and merged enlargements of boxed regions. Scale bar: 10 μm. ( m ) Confocal image of a region of an AP-2 α-subunit siRNA transfected HeLa cell also expressing GFP-FCHO1. Prior to fixation and staining with an anti-clathrin HC mAb, the silenced cells were incubated at 37°C with 25 μg/ml Alexa633-labeled transferrin. ( n ) Biochemical assessment of AP-2 siRNA silencing in HeLa cell lysates by immunoblotting. ( o - r ) Representative confocal images of the intracellular localization of full-length (1-889), EFC domain (1-275), ΔμHD (1-609) or μHD (609-889) GFP-tagged FCHO1 compared with AP-2 (α subunit). Insets provide color-separated and merged enlargements of boxed regions. Scale bar: 10 μm.
    Figure Legend Snippet: Binding properties of FCHO1 ( a ) Cartoon of FCHO1 with the location and relative binding properties of the various truncations tested. ( b ) Coomassie-stained gel and blot of supernatant (S) and pellet (P) fractions of a GST pull-down assay with brain cytosol and immobilized GST or the indicated GST-FCHO1 or GST-ARH fragments. Immunoblotted with anti-clathrin heavy chain (HC) and AP-1/2 β1/2-subunit antibodies. Molecular mass standards (kDa) are shown, and large adaptor subunits (arrowheads) indicated. ( c ) Pull-down assay with FCHO1 overexpressing HeLa cell lysate and immobilized GST or the indicated GST-fusion proteins. Two independent anti-FCHO1 antibodies used for detection. Non-specific bands (asterisks) are indicated. ( d ) Pull-down assay with FCHO1 overexpressing HeLa lysates and immobilized GST, GST-α C appendage or the indicated α C appendage point mutants. ( e - l ) Mock or AP-2 α-subunit siRNA-treated HeLa cells transfected with either GFP-FCHO1 or GFP-FCHO2 as indicated were incubated with Alexa568 transferrin on ice before fixation. Representative confocal images show AP-2 silencing leads to loss of transferrin clusters on the surface. Insets (f,h,i,l) provide color-separated and merged enlargements of boxed regions. Scale bar: 10 μm. ( m ) Confocal image of a region of an AP-2 α-subunit siRNA transfected HeLa cell also expressing GFP-FCHO1. Prior to fixation and staining with an anti-clathrin HC mAb, the silenced cells were incubated at 37°C with 25 μg/ml Alexa633-labeled transferrin. ( n ) Biochemical assessment of AP-2 siRNA silencing in HeLa cell lysates by immunoblotting. ( o - r ) Representative confocal images of the intracellular localization of full-length (1-889), EFC domain (1-275), ΔμHD (1-609) or μHD (609-889) GFP-tagged FCHO1 compared with AP-2 (α subunit). Insets provide color-separated and merged enlargements of boxed regions. Scale bar: 10 μm.

    Techniques Used: Binding Assay, Staining, Pull Down Assay, Transfection, Incubation, Expressing, Labeling

    40) Product Images from "Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells"

    Article Title: Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200906083

    Effects of OMA1 siRNA on apoptosis. (A) HeLa cells were transfected with siRNA oligonucleotides followed by 0, 3, and 6 h with staurosporine (STS). Extracts were blotted and probed with OPA1 antibody. Staurosporine converts band b to band e, similar to CCCP-induced cleavage. OMA1 siRNA slows this process, whereas DRP1 siRNA does not, even though DRP1 siRNA inhibits apoptosis (detected with PARP and caspase cleavage; not depicted). The black line indicates that intervening lanes have been spliced out. (B) The effects of OMA1 siRNA on staurosporine-induced apoptosis were determined by counting the numbers of pyknotic nuclei in 300 cells for each time point. Mean values for three independent experiments are given with SD. The significance of differences between scrambled (closed bars) and OMA1 siRNA (open bars) was determined with an unpaired Student's t test.
    Figure Legend Snippet: Effects of OMA1 siRNA on apoptosis. (A) HeLa cells were transfected with siRNA oligonucleotides followed by 0, 3, and 6 h with staurosporine (STS). Extracts were blotted and probed with OPA1 antibody. Staurosporine converts band b to band e, similar to CCCP-induced cleavage. OMA1 siRNA slows this process, whereas DRP1 siRNA does not, even though DRP1 siRNA inhibits apoptosis (detected with PARP and caspase cleavage; not depicted). The black line indicates that intervening lanes have been spliced out. (B) The effects of OMA1 siRNA on staurosporine-induced apoptosis were determined by counting the numbers of pyknotic nuclei in 300 cells for each time point. Mean values for three independent experiments are given with SD. The significance of differences between scrambled (closed bars) and OMA1 siRNA (open bars) was determined with an unpaired Student's t test.

    Techniques Used: Transfection

    Effects of OMA1 on CCCP-induced cleavage of OPA1 and recovery of fusion competence after CCCP washout. (A) HeLa cells were transfected with OMA1 siRNA or scrambled oligonucleotides followed by 30 min with 10 µM CCCP (OMA1 RNA was reduced by 90%). CCCP-induced cleavage normally converts band b to band e, but this cleavage is inhibited by OMA1 siRNA (arrow). (B) Effects of Oma1 siRNA on CCCP-induced cleavage are suppressed by cotransfected wild-type OMA1 but not by the OMA1(H331A) mutant. Cotransfected OPA1 isoform 1 was detected with an myc tag ( Griparic et al., 2007 ). Isoform 1 is not cleaved by YME1L, but it is cleaved by OMA1, showing a CCCP-induced shift from band b to band e. OMA1 expression and the shift from 40- to 60-kD proteins are shown on the bottom. Black lines indicate that intervening lanes have been spliced out. (C–I) Effects of OMA1 siRNA on recovery from CCCP-induced mitochondrial fragmentation. HeLa cells were transfected with scrambled (C–E) or OMA1 siRNA (F–H). Mitochondria are normally filamentous (C and F) but fragment after 30 min with CCCP (D and G). At 90 min after CCCP washout, the filamentous morphology of mitochondria returns more quickly in OMA1 siRNA cells (H) than in control cells (E). (I) Percentages of cells with fully fragmented mitochondria (open bars) or with partial or full recovery of tubular mitochondria (closed bars) are shown at 60 or 90 min after washout. Means and SD were determined with six plates in two independent experiments (400 cells per plate). P-values were determined with an unpaired Student's t test. OMA1 expression was reduced by 70% (means with real-time PCR). Bar, 10 µm.
    Figure Legend Snippet: Effects of OMA1 on CCCP-induced cleavage of OPA1 and recovery of fusion competence after CCCP washout. (A) HeLa cells were transfected with OMA1 siRNA or scrambled oligonucleotides followed by 30 min with 10 µM CCCP (OMA1 RNA was reduced by 90%). CCCP-induced cleavage normally converts band b to band e, but this cleavage is inhibited by OMA1 siRNA (arrow). (B) Effects of Oma1 siRNA on CCCP-induced cleavage are suppressed by cotransfected wild-type OMA1 but not by the OMA1(H331A) mutant. Cotransfected OPA1 isoform 1 was detected with an myc tag ( Griparic et al., 2007 ). Isoform 1 is not cleaved by YME1L, but it is cleaved by OMA1, showing a CCCP-induced shift from band b to band e. OMA1 expression and the shift from 40- to 60-kD proteins are shown on the bottom. Black lines indicate that intervening lanes have been spliced out. (C–I) Effects of OMA1 siRNA on recovery from CCCP-induced mitochondrial fragmentation. HeLa cells were transfected with scrambled (C–E) or OMA1 siRNA (F–H). Mitochondria are normally filamentous (C and F) but fragment after 30 min with CCCP (D and G). At 90 min after CCCP washout, the filamentous morphology of mitochondria returns more quickly in OMA1 siRNA cells (H) than in control cells (E). (I) Percentages of cells with fully fragmented mitochondria (open bars) or with partial or full recovery of tubular mitochondria (closed bars) are shown at 60 or 90 min after washout. Means and SD were determined with six plates in two independent experiments (400 cells per plate). P-values were determined with an unpaired Student's t test. OMA1 expression was reduced by 70% (means with real-time PCR). Bar, 10 µm.

    Techniques Used: Transfection, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction

    Related Articles

    Transduction:

    Article Title: Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk
    Article Snippet: .. HeLa cells (parental strain or stably transduced with LC3-GFP) were reverse transfected with 5 pmol pooled siRNA (three siRNAs targeting one specific human gene per well) against selected genes within IBD susceptibility loci from the Silencer Select Human Druggable Genome siRNA Library (Ambion) available in 96-well glass-bottomed plates at a density of 6 × 103 cells per well for imaging analysis. .. S. Typhimurium overnight cultures were subcultured 1:33 for 3 hr and then infected at MOI 100:1 for DsRed S. Typhimurium and bioluminescent S. Typhimurium (Perkin Elmer).

    Transfection:

    Article Title: A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
    Article Snippet: .. siRNA oligonucleotide transfections β-K562 cells were transfected with siRNA oligonucleotides (H-Silencer Select Druggable Genome siRNA Library V4, Ambion). .. A siRNA oligo targeting the proteasome subunit PSMC3 and a non-targeting oligo (siNTO) were used as positive and negative controls for transfection (siRNA sequences are listed in ).

    Article Title: Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk
    Article Snippet: .. HeLa cells (parental strain or stably transduced with LC3-GFP) were reverse transfected with 5 pmol pooled siRNA (three siRNAs targeting one specific human gene per well) against selected genes within IBD susceptibility loci from the Silencer Select Human Druggable Genome siRNA Library (Ambion) available in 96-well glass-bottomed plates at a density of 6 × 103 cells per well for imaging analysis. .. S. Typhimurium overnight cultures were subcultured 1:33 for 3 hr and then infected at MOI 100:1 for DsRed S. Typhimurium and bioluminescent S. Typhimurium (Perkin Elmer).

    High Throughput Screening Assay:

    Article Title: Identification of genes that are essential to restrict genome duplication to once per cell division
    Article Snippet: .. siRNA screen A high throughput screen (HTS) that was originally developed to identify small molecules that induced accumulation of excess DNA in cancer cells [ ] was adapted to screen the Ambion ‘Silencer Select Human Genome siRNA Library V4′ for genes that are essential to prevent EDR in human cells [ ]. ..

    Stable Transfection:

    Article Title: Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk
    Article Snippet: .. HeLa cells (parental strain or stably transduced with LC3-GFP) were reverse transfected with 5 pmol pooled siRNA (three siRNAs targeting one specific human gene per well) against selected genes within IBD susceptibility loci from the Silencer Select Human Druggable Genome siRNA Library (Ambion) available in 96-well glass-bottomed plates at a density of 6 × 103 cells per well for imaging analysis. .. S. Typhimurium overnight cultures were subcultured 1:33 for 3 hr and then infected at MOI 100:1 for DsRed S. Typhimurium and bioluminescent S. Typhimurium (Perkin Elmer).

    Purification:

    Article Title: Putative ligand binding sites of two functionally characterized bark beetle odorant receptors
    Article Snippet: .. The PCR products were purified as described above, and then included in a second PCR reaction, but this time using extended primers to add a 5’ NotI recognition site, a Kozak sequence (‘cacc’) and an N-terminal epitope tag (c-Myc for ItypOrco and V5 for ItypORs), as well as a 3’ ApaI recognition site. .. The PCR products were purified and then digested using NotI and ApaI restriction enzymes (NEB, Ipswich, MA, USA).

    Polymerase Chain Reaction:

    Article Title: Putative ligand binding sites of two functionally characterized bark beetle odorant receptors
    Article Snippet: .. The PCR products were purified as described above, and then included in a second PCR reaction, but this time using extended primers to add a 5’ NotI recognition site, a Kozak sequence (‘cacc’) and an N-terminal epitope tag (c-Myc for ItypOrco and V5 for ItypORs), as well as a 3’ ApaI recognition site. .. The PCR products were purified and then digested using NotI and ApaI restriction enzymes (NEB, Ipswich, MA, USA).

    Imaging:

    Article Title: Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk
    Article Snippet: .. HeLa cells (parental strain or stably transduced with LC3-GFP) were reverse transfected with 5 pmol pooled siRNA (three siRNAs targeting one specific human gene per well) against selected genes within IBD susceptibility loci from the Silencer Select Human Druggable Genome siRNA Library (Ambion) available in 96-well glass-bottomed plates at a density of 6 × 103 cells per well for imaging analysis. .. S. Typhimurium overnight cultures were subcultured 1:33 for 3 hr and then infected at MOI 100:1 for DsRed S. Typhimurium and bioluminescent S. Typhimurium (Perkin Elmer).

    Genome Wide:

    Article Title: Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants
    Article Snippet: .. The primary genome-wide siRNA screen was conducted using the Ambion Silencer Select Human Genome siRNA Library v4, which consists of three unique, nonoverlapping, nonpooled siRNAs for ∼21,584 gene targets. .. The secondary screen was conducted using three independent siRNAs from the Ambion Silencer library.

    Sequencing:

    Article Title: Putative ligand binding sites of two functionally characterized bark beetle odorant receptors
    Article Snippet: .. The PCR products were purified as described above, and then included in a second PCR reaction, but this time using extended primers to add a 5’ NotI recognition site, a Kozak sequence (‘cacc’) and an N-terminal epitope tag (c-Myc for ItypOrco and V5 for ItypORs), as well as a 3’ ApaI recognition site. .. The PCR products were purified and then digested using NotI and ApaI restriction enzymes (NEB, Ipswich, MA, USA).

    HTRF Assay:

    Article Title: An Integrated Systems Biology Approach Identifies the Proteasome as a Critical Host Machinery for ZIKV and DENV Replication
    Article Snippet: .. siRNA screening RNAi screening was conducted using the Ambion Silencer® Select Human Druggable Genome siRNA Library Version 4 as described previously [ ] and the HTRF assay for NS1 antigen was performed as described above. .. The HTRF signal was for each unique non-overlapping siRNA against the target genes was normalized to a negative control targeting siRNA.

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  • 92
    Thermo Fisher bugz sirna oligonucleotide
    Effects of <t>BuGZ</t> on AurA kinase activity in mitosis. (A–D) Cells were transfected with control, BuGZ, or TPX2 <t>siRNA</t> (si) or were treated with AurA inhibitor MLN8237 (100 nM) followed by staining with antibodies to total AurA (A) or p-AurA (B). Bars, 10 µm. The immunostaining intensity of total AurA (C) and p-AurA (D) in control siRNA–treated cells in metaphase or prometaphase (ProM, containing misaligned chromosomes or thick chromosome bars) and cells in the experimental groups containing misaligned chromosomes were quantified. 75–152 total cells from three independent experiments in each experiment were measured and quantified. Error bars indicate SEM. One-way ANOVA: *, P
    Bugz Sirna Oligonucleotide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sirna oligonucleotides
    RASSF1A regulation is dependent on FoxM1. <t>T84</t> and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with <t>siRNA</t> for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p
    Sirna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The mechanism by which SREBP1 causes highly active DNFA gene expression in melanoma cells. a , mRNAs of HT-144 cells were sequenced after ASOs (5 nM) or pooled <t>siRNAs</t> (50 nM) treatment in 1% ITS medium for three days. RNA-Seq data were analyzed with DESeq2 and principal component analysis (PCA). b , c , top 20 enriched signaling and metabolic KEGG pathways were discovered from differentially expressed genes (siSREBF1 vs siNegative group) in RNA-Seq analysis using Generally Applicable Gene-set Enrichment (GAGE). Red dash line marks P value = 0.05. d-f , HT-144 cells were <t>transfected</t> with ASO-4 (5 nM) or control ASO (5 nM), cultured in 1% ITS medium. Percentage of input DNA was compared between two treatments for the indicated antibodies at the 5’ promoter region of the SCD gene. g-i , ChIP-qPCR analyses detected DNA pulldown using indicated antibodies at the SCD gene in HT-144 cells. ChIP-qPCR signals were compared between cells cultured in 10% FBS and 1% ITS medium condition. Data were presented as mean ± SD and quantified from 3 triplicates. Two-way ANOVA tests were performed. ns, not significant; *, P
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    Thermo Fisher cul4b specific shrna
    HIV-2/SIV mac239 Vpr can trigger G 2 cell cycle arrest through either CUL4A or <t>CUL4B.</t> (A) HEK293T cells stably expressing shRNA specific for either CUL4A or CUL4B mRNA were transfected with either an empty expression vector or one for FLAG–HIV-2
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    Effects of BuGZ on AurA kinase activity in mitosis. (A–D) Cells were transfected with control, BuGZ, or TPX2 siRNA (si) or were treated with AurA inhibitor MLN8237 (100 nM) followed by staining with antibodies to total AurA (A) or p-AurA (B). Bars, 10 µm. The immunostaining intensity of total AurA (C) and p-AurA (D) in control siRNA–treated cells in metaphase or prometaphase (ProM, containing misaligned chromosomes or thick chromosome bars) and cells in the experimental groups containing misaligned chromosomes were quantified. 75–152 total cells from three independent experiments in each experiment were measured and quantified. Error bars indicate SEM. One-way ANOVA: *, P

    Journal: The Journal of Cell Biology

    Article Title: Aurora A activation in mitosis promoted by BuGZ

    doi: 10.1083/jcb.201706103

    Figure Lengend Snippet: Effects of BuGZ on AurA kinase activity in mitosis. (A–D) Cells were transfected with control, BuGZ, or TPX2 siRNA (si) or were treated with AurA inhibitor MLN8237 (100 nM) followed by staining with antibodies to total AurA (A) or p-AurA (B). Bars, 10 µm. The immunostaining intensity of total AurA (C) and p-AurA (D) in control siRNA–treated cells in metaphase or prometaphase (ProM, containing misaligned chromosomes or thick chromosome bars) and cells in the experimental groups containing misaligned chromosomes were quantified. 75–152 total cells from three independent experiments in each experiment were measured and quantified. Error bars indicate SEM. One-way ANOVA: *, P

    Article Snippet: For BuGZ knockdown, we used our previously published BuGZ siRNA oligonucleotide (5′-GCCUGCUACACUUACAACAACUAGU-3′; , ), control oligonucleotide (12935-300; Thermo Fisher Scientific), and a TPX2 oligonucleotide (SI02665082; QIAGEN).

    Techniques: Activity Assay, Transfection, Staining, Immunostaining

    RASSF1A regulation is dependent on FoxM1. T84 and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with siRNA for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p

    Journal: Cancers

    Article Title: Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer

    doi: 10.3390/cancers11020199

    Figure Lengend Snippet: RASSF1A regulation is dependent on FoxM1. T84 and Colo 205 cells were transfected with plasmid for overexpression of FoxM1. ( A ) Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of FoxM1, RASSF1A. Expression is normalized against GAPDH. The right panel of ( A ) represents the densitometric analysis of FoxM1 and RASSF1A. ( B ) T84 and Colo 205 cells were transfected with siRNA for FoxM1 or control siRNA for 48 h. Cell lysates were evaluated for FoxM1 (( B ), 1st lane), RASSF1A (( B ), 2nd lane) by immunoblot and quantified by densitometry. The results are from three independent experiments. (** p

    Article Snippet: T84 and Colo 205 cells were transfected with siRNA oligonucleotides using TurboFect (Thermo Scientific, #R0533) according to the manufacturer’s recommendations.

    Techniques: Transfection, Plasmid Preparation, Over Expression, Expressing

    The mechanism by which SREBP1 causes highly active DNFA gene expression in melanoma cells. a , mRNAs of HT-144 cells were sequenced after ASOs (5 nM) or pooled siRNAs (50 nM) treatment in 1% ITS medium for three days. RNA-Seq data were analyzed with DESeq2 and principal component analysis (PCA). b , c , top 20 enriched signaling and metabolic KEGG pathways were discovered from differentially expressed genes (siSREBF1 vs siNegative group) in RNA-Seq analysis using Generally Applicable Gene-set Enrichment (GAGE). Red dash line marks P value = 0.05. d-f , HT-144 cells were transfected with ASO-4 (5 nM) or control ASO (5 nM), cultured in 1% ITS medium. Percentage of input DNA was compared between two treatments for the indicated antibodies at the 5’ promoter region of the SCD gene. g-i , ChIP-qPCR analyses detected DNA pulldown using indicated antibodies at the SCD gene in HT-144 cells. ChIP-qPCR signals were compared between cells cultured in 10% FBS and 1% ITS medium condition. Data were presented as mean ± SD and quantified from 3 triplicates. Two-way ANOVA tests were performed. ns, not significant; *, P

    Journal: bioRxiv

    Article Title: Elevated de novo fatty acid biosynthesis gene expression promotes melanoma cell survival and drug resistance

    doi: 10.1101/441303

    Figure Lengend Snippet: The mechanism by which SREBP1 causes highly active DNFA gene expression in melanoma cells. a , mRNAs of HT-144 cells were sequenced after ASOs (5 nM) or pooled siRNAs (50 nM) treatment in 1% ITS medium for three days. RNA-Seq data were analyzed with DESeq2 and principal component analysis (PCA). b , c , top 20 enriched signaling and metabolic KEGG pathways were discovered from differentially expressed genes (siSREBF1 vs siNegative group) in RNA-Seq analysis using Generally Applicable Gene-set Enrichment (GAGE). Red dash line marks P value = 0.05. d-f , HT-144 cells were transfected with ASO-4 (5 nM) or control ASO (5 nM), cultured in 1% ITS medium. Percentage of input DNA was compared between two treatments for the indicated antibodies at the 5’ promoter region of the SCD gene. g-i , ChIP-qPCR analyses detected DNA pulldown using indicated antibodies at the SCD gene in HT-144 cells. ChIP-qPCR signals were compared between cells cultured in 10% FBS and 1% ITS medium condition. Data were presented as mean ± SD and quantified from 3 triplicates. Two-way ANOVA tests were performed. ns, not significant; *, P

    Article Snippet: Melanoma cell lines were transfected with siRNAs or ASOs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and the reverse transfection protocol suggested by the manufacture.

    Techniques: Expressing, RNA Sequencing Assay, Transfection, Allele-specific Oligonucleotide, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    SREBP1 regulates the elevated DNFA gene expression in melanoma cells. a , HT-144 cells were treated with ASOs, individual siRNA agents or pooled siRNAs (all individual agents combined) to deplete SREBP1 in 1% ITS medium. Total cell lysates were assayed with immunoblot by the indicated antibodies. HT-144 cells were transfected with ( b ) ASOs, ( c ) individual siRNA agents or pooled siRNAs in 1% ITS medium. Nuclear and cytoplasmic extracts were isolated for Western blot analysis for full length (fl) and nuclear (n) SREBP1 protein after treatment. d , e , HT-144 cells were transfected with the pooled siRNAs (50 nM) in 1% ITS medium for three days to deplete SREBF1 , SREBF2 , MED15 or CREBBP. RT-qPCR assay of mRNA shows relative expression of DNFA enzymes from siRNAs treatment groups to that of negative control siRNA treatment (siNegative). f-h , HT-144 cells were transfected with plasmids carrying the transcriptionally active N-terminal portion of SREBP1a (nSREBP1a), N-terminal portion of SREBP1c (nSREBP1c) or empty vector (pcDNA3) for two days. f , the total cell lysate was analyzed by Western blot assay using the indicated antibodies. g , h , the mRNAs were analyzed with RT-qPCR assay. The bar graphs show the relative expression of DNFA enzymes to that of pcDNA3 (control) transfection group. Data are expressed as mean ± SD and quantified from triplicates. One-way ANOVA tests were performed. ns, not significant; *, P

    Journal: bioRxiv

    Article Title: Elevated de novo fatty acid biosynthesis gene expression promotes melanoma cell survival and drug resistance

    doi: 10.1101/441303

    Figure Lengend Snippet: SREBP1 regulates the elevated DNFA gene expression in melanoma cells. a , HT-144 cells were treated with ASOs, individual siRNA agents or pooled siRNAs (all individual agents combined) to deplete SREBP1 in 1% ITS medium. Total cell lysates were assayed with immunoblot by the indicated antibodies. HT-144 cells were transfected with ( b ) ASOs, ( c ) individual siRNA agents or pooled siRNAs in 1% ITS medium. Nuclear and cytoplasmic extracts were isolated for Western blot analysis for full length (fl) and nuclear (n) SREBP1 protein after treatment. d , e , HT-144 cells were transfected with the pooled siRNAs (50 nM) in 1% ITS medium for three days to deplete SREBF1 , SREBF2 , MED15 or CREBBP. RT-qPCR assay of mRNA shows relative expression of DNFA enzymes from siRNAs treatment groups to that of negative control siRNA treatment (siNegative). f-h , HT-144 cells were transfected with plasmids carrying the transcriptionally active N-terminal portion of SREBP1a (nSREBP1a), N-terminal portion of SREBP1c (nSREBP1c) or empty vector (pcDNA3) for two days. f , the total cell lysate was analyzed by Western blot assay using the indicated antibodies. g , h , the mRNAs were analyzed with RT-qPCR assay. The bar graphs show the relative expression of DNFA enzymes to that of pcDNA3 (control) transfection group. Data are expressed as mean ± SD and quantified from triplicates. One-way ANOVA tests were performed. ns, not significant; *, P

    Article Snippet: Melanoma cell lines were transfected with siRNAs or ASOs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and the reverse transfection protocol suggested by the manufacture.

    Techniques: Expressing, Transfection, Isolation, Western Blot, Quantitative RT-PCR, Negative Control, Plasmid Preparation

    HIV-2/SIV mac239 Vpr can trigger G 2 cell cycle arrest through either CUL4A or CUL4B. (A) HEK293T cells stably expressing shRNA specific for either CUL4A or CUL4B mRNA were transfected with either an empty expression vector or one for FLAG–HIV-2

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: HIV-2/SIV mac239 Vpr can trigger G 2 cell cycle arrest through either CUL4A or CUL4B. (A) HEK293T cells stably expressing shRNA specific for either CUL4A or CUL4B mRNA were transfected with either an empty expression vector or one for FLAG–HIV-2

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Stable Transfection, Expressing, shRNA, Transfection, Plasmid Preparation

    Vpx can initiate SAMHD1 depletion through either CUL4A- or CUL4B-containing CRL4 complexes, but both CUL4 types are required for maximal SAMHD1 depletion in primary macrophages. (A) HEK293T cells that stably express a control shRNA or shRNAs specific

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: Vpx can initiate SAMHD1 depletion through either CUL4A- or CUL4B-containing CRL4 complexes, but both CUL4 types are required for maximal SAMHD1 depletion in primary macrophages. (A) HEK293T cells that stably express a control shRNA or shRNAs specific

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Stable Transfection, shRNA

    HIV-1 Vpr-mediated UNG2 degradation and constitutive UNG2 turnover are not specifically dependent on CUL4A or CUL4B. (A) HEK293T cells that stably express a control shRNA were transfected with an expression vector for UNG2-2HA. HEK293T cells that stably

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: HIV-1 Vpr-mediated UNG2 degradation and constitutive UNG2 turnover are not specifically dependent on CUL4A or CUL4B. (A) HEK293T cells that stably express a control shRNA were transfected with an expression vector for UNG2-2HA. HEK293T cells that stably

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Stable Transfection, shRNA, Transfection, Expressing, Plasmid Preparation

    HIV-1 Vpr can trigger G 2 cell cycle arrest in the absence of either CUL4A or CUL4B but not both. (A) Cultures of HEK293T cells or HEK293T cells that stably express shRNA against firefly luciferase (control), CUL4A, or CUL4B were tested for CUL4-specific

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: HIV-1 Vpr can trigger G 2 cell cycle arrest in the absence of either CUL4A or CUL4B but not both. (A) Cultures of HEK293T cells or HEK293T cells that stably express shRNA against firefly luciferase (control), CUL4A, or CUL4B were tested for CUL4-specific

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Stable Transfection, shRNA, Luciferase

    Intracellular CUL4A and CUL4B distributions can vary by cell type. (A) Cultures of HEK293T cells, primary T cells, and primary hMDMs were separated into cytosolic and nuclear fractions by cell membrane lysis and centrifugation of nuclei through a sucrose

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: Intracellular CUL4A and CUL4B distributions can vary by cell type. (A) Cultures of HEK293T cells, primary T cells, and primary hMDMs were separated into cytosolic and nuclear fractions by cell membrane lysis and centrifugation of nuclei through a sucrose

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Lysis, Centrifugation

    CUL4A and CUL4B are both coisolated with Vpr and Vpx. Cultures of HEK293T cells were transfected with expression vectors for untagged HIV-1 Vpr or FLAG-epitope-tagged HIV-1 Vpr, SIV Vpr, or SIV Vpx. Twenty-four hours after transfection, the cells were

    Journal: Journal of Virology

    Article Title: Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

    doi: 10.1128/JVI.00241-14

    Figure Lengend Snippet: CUL4A and CUL4B are both coisolated with Vpr and Vpx. Cultures of HEK293T cells were transfected with expression vectors for untagged HIV-1 Vpr or FLAG-epitope-tagged HIV-1 Vpr, SIV Vpr, or SIV Vpx. Twenty-four hours after transfection, the cells were

    Article Snippet: The following plasmids were used to generate lentiviral vectors for use in establishing stable cell lines: nontargeting (catalog no. RHS4743), CUL4A-specific (clone V2THS_32527), CUL4B-specific (clone V2THS_32515]), DCAF1/VprBP-specific (clone V2THS_74081), and DDB1-specific (clone V2THS_151130) pTRIPZ short hairpin RNA (shRNA) expression vectors for inducible transduction (Thermo Scientific Life Science Research) and firefly luciferase-specific shRNA (control shRNA) (pSicoRshluc was a gift from Tyler Jacks) (Addgene plasmid 14782), CUL4A-specific shRNA (catalog no. RHS4430-99165652, oligonucleotide V2LHS_32529; Thermo Scientific Life Science Research), and CUL4B-specific shRNA (catalog no. RHS4430-98475972, oligonucleotide V2LHS_32515; Thermo Scientific Life Science Research) for constitutive transduction.

    Techniques: Transfection, Expressing, FLAG-tag