g418  (Millipore)

 
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    Name:
    G 418 Solution
    Description:
    O 2 Amino 2 7 dideoxy δ glycero α δ glucoheptopyranosyl 1 4 O 3 deoxy 4C methyl 3 methyl amino β L arabinopyranosyl δ streptamine disulfate salt G 418 an aminoglycoside antibiotic is used as a dominant selection agent in transfection experiments The antibiotic interferes with the function of 80S ribosomes and blocks protein synthesis in eukaryotic cells Transfection of neomycin resistance genes into cells results in resistance to G 418 and enables the cells to grow in culture media containing G 418
    Catalog Number:
    G418-RO
    Price:
    None
    Applications:
    G-418 Solution is used to select eukaryotic cells that are stably transfected with the neomycin resistance gene (neo), and to maintain the phenotype (neor) of resistant cells.G-418 is also used to eliminate contaminating fibroblasts from mixed cultures.
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    Structured Review

    Millipore g418
    G 418 Solution
    O 2 Amino 2 7 dideoxy δ glycero α δ glucoheptopyranosyl 1 4 O 3 deoxy 4C methyl 3 methyl amino β L arabinopyranosyl δ streptamine disulfate salt G 418 an aminoglycoside antibiotic is used as a dominant selection agent in transfection experiments The antibiotic interferes with the function of 80S ribosomes and blocks protein synthesis in eukaryotic cells Transfection of neomycin resistance genes into cells results in resistance to G 418 and enables the cells to grow in culture media containing G 418
    https://www.bioz.com/result/g418/product/Millipore
    Average 97 stars, based on 1 article reviews
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    g418 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation"

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006500

    EBNA3C promotes cellular proliferation by upregulating Bcl6-targeted Bcl2 expression. A) Saos-2 cells were transfected with the indicated plasmids in combination with the GFP vector and selected with G418 antibiotic for 2 weeks. The GFP fluorescence of each plate was scanned by PhosphorImager and the colonies were calculated by Image J software. B) 10 5 selected cells from the former experiment were plated and cultured for 10 days. Viable cells were counted at the indicated time using trypan blue staining. C) HEK293 cells were transfected, selected and counted the numbers. D) 10 million LCL1 cells were treated with 50 μg/ml (110 μM) Bcl6 inhibitor for 15 hours. Then cells were harvested, and western blot analysis was performed with indicated antibodies. These results shown are representative of three independent experiments.
    Figure Legend Snippet: EBNA3C promotes cellular proliferation by upregulating Bcl6-targeted Bcl2 expression. A) Saos-2 cells were transfected with the indicated plasmids in combination with the GFP vector and selected with G418 antibiotic for 2 weeks. The GFP fluorescence of each plate was scanned by PhosphorImager and the colonies were calculated by Image J software. B) 10 5 selected cells from the former experiment were plated and cultured for 10 days. Viable cells were counted at the indicated time using trypan blue staining. C) HEK293 cells were transfected, selected and counted the numbers. D) 10 million LCL1 cells were treated with 50 μg/ml (110 μM) Bcl6 inhibitor for 15 hours. Then cells were harvested, and western blot analysis was performed with indicated antibodies. These results shown are representative of three independent experiments.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Fluorescence, Software, Cell Culture, Staining, Western Blot

    2) Product Images from "Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1"

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1

    Journal: Molecular and Cellular Biology

    doi:

    Ectopic expression of p120 E4F reduces the growth rate of NIH 3T3 fibroblasts. (A) NIH 3T3 cell lines that express E4F cDNA or an amino-terminal E4F fragment were plated at 5 × 10 4 cells per 35-mm-diameter well, and cell counts were determined after the indicated times of growth. Cell lines included the parental line (NIH 3T3), a G418-resistant control (3T3/neo 23-1), three lines that express p120 E4F from E4F cDNA (E4F2.5K/3T3-4, -5, and -7), and two lines that express the first 262 amino acids from E4F cDNA (E4F252/3T3 25-2 and 26-2). Growth rates of the indicated lines were measured in parallel in three independent experiments. Growth curves shown are from a single representative experiment, with each point being the average from duplicate wells; the standard error between duplicates was less than 4% at all points. (B) Expression levels of ectopically expressed p120 E4F in E4F2.5K/3T3 and control cell lines were determined by precipitation with S-protein–agarose and Western blotting using α-E4F-Nterm antiserum after separation of proteins by SDS–10% PAGE. The position of p120 E4F is indicated by the arrow. The positions of molecular mass markers (in kilodaltons) are also shown. (C) Expression levels of the ectopically expressed p50 E4F -like E4F amino-terminal fragment in E4F262 cell lines were determined by Western blotting as described for panel B. The position of the E4F262 protein is indicated by the arrow.
    Figure Legend Snippet: Ectopic expression of p120 E4F reduces the growth rate of NIH 3T3 fibroblasts. (A) NIH 3T3 cell lines that express E4F cDNA or an amino-terminal E4F fragment were plated at 5 × 10 4 cells per 35-mm-diameter well, and cell counts were determined after the indicated times of growth. Cell lines included the parental line (NIH 3T3), a G418-resistant control (3T3/neo 23-1), three lines that express p120 E4F from E4F cDNA (E4F2.5K/3T3-4, -5, and -7), and two lines that express the first 262 amino acids from E4F cDNA (E4F252/3T3 25-2 and 26-2). Growth rates of the indicated lines were measured in parallel in three independent experiments. Growth curves shown are from a single representative experiment, with each point being the average from duplicate wells; the standard error between duplicates was less than 4% at all points. (B) Expression levels of ectopically expressed p120 E4F in E4F2.5K/3T3 and control cell lines were determined by precipitation with S-protein–agarose and Western blotting using α-E4F-Nterm antiserum after separation of proteins by SDS–10% PAGE. The position of p120 E4F is indicated by the arrow. The positions of molecular mass markers (in kilodaltons) are also shown. (C) Expression levels of the ectopically expressed p50 E4F -like E4F amino-terminal fragment in E4F262 cell lines were determined by Western blotting as described for panel B. The position of the E4F262 protein is indicated by the arrow.

    Techniques Used: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis

    3) Product Images from "Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism"

    Article Title: Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006117

    RAD50 gene conditional inactivation in L . infantum . (A) Schematic representation of the RAD50 locus in L . infantum before and after integration of the inactivation cassettes blasticidin-S deaminase (5’- BLAST-3’ ), puromycin acetyltransferase (5’- PURO-3’ ) and transfection construct Psp- NEO - RAD50 . S, SacI restriction sites. (B, C) Southern blot analysis with genomic DNAs digested with SacI were hybridized with probes covering either the 5’ flanking region of RAD50 (B) or the RAD50 ORF (C). (D) PCR analysis with primers set aa’ and bb’ the chromosomal copies of the MRE11 and RAD50 genes respectively. Lanes: 1, L . infantum WT; 2, WT Psp- NEO-RAD50 ; 3, WT Rev Psp- NEO - RAD50 ; 4, RAD50 -/- Psp- NEO - RAD50 ; 5, RAD50 -/- Rev Psp- NEO - RAD50 grown for 55 passages in absence of G418; 6, MRE11 -/- and 7, MRE11 -/- RAD50 -/- .
    Figure Legend Snippet: RAD50 gene conditional inactivation in L . infantum . (A) Schematic representation of the RAD50 locus in L . infantum before and after integration of the inactivation cassettes blasticidin-S deaminase (5’- BLAST-3’ ), puromycin acetyltransferase (5’- PURO-3’ ) and transfection construct Psp- NEO - RAD50 . S, SacI restriction sites. (B, C) Southern blot analysis with genomic DNAs digested with SacI were hybridized with probes covering either the 5’ flanking region of RAD50 (B) or the RAD50 ORF (C). (D) PCR analysis with primers set aa’ and bb’ the chromosomal copies of the MRE11 and RAD50 genes respectively. Lanes: 1, L . infantum WT; 2, WT Psp- NEO-RAD50 ; 3, WT Rev Psp- NEO - RAD50 ; 4, RAD50 -/- Psp- NEO - RAD50 ; 5, RAD50 -/- Rev Psp- NEO - RAD50 grown for 55 passages in absence of G418; 6, MRE11 -/- and 7, MRE11 -/- RAD50 -/- .

    Techniques Used: Transfection, Construct, Southern Blot, Polymerase Chain Reaction

    4) Product Images from "Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer"

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccr.2008.05.017

    Fbx4 serine 12 is required for Cyclin D1 ubiquitination and proteolysis A. 293T cells were transfected with plasmids encoding Flag-tagged Fbx4 mutants and wild type Myc-tagged Fbx4. Complexes were isolated by affinity chromatography using the M2 conjugated agarose and individual components detected by immunoblot with Fbx4 and Skp1 antibodies. B. NIH-3T3 cells, wherein Fbx4 had previously been knocked down by shRNA, were transfected with wt, S12A or S12E Fbx4 pcDNA3 followed by G418 selection of stably expressing clones. Asynchronous cells were harvested and subjected to Western blotting with cyclin D1, TRF1 and Fbx4 antibodies. C. Quantification of B. Error bars indicate +/−SD. D. Ligase complexes (purified protein shown in bottom panel) were purified from stable cell lines expressing wt, S12E and S12A Fbx4 (described in B). In vitro ubiquitination reactions were performed using GST-tagged purified cyclin D1. Ubiquitin-conjugated cyclin D1 was detected by immunoblot. Non-specific complexes are denoted by asterisk. E. NIH3T3 cells were serum starved for 48 hrs and released into 10% FBS containing media for the indicated intervals. Immunoblot analysis was performed using pS11/12-Fbx4 and a total Fbx4 antibody. F. Cells synchronized via a nocodazole block were harvested at the indicated intervals following nocodazole release. Fbx4 phosphorylation was detected by precipitation with the pS11/12 antibody and immunoblot with the total Fbx4 antibody. Total cyclin D1 and Fbx4 levels were assessed by direct western.
    Figure Legend Snippet: Fbx4 serine 12 is required for Cyclin D1 ubiquitination and proteolysis A. 293T cells were transfected with plasmids encoding Flag-tagged Fbx4 mutants and wild type Myc-tagged Fbx4. Complexes were isolated by affinity chromatography using the M2 conjugated agarose and individual components detected by immunoblot with Fbx4 and Skp1 antibodies. B. NIH-3T3 cells, wherein Fbx4 had previously been knocked down by shRNA, were transfected with wt, S12A or S12E Fbx4 pcDNA3 followed by G418 selection of stably expressing clones. Asynchronous cells were harvested and subjected to Western blotting with cyclin D1, TRF1 and Fbx4 antibodies. C. Quantification of B. Error bars indicate +/−SD. D. Ligase complexes (purified protein shown in bottom panel) were purified from stable cell lines expressing wt, S12E and S12A Fbx4 (described in B). In vitro ubiquitination reactions were performed using GST-tagged purified cyclin D1. Ubiquitin-conjugated cyclin D1 was detected by immunoblot. Non-specific complexes are denoted by asterisk. E. NIH3T3 cells were serum starved for 48 hrs and released into 10% FBS containing media for the indicated intervals. Immunoblot analysis was performed using pS11/12-Fbx4 and a total Fbx4 antibody. F. Cells synchronized via a nocodazole block were harvested at the indicated intervals following nocodazole release. Fbx4 phosphorylation was detected by precipitation with the pS11/12 antibody and immunoblot with the total Fbx4 antibody. Total cyclin D1 and Fbx4 levels were assessed by direct western.

    Techniques Used: Transfection, Isolation, Affinity Chromatography, shRNA, Selection, Stable Transfection, Expressing, Clone Assay, Western Blot, Purification, In Vitro, Blocking Assay

    5) Product Images from "Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice"

    Article Title: Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-44-47

    Establishment of transgenic monoclonal colonies and the development of MTCL. The shRNA-expressing plasmids PB-EN3D2B or PB-ENlacZNH21 were integrated into the genome of IBRS-2 cells using the piggyBac transposon system. Monoclonal colonies with both G418 resistance and EGFP expression were selected ( A , magnification is 100×). The colonies were then cultured to generate cell lines. All of the cells expressed EGFP ( B , magnification is 200×). The images were adjusted for contrast and dpi (dot per inch) to improve their appearance.
    Figure Legend Snippet: Establishment of transgenic monoclonal colonies and the development of MTCL. The shRNA-expressing plasmids PB-EN3D2B or PB-ENlacZNH21 were integrated into the genome of IBRS-2 cells using the piggyBac transposon system. Monoclonal colonies with both G418 resistance and EGFP expression were selected ( A , magnification is 100×). The colonies were then cultured to generate cell lines. All of the cells expressed EGFP ( B , magnification is 200×). The images were adjusted for contrast and dpi (dot per inch) to improve their appearance.

    Techniques Used: Transgenic Assay, shRNA, Expressing, Cell Culture

    6) Product Images from "Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb"

    Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb

    Journal: Journal of neuroscience methods

    doi: 10.1016/j.jneumeth.2008.01.028

    Assessment of telomerase activity in immortalized and primary OB cell cultures. Cellular lysates were subjected to TRAP assays and the resultant products, separated by non-denaturing 12% polyacrylamide gel electrophoresis, were detected by using autoradiography. A: Lysates of early-subpassaged OBGF400 (hTERT-immortalized) cells (lane 1), primary L-OB hTERT - cells (lane 3) and their respective heat-inactivated counterparts (lanes 2 and 4). Controls consisted of a telomerase positive extract (lane 5), a primer-dimer PCR contamination control (lane 6) as well as 2 μl and 1 μl of a telomerase quantification control template, TSR8 (lanes 7 and 8, respectively). B: Lysates of late subpassages of OBGF400 (hTERT-immortalized) cells with (lane 1) or without (lane 3) G418 re-exposure and their respective heat-inactivated counterparts (lanes 2 and 4). The telomerase positive extract (lane 5) as well as 1 μl of a telomerase quantification control template, TSR8 (lane 6), are shown. The presence of active telomerase is indicated by a ladder of amplicons (TRAP products) with 6-base pair (bp) increments starting at 50 bp. Each reaction also produced a 36 bp internal amplification control (IAC) and contained unincorporated oligonucleotides (oligont).
    Figure Legend Snippet: Assessment of telomerase activity in immortalized and primary OB cell cultures. Cellular lysates were subjected to TRAP assays and the resultant products, separated by non-denaturing 12% polyacrylamide gel electrophoresis, were detected by using autoradiography. A: Lysates of early-subpassaged OBGF400 (hTERT-immortalized) cells (lane 1), primary L-OB hTERT - cells (lane 3) and their respective heat-inactivated counterparts (lanes 2 and 4). Controls consisted of a telomerase positive extract (lane 5), a primer-dimer PCR contamination control (lane 6) as well as 2 μl and 1 μl of a telomerase quantification control template, TSR8 (lanes 7 and 8, respectively). B: Lysates of late subpassages of OBGF400 (hTERT-immortalized) cells with (lane 1) or without (lane 3) G418 re-exposure and their respective heat-inactivated counterparts (lanes 2 and 4). The telomerase positive extract (lane 5) as well as 1 μl of a telomerase quantification control template, TSR8 (lane 6), are shown. The presence of active telomerase is indicated by a ladder of amplicons (TRAP products) with 6-base pair (bp) increments starting at 50 bp. Each reaction also produced a 36 bp internal amplification control (IAC) and contained unincorporated oligonucleotides (oligont).

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Polymerase Chain Reaction, Produced, Amplification

    7) Product Images from "Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection"

    Article Title: Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection

    Journal: Virology Journal

    doi: 10.1186/1743-422X-3-56

    Arbidol inhibits chronic HCV replication . FL-Neo replicon cells were passaged without G418 in the presence of 6 μg/ml (11 μM) ARB for 3 days (lane 3d), or 1, 2, 3 or 4 weeks (lanes 1w to 4w). Huh7 cells, as a control for non HCV-replicating cells, were treated (lane ARB +) or not (lane ARB -) at 6 μg/ml for 3 days. The rightmost lane labeled ARB- represents proteins from control FL-Neo cells, treated without ARB for 4 weeks. FL-Neo cells were separately treated with 100 U/ml IFN-α for 48 h (lane IFN +) or not (lane IFN -). Cells were then lysed and treated as described in Materials and Methods. Ten μg of total cell protein of each sample extract were submitted to SDS-PAGE, followed by western blotting with murine monoclonal antibodies to NS5A or core proteins, or polyclonal antiserum to GAPDH.
    Figure Legend Snippet: Arbidol inhibits chronic HCV replication . FL-Neo replicon cells were passaged without G418 in the presence of 6 μg/ml (11 μM) ARB for 3 days (lane 3d), or 1, 2, 3 or 4 weeks (lanes 1w to 4w). Huh7 cells, as a control for non HCV-replicating cells, were treated (lane ARB +) or not (lane ARB -) at 6 μg/ml for 3 days. The rightmost lane labeled ARB- represents proteins from control FL-Neo cells, treated without ARB for 4 weeks. FL-Neo cells were separately treated with 100 U/ml IFN-α for 48 h (lane IFN +) or not (lane IFN -). Cells were then lysed and treated as described in Materials and Methods. Ten μg of total cell protein of each sample extract were submitted to SDS-PAGE, followed by western blotting with murine monoclonal antibodies to NS5A or core proteins, or polyclonal antiserum to GAPDH.

    Techniques Used: Labeling, SDS Page, Western Blot

    8) Product Images from "Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells"

    Article Title: Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells

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

    doi:

    Establishment of TLS-ERG- and NEO-transduced CD34 + Lin − human CB cells in erythroid liquid culture assays. ( A , upper ). ( Lower ) Flow cytometric analysis of TLS-ERG- and NEO-transduced cells in liquid culture in the presence of G418 (1 mg/ml) after 20 days under conditions that promote erythroid differentiation. ( B ) Percentage of CD33-expressing cells during erythropoiesis culture.
    Figure Legend Snippet: Establishment of TLS-ERG- and NEO-transduced CD34 + Lin − human CB cells in erythroid liquid culture assays. ( A , upper ). ( Lower ) Flow cytometric analysis of TLS-ERG- and NEO-transduced cells in liquid culture in the presence of G418 (1 mg/ml) after 20 days under conditions that promote erythroid differentiation. ( B ) Percentage of CD33-expressing cells during erythropoiesis culture.

    Techniques Used: Flow Cytometry, Expressing

    Comparison of the frequency and distribution of TLS-ERG- and NEO-transduced progenitors. Following transduction of TLS-ERG and NEO, CD34 + Lin − ). The transduced progenitors were selected in 1500 μg/ml G418 ( A ) or not ( B ). The frequency and distribution of myeloid (CFU-GM, CFU-G, CFU-M), erythroid (BFU-E), and myeloid/erythroid (CFU-Mix) progenitor colonies was scored after 14 days. Colony designations were confirmed by May–Grünwald–Giemsa staining of cytospin preparations. Mean frequencies and standard deviations are graphically represented from seven independent experiments performed with seven different CB samples.
    Figure Legend Snippet: Comparison of the frequency and distribution of TLS-ERG- and NEO-transduced progenitors. Following transduction of TLS-ERG and NEO, CD34 + Lin − ). The transduced progenitors were selected in 1500 μg/ml G418 ( A ) or not ( B ). The frequency and distribution of myeloid (CFU-GM, CFU-G, CFU-M), erythroid (BFU-E), and myeloid/erythroid (CFU-Mix) progenitor colonies was scored after 14 days. Colony designations were confirmed by May–Grünwald–Giemsa staining of cytospin preparations. Mean frequencies and standard deviations are graphically represented from seven independent experiments performed with seven different CB samples.

    Techniques Used: Transduction, Staining

    Individual TLS-ERG-transduced CFU-GM colonies self-renew and form secondary colonies upon replating. Well isolated TLS-ERG- and NEO-transduced colonies derived from day 12 primary G418-selected methylcellulose cultures were randomly plucked and replated in secondary cultures under the same conditions used to establish the primary cultures. Each open dot represents the number of secondary colonies generated per primary colony. The horizontal line indicates the mean.
    Figure Legend Snippet: Individual TLS-ERG-transduced CFU-GM colonies self-renew and form secondary colonies upon replating. Well isolated TLS-ERG- and NEO-transduced colonies derived from day 12 primary G418-selected methylcellulose cultures were randomly plucked and replated in secondary cultures under the same conditions used to establish the primary cultures. Each open dot represents the number of secondary colonies generated per primary colony. The horizontal line indicates the mean.

    Techniques Used: Isolation, Derivative Assay, Generated

    TLS-ERG-transduced CFU-GM colonies proliferate in liquid culture. ( A , upper ) TLS-ERG-transduced G418-resistant CFU-GM colonies (from six independent experiments) were placed in liquid culture containing 300 ng/ml SCF, 300 ng/ml GM-CSF, and 50 ng/ml IL-3 and allowed to progress through 4 proliferation levels in the absence of G418. ( Lower ) Bar graph depicting the percentage of 204 colonies attaining a given proliferation level. ( B ) RT-PCR ( Upper ) and Western blot ( Lower ) analysis revealing the presence of TLS-ERG transcripts and protein in nonproliferating level 4 clones, respectively. Although H 2 O represents the reagent control for RT-PCR, the negative (−) and positive (+) controls for RT-PCR and Western blot analyses are from PG13MSCVNEO and PG13MSCVTLS-ERG retroviral producer cells, respectively. ( C ) May–Grünwald–Giemsa stained cytospin preparation of TLS-ERG-transduced CFU-GM-derived cells that had been proliferating in liquid culture for 3 weeks.
    Figure Legend Snippet: TLS-ERG-transduced CFU-GM colonies proliferate in liquid culture. ( A , upper ) TLS-ERG-transduced G418-resistant CFU-GM colonies (from six independent experiments) were placed in liquid culture containing 300 ng/ml SCF, 300 ng/ml GM-CSF, and 50 ng/ml IL-3 and allowed to progress through 4 proliferation levels in the absence of G418. ( Lower ) Bar graph depicting the percentage of 204 colonies attaining a given proliferation level. ( B ) RT-PCR ( Upper ) and Western blot ( Lower ) analysis revealing the presence of TLS-ERG transcripts and protein in nonproliferating level 4 clones, respectively. Although H 2 O represents the reagent control for RT-PCR, the negative (−) and positive (+) controls for RT-PCR and Western blot analyses are from PG13MSCVNEO and PG13MSCVTLS-ERG retroviral producer cells, respectively. ( C ) May–Grünwald–Giemsa stained cytospin preparation of TLS-ERG-transduced CFU-GM-derived cells that had been proliferating in liquid culture for 3 weeks.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Staining, Derivative Assay

    9) Product Images from "Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus"

    Article Title: Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.3.1378-1386.2001

    ). Bottom, components of pQ152, which was used to construct recombinant virus. An expanded segment of the KSHV genome shows the 4.8-kb Bam HI fragment containing ORFs 57 and K9. This fragment was used for the insertion of the GFP/Neo cassette between the polyadenylation sites for ORFs 57 and K9. (B) Photomicrographs of BCBL-1 cells that were transfected with pQ152 and grown with G418 selection to generate recombinant rKSHV.152 virus 5 weeks postelectroporation (×100). Panel 1, phase contrast; panel 2, fluorescence. (C) Hybridization analysis, following gel electrophoresis, of DNA isolated from BCBL-1, and BCBL-1 with rKSHV.152, digested with Bam HI, Hin dIII, or Pst I. Left panel: analysis with the 4.8-kb Bam HI fragment labeled with 32 P as a probe. Right panel: analysis of BCBL-1 with rKSHV.152 with the GFP/Neo construct labeled with 32 P used as a probe. B, Bam HI; H, Hin dIII; P, Pst ) are as follows: Bam HI, 4,774 bp; Hin dIII, 2,030 and 3,001 bp; and Pst I, 5,907 bp. The predicted bands from a correct recombination event with the addition of the 2.7-kb GFP/Neo cassette are marked by an (∗). (D) Hybridization analysis of DNA isolated from HF infected with rKSHV.152 following digestion with Bam HI, Hin dIII, or Pst I and gel electrophoresis. Left panel: autoradiogram of HF/rKSHV.152 DNA hybridized with the 32 P-labeled KSHV Bam HI 4.8-kb fragment. Right panel: autoradiogram of HF/rKSHV.152 DNA hybridized with the 32 P-labeled GFP/Neo as a probe. B, Bam HI; H, Hin dIII; P, Pst I. Fragment sizes expected for a correct recombination event would be 7.5 kb for Bam HI, 5.7 kb for Hin dIII, and 8.6 kb for Pst ). (E) 293 cells inoculated with virus isolated from BCGL-1 cells containing rKSHV.152. Infected 293 cells were examined for GFP expression and the expression of ORF 59 protein using the MAb 11D1 visualized with Alexa 594 (red). Shown are photomicrographs of infected 293 cells 2 dpi (×100). Panel 1, phase contrast; panel 2, fluorescence with filters for GFP; panel 3, fluorescence with filters for Alexa 594 (red); panel 4, merged image from the green and red filters.
    Figure Legend Snippet: ). Bottom, components of pQ152, which was used to construct recombinant virus. An expanded segment of the KSHV genome shows the 4.8-kb Bam HI fragment containing ORFs 57 and K9. This fragment was used for the insertion of the GFP/Neo cassette between the polyadenylation sites for ORFs 57 and K9. (B) Photomicrographs of BCBL-1 cells that were transfected with pQ152 and grown with G418 selection to generate recombinant rKSHV.152 virus 5 weeks postelectroporation (×100). Panel 1, phase contrast; panel 2, fluorescence. (C) Hybridization analysis, following gel electrophoresis, of DNA isolated from BCBL-1, and BCBL-1 with rKSHV.152, digested with Bam HI, Hin dIII, or Pst I. Left panel: analysis with the 4.8-kb Bam HI fragment labeled with 32 P as a probe. Right panel: analysis of BCBL-1 with rKSHV.152 with the GFP/Neo construct labeled with 32 P used as a probe. B, Bam HI; H, Hin dIII; P, Pst ) are as follows: Bam HI, 4,774 bp; Hin dIII, 2,030 and 3,001 bp; and Pst I, 5,907 bp. The predicted bands from a correct recombination event with the addition of the 2.7-kb GFP/Neo cassette are marked by an (∗). (D) Hybridization analysis of DNA isolated from HF infected with rKSHV.152 following digestion with Bam HI, Hin dIII, or Pst I and gel electrophoresis. Left panel: autoradiogram of HF/rKSHV.152 DNA hybridized with the 32 P-labeled KSHV Bam HI 4.8-kb fragment. Right panel: autoradiogram of HF/rKSHV.152 DNA hybridized with the 32 P-labeled GFP/Neo as a probe. B, Bam HI; H, Hin dIII; P, Pst I. Fragment sizes expected for a correct recombination event would be 7.5 kb for Bam HI, 5.7 kb for Hin dIII, and 8.6 kb for Pst ). (E) 293 cells inoculated with virus isolated from BCGL-1 cells containing rKSHV.152. Infected 293 cells were examined for GFP expression and the expression of ORF 59 protein using the MAb 11D1 visualized with Alexa 594 (red). Shown are photomicrographs of infected 293 cells 2 dpi (×100). Panel 1, phase contrast; panel 2, fluorescence with filters for GFP; panel 3, fluorescence with filters for Alexa 594 (red); panel 4, merged image from the green and red filters.

    Techniques Used: Construct, Recombinant, Transfection, Selection, Fluorescence, Hybridization, Nucleic Acid Electrophoresis, Isolation, Labeling, Infection, Expressing

    Analysis of ORF73/LANA and viral DNA in long-term rKSHV.152-infected cells. (A) Cultures of T24, DU145, and HF infected with rKSHV.152, selected with G418, and photographed with phase contrast (vis) and fluorescence (uv) (×100). (B) IFA detection of KSHV ORF 73 protein nuclear expression in HF with a rabbit polyclonal antibody to ORF 73 protein and visualized with a biotinylated anti-rabbit antibody reacted with strepavidin Alexa 594 (red). Cells were counterstained with DAPI, resulting in the blue nuclei (×2,000). (C) Gardella gel analysis of viral DNA present in long-term rKSHV.152-infected cultures. Lane 1, KSHV virion isolated from the media of TPA-induced BCBL-1 cells; lane 2, BCBL-1 cells; lane 3, HF; lane 4, HF/rKSHV.152; lane 5, T24; lane 6, T24/rKSHV.152; lane 7, DU145; lane 8, DU145/rKSHV.152.
    Figure Legend Snippet: Analysis of ORF73/LANA and viral DNA in long-term rKSHV.152-infected cells. (A) Cultures of T24, DU145, and HF infected with rKSHV.152, selected with G418, and photographed with phase contrast (vis) and fluorescence (uv) (×100). (B) IFA detection of KSHV ORF 73 protein nuclear expression in HF with a rabbit polyclonal antibody to ORF 73 protein and visualized with a biotinylated anti-rabbit antibody reacted with strepavidin Alexa 594 (red). Cells were counterstained with DAPI, resulting in the blue nuclei (×2,000). (C) Gardella gel analysis of viral DNA present in long-term rKSHV.152-infected cultures. Lane 1, KSHV virion isolated from the media of TPA-induced BCBL-1 cells; lane 2, BCBL-1 cells; lane 3, HF; lane 4, HF/rKSHV.152; lane 5, T24; lane 6, T24/rKSHV.152; lane 7, DU145; lane 8, DU145/rKSHV.152.

    Techniques Used: Infection, Fluorescence, Immunofluorescence, Expressing, Isolation

    10) Product Images from "Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb"

    Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb

    Journal: Journal of neuroscience methods

    doi: 10.1016/j.jneumeth.2008.01.028

    Assessment of telomerase activity in immortalized and primary OB cell cultures. Cellular lysates were subjected to TRAP assays and the resultant products, separated by non-denaturing 12% polyacrylamide gel electrophoresis, were detected by using autoradiography. A: Lysates of early-subpassaged OBGF400 (hTERT-immortalized) cells (lane 1), primary L-OB hTERT - cells (lane 3) and their respective heat-inactivated counterparts (lanes 2 and 4). Controls consisted of a telomerase positive extract (lane 5), a primer-dimer PCR contamination control (lane 6) as well as 2 μl and 1 μl of a telomerase quantification control template, TSR8 (lanes 7 and 8, respectively). B: Lysates of late subpassages of OBGF400 (hTERT-immortalized) cells with (lane 1) or without (lane 3) G418 re-exposure and their respective heat-inactivated counterparts (lanes 2 and 4). The telomerase positive extract (lane 5) as well as 1 μl of a telomerase quantification control template, TSR8 (lane 6), are shown. The presence of active telomerase is indicated by a ladder of amplicons (TRAP products) with 6-base pair (bp) increments starting at 50 bp. Each reaction also produced a 36 bp internal amplification control (IAC) and contained unincorporated oligonucleotides (oligont).
    Figure Legend Snippet: Assessment of telomerase activity in immortalized and primary OB cell cultures. Cellular lysates were subjected to TRAP assays and the resultant products, separated by non-denaturing 12% polyacrylamide gel electrophoresis, were detected by using autoradiography. A: Lysates of early-subpassaged OBGF400 (hTERT-immortalized) cells (lane 1), primary L-OB hTERT - cells (lane 3) and their respective heat-inactivated counterparts (lanes 2 and 4). Controls consisted of a telomerase positive extract (lane 5), a primer-dimer PCR contamination control (lane 6) as well as 2 μl and 1 μl of a telomerase quantification control template, TSR8 (lanes 7 and 8, respectively). B: Lysates of late subpassages of OBGF400 (hTERT-immortalized) cells with (lane 1) or without (lane 3) G418 re-exposure and their respective heat-inactivated counterparts (lanes 2 and 4). The telomerase positive extract (lane 5) as well as 1 μl of a telomerase quantification control template, TSR8 (lane 6), are shown. The presence of active telomerase is indicated by a ladder of amplicons (TRAP products) with 6-base pair (bp) increments starting at 50 bp. Each reaction also produced a 36 bp internal amplification control (IAC) and contained unincorporated oligonucleotides (oligont).

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Polymerase Chain Reaction, Produced, Amplification

    11) Product Images from "Efficient Replication of Genotype 3a and 4a Hepatitis C Virus Replicons in Human Hepatoma Cells"

    Article Title: Efficient Replication of Genotype 3a and 4a Hepatitis C Virus Replicons in Human Hepatoma Cells

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01256-12

    Replication of HCV-Feo replicons in Huh-7.5 cells. After expanding the replicon cell clones, cells were harvested twice as described in Materials and Methods. (A) HCV RNA copies in G418-selected replicon cells were measured by qRT-PCR. The means and SDs of the RNA measurement are shown. (B) One million cells were fixed in 2% paraformaldehyde and stained with anti-NS5A antibodies. Mean fluorescence intensity of NS5A is shown at the top of each box. (C) Expression of firefly luciferase in the cells carrying Feo replicons. Data are the means and SDs of luciferase activity/cell. S52/SG-Feo(AI), S52/SG-Feo carrying the T1062A and T1435I mutations; S52/SG-Feo(SH), S52/SG-Feo carrying the P1226S and D1437H mutations; ED43/SG-Feo(K), ED43/SG-Feo carrying the T1369K mutation; ED43/SG-Feo(Y), ED43/SG-Feo carrying the D1431Y mutation.
    Figure Legend Snippet: Replication of HCV-Feo replicons in Huh-7.5 cells. After expanding the replicon cell clones, cells were harvested twice as described in Materials and Methods. (A) HCV RNA copies in G418-selected replicon cells were measured by qRT-PCR. The means and SDs of the RNA measurement are shown. (B) One million cells were fixed in 2% paraformaldehyde and stained with anti-NS5A antibodies. Mean fluorescence intensity of NS5A is shown at the top of each box. (C) Expression of firefly luciferase in the cells carrying Feo replicons. Data are the means and SDs of luciferase activity/cell. S52/SG-Feo(AI), S52/SG-Feo carrying the T1062A and T1435I mutations; S52/SG-Feo(SH), S52/SG-Feo carrying the P1226S and D1437H mutations; ED43/SG-Feo(K), ED43/SG-Feo carrying the T1369K mutation; ED43/SG-Feo(Y), ED43/SG-Feo carrying the D1431Y mutation.

    Techniques Used: Clone Assay, Quantitative RT-PCR, Staining, Fluorescence, Expressing, Luciferase, Activity Assay, Mutagenesis

    12) Product Images from "Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression"

    Article Title: Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137887

    Stable cell lines with miR-19a target genes exhibit suppression of cell growth, migration, and invasion. (A) Cell growth of G418-resistant stable cells of A549 (upper) and LK79 (lower) transfected with miR-19a target cDNA expression vectors or empty vector (Ctrl). Cells were counted using Hoechst 33342 staining at 24, 48, and 72 h. The number of each miR-19a target expression cell was significantly less than control cells at 24, 48, and 72 h. (B) Migration assays used A549 (upper) and LK79 (lower) stable cells with miR-19a target genes. Scale bar, 100 μm. (C) In vitro invasion assay used A549 (upper) and LK79 (lower) stable cells with miR-19a target genes. (D) Average number of invading A549 (left) and LK79 (right) stable cells with miR-19a target genes for the invasion assay shown in (C). **, p
    Figure Legend Snippet: Stable cell lines with miR-19a target genes exhibit suppression of cell growth, migration, and invasion. (A) Cell growth of G418-resistant stable cells of A549 (upper) and LK79 (lower) transfected with miR-19a target cDNA expression vectors or empty vector (Ctrl). Cells were counted using Hoechst 33342 staining at 24, 48, and 72 h. The number of each miR-19a target expression cell was significantly less than control cells at 24, 48, and 72 h. (B) Migration assays used A549 (upper) and LK79 (lower) stable cells with miR-19a target genes. Scale bar, 100 μm. (C) In vitro invasion assay used A549 (upper) and LK79 (lower) stable cells with miR-19a target genes. (D) Average number of invading A549 (left) and LK79 (right) stable cells with miR-19a target genes for the invasion assay shown in (C). **, p

    Techniques Used: Stable Transfection, Migration, Transfection, Expressing, Plasmid Preparation, Staining, In Vitro, Invasion Assay

    Effect of miR-19a target genes on lung cancer cells. (A) Relative cell viability of A549 cells 24 h after transfection of expression plasmids with miR-19a target gene cDNAs. (B) Relative cell viability of LK79 cells 24 h after transfection of expression plasmids with miR-19a target gene cDNAs. (C) Colony formation of A549 cells ( upper ) and LK79 cells (lower) 3 weeks after transfection with miR-19a target cDNA plasmids and G418 selection. Each assay was performed in 3 wells of 6-well plates, and the representative images of the results are shown. (D) Average colony number of A549 cells ( left ) and LK79 cells (right) in the colony formation assay. **, p
    Figure Legend Snippet: Effect of miR-19a target genes on lung cancer cells. (A) Relative cell viability of A549 cells 24 h after transfection of expression plasmids with miR-19a target gene cDNAs. (B) Relative cell viability of LK79 cells 24 h after transfection of expression plasmids with miR-19a target gene cDNAs. (C) Colony formation of A549 cells ( upper ) and LK79 cells (lower) 3 weeks after transfection with miR-19a target cDNA plasmids and G418 selection. Each assay was performed in 3 wells of 6-well plates, and the representative images of the results are shown. (D) Average colony number of A549 cells ( left ) and LK79 cells (right) in the colony formation assay. **, p

    Techniques Used: Transfection, Expressing, Selection, Colony Assay

    13) Product Images from "Apoptotic and Growth-Promoting Activity of E2F Modulated by MDM2"

    Article Title: Apoptotic and Growth-Promoting Activity of E2F Modulated by MDM2

    Journal: Molecular and Cellular Biology

    doi:

    hDM2 regulation of E2F promotes DNA synthesis and confers a survival advantage. (a) SAOS2 cells were transfected as indicated with expression vectors encoding E2F-1 (6 μg; bars 2, 3, 4, and 5) or DP-1 (6 μg; bars 4, 5, and 6) together with hDM2 (6 μg; bars 1, 3, and 5). After 6 h, the cells were washed and grown for ∼18 h in 0.2% fetal calf serum in DMEM. BrdU incorporation was assayed at ∼18 h as described in the text, and immunostaining was performed in parallel to determine transfection efficiency (data not shown). The BrdU incorporation was determined relative to the transfection efficiency, and the data shown represent the average values (+ standard deviations) from three independent experiments where the BrdU incorporation is expressed relative to the control treatment of pcDNA3-transfected cells. (b) SAOS2 cells were transfected as indicated with expression vectors encoding E2F-1 (12 μg; bars 2 and 3), DP-1 (12 μg; bars 3 and 4), and hDM2 (12 μg; bars 1, 2, 3, and 4). After 6 h, the cells were washed and grown in either 0.2% (■) or 10% (□) fetal calf serum for ∼18 h, after which the cells were placed under G418 selection under normal culture conditions; each bar represents the average data from a single experiment involving duplicate treatments. The average number of colonies over three distinct experiments obtained after transfection with pcDNA3 was 588. The colonies were stained with crystal violet, and colony-forming activity is presented as the increase in colony numbers in the presence of hDM2 relative to the numbers in the absence of hDM2 in cells treated with the indicated expression vectors. +, present; −, absent.
    Figure Legend Snippet: hDM2 regulation of E2F promotes DNA synthesis and confers a survival advantage. (a) SAOS2 cells were transfected as indicated with expression vectors encoding E2F-1 (6 μg; bars 2, 3, 4, and 5) or DP-1 (6 μg; bars 4, 5, and 6) together with hDM2 (6 μg; bars 1, 3, and 5). After 6 h, the cells were washed and grown for ∼18 h in 0.2% fetal calf serum in DMEM. BrdU incorporation was assayed at ∼18 h as described in the text, and immunostaining was performed in parallel to determine transfection efficiency (data not shown). The BrdU incorporation was determined relative to the transfection efficiency, and the data shown represent the average values (+ standard deviations) from three independent experiments where the BrdU incorporation is expressed relative to the control treatment of pcDNA3-transfected cells. (b) SAOS2 cells were transfected as indicated with expression vectors encoding E2F-1 (12 μg; bars 2 and 3), DP-1 (12 μg; bars 3 and 4), and hDM2 (12 μg; bars 1, 2, 3, and 4). After 6 h, the cells were washed and grown in either 0.2% (■) or 10% (□) fetal calf serum for ∼18 h, after which the cells were placed under G418 selection under normal culture conditions; each bar represents the average data from a single experiment involving duplicate treatments. The average number of colonies over three distinct experiments obtained after transfection with pcDNA3 was 588. The colonies were stained with crystal violet, and colony-forming activity is presented as the increase in colony numbers in the presence of hDM2 relative to the numbers in the absence of hDM2 in cells treated with the indicated expression vectors. +, present; −, absent.

    Techniques Used: DNA Synthesis, Transfection, Expressing, BrdU Incorporation Assay, Immunostaining, Selection, Staining, Activity Assay

    14) Product Images from "The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells"

    Article Title: The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddn241

    Expression of the OA1 cDNA rescues the Oa1 -KO phenotype. Oa1 −/− melanocytes were transfected with a plasmid vector ( A ) or infected with a retroviral vector ( B ) expressing either wild-type (pR/OA1wt and LOA1SN) or mutant (pR/OA1T232K or LOA1Δ18SN; both these mutants display a subcellular distribution indistinguishable from wild-type) human OA1. Expression of the recombinant proteins was analyzed 24 h after transfection (A), or following G418 selection through several passages (B), by indirect immunofluorescence with antibodies specific to human OA1. Transient expression of wild-type OA1 is not sufficient to eliminate the giant melanosomes (A, pR/OA1wt; arrows), which disappear only upon stable transduction (B, LOA1SN). Redistribution of melanosomes towards the perinuclear/Golgi area, where OA1 is also normally enriched, is induced by wild-type OA1 both in transiently and stably transduced Oa1 −/− cells (A, pR/OA1wt, and B, LOA1SN; arrowheads). No correction of melanosomal size or distribution was observed with mutant OA1 at any time (A, pR/OA1T232K, and B, LOA1Δ18SN; arrowheads). Bars, 15 µm.
    Figure Legend Snippet: Expression of the OA1 cDNA rescues the Oa1 -KO phenotype. Oa1 −/− melanocytes were transfected with a plasmid vector ( A ) or infected with a retroviral vector ( B ) expressing either wild-type (pR/OA1wt and LOA1SN) or mutant (pR/OA1T232K or LOA1Δ18SN; both these mutants display a subcellular distribution indistinguishable from wild-type) human OA1. Expression of the recombinant proteins was analyzed 24 h after transfection (A), or following G418 selection through several passages (B), by indirect immunofluorescence with antibodies specific to human OA1. Transient expression of wild-type OA1 is not sufficient to eliminate the giant melanosomes (A, pR/OA1wt; arrows), which disappear only upon stable transduction (B, LOA1SN). Redistribution of melanosomes towards the perinuclear/Golgi area, where OA1 is also normally enriched, is induced by wild-type OA1 both in transiently and stably transduced Oa1 −/− cells (A, pR/OA1wt, and B, LOA1SN; arrowheads). No correction of melanosomal size or distribution was observed with mutant OA1 at any time (A, pR/OA1T232K, and B, LOA1Δ18SN; arrowheads). Bars, 15 µm.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Infection, Mutagenesis, Recombinant, Selection, Immunofluorescence, Transduction, Stable Transfection

    15) Product Images from "Association of 15-hydroxyprostaglandin dehydrogenate and poor prognosis of obese breast cancer patients"

    Article Title: Association of 15-hydroxyprostaglandin dehydrogenate and poor prognosis of obese breast cancer patients

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15280

    Down regulation of HPGD expression could increase the migration and proliferation ability of breast cancer cell line MCF-7 ( A ) MCF-7 cell lines were transfected with either HPGD-directed siRNA (HPGD-siRNA) or the control siRNA (Con-siRNA). Forty-eight hours after transfection, cells were lysed and immunoblotted with antibody to HPGD, immunoblotting with β-actin antibody was used for loading control. ( B ) Stretch assay showed that HPGD impairment could increase the migration ability of MCF-7 cell lines. Both MCF-7 cell lines with stable silencing of HPGD or stable transfected with Con-siRNA were plated and incubated until confluent. A wound was scratched across each well and wound closure was monitored hourly for 6 h. Wound closure was determined as a percentage of wound confluence compared with the respective negative control (regarded as 100%). ( C ) Two days following transfection with the indicated plasmids (Con-siRNA or HPGD-siRNA), G418 (500 μg/mL) was added to the culture medium, and at day 14, the cells were stained using gentian violet. Untransfected cells were treated similarly, and all died within 2 weeks of culture in the selection medium. Colony formation assays indicated that silencing of HPGD could increase the proliferation ability of MCF-7. ( D ) Quantification was done using AlphaImager 2000. The number of colonies of HPGD-siRNA group compared with the Con-siRNA group (regarded as 100%) was 244%. Columns, mean of three independent experiments; bars, SD; * p
    Figure Legend Snippet: Down regulation of HPGD expression could increase the migration and proliferation ability of breast cancer cell line MCF-7 ( A ) MCF-7 cell lines were transfected with either HPGD-directed siRNA (HPGD-siRNA) or the control siRNA (Con-siRNA). Forty-eight hours after transfection, cells were lysed and immunoblotted with antibody to HPGD, immunoblotting with β-actin antibody was used for loading control. ( B ) Stretch assay showed that HPGD impairment could increase the migration ability of MCF-7 cell lines. Both MCF-7 cell lines with stable silencing of HPGD or stable transfected with Con-siRNA were plated and incubated until confluent. A wound was scratched across each well and wound closure was monitored hourly for 6 h. Wound closure was determined as a percentage of wound confluence compared with the respective negative control (regarded as 100%). ( C ) Two days following transfection with the indicated plasmids (Con-siRNA or HPGD-siRNA), G418 (500 μg/mL) was added to the culture medium, and at day 14, the cells were stained using gentian violet. Untransfected cells were treated similarly, and all died within 2 weeks of culture in the selection medium. Colony formation assays indicated that silencing of HPGD could increase the proliferation ability of MCF-7. ( D ) Quantification was done using AlphaImager 2000. The number of colonies of HPGD-siRNA group compared with the Con-siRNA group (regarded as 100%) was 244%. Columns, mean of three independent experiments; bars, SD; * p

    Techniques Used: Expressing, Migration, Transfection, Incubation, Negative Control, Staining, Selection

    16) Product Images from "In vivo selection for spine-derived highly metastatic lung cancer cells is associated with increased migration, inflammation and decreased adhesion"

    Article Title: In vivo selection for spine-derived highly metastatic lung cancer cells is associated with increased migration, inflammation and decreased adhesion

    Journal: Oncotarget

    doi:

    Establishment of the spinal metastasis model A. In vivo selection scheme for the spinal metastatic subpopulations from the A549 lung adenocarcinoma cell line. Luciferase-labeled A549 cells (A549L0) were intracardially inoculated into nude mice. Spinal metastases were monitored by BLI after injection. Tumor cells were extracted from spinal metastases and selected by G418, then reinoculated after expansion in culture. After three rounds of in vivo selection, ten subclones including A549L6 were selected and re-inoculated into mice to evaluate their metastatic phenotype. B. Nude mice were intracardially inoculated with 1×10 5 A549L6 cells and monitored using bioluminescence imaging ( n = 20). 10 representative mice are shown. C. Kaplan-Meier analysis of spinal metastases-free survival according to BLI. D. The target organs of the original A549 cells (A549L0), the 3 rd round spine metastasis cancer cells (A549L3M), and the spinal metastatic cancer cells (A549L6) in nude mice.
    Figure Legend Snippet: Establishment of the spinal metastasis model A. In vivo selection scheme for the spinal metastatic subpopulations from the A549 lung adenocarcinoma cell line. Luciferase-labeled A549 cells (A549L0) were intracardially inoculated into nude mice. Spinal metastases were monitored by BLI after injection. Tumor cells were extracted from spinal metastases and selected by G418, then reinoculated after expansion in culture. After three rounds of in vivo selection, ten subclones including A549L6 were selected and re-inoculated into mice to evaluate their metastatic phenotype. B. Nude mice were intracardially inoculated with 1×10 5 A549L6 cells and monitored using bioluminescence imaging ( n = 20). 10 representative mice are shown. C. Kaplan-Meier analysis of spinal metastases-free survival according to BLI. D. The target organs of the original A549 cells (A549L0), the 3 rd round spine metastasis cancer cells (A549L3M), and the spinal metastatic cancer cells (A549L6) in nude mice.

    Techniques Used: In Vivo, Selection, Luciferase, Labeling, Mouse Assay, Injection, Imaging

    17) Product Images from "The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment"

    Article Title: The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment

    Journal: Cell Journal (Yakhteh)

    doi:

    Generation of knock-in mouse ESCs by a single homology arm donor plasmid. A. Schematic presentation of targeted locus and genotyping primers, B. PCR genotyping of 16 ESC colonies transfected with the donor and Cas9n/sgPdx1 expressing plasmids, selected by G418 treatment. Three positive colonies for F1-R1 genotyping were tested by F2-R2 primers and C. Sequencing results for F1-R1 (left) and F2-R2 (right) PCR products from all three positive clones. ESCs; Embryonic stem cells and PCR; Polymerase chain reaction.
    Figure Legend Snippet: Generation of knock-in mouse ESCs by a single homology arm donor plasmid. A. Schematic presentation of targeted locus and genotyping primers, B. PCR genotyping of 16 ESC colonies transfected with the donor and Cas9n/sgPdx1 expressing plasmids, selected by G418 treatment. Three positive colonies for F1-R1 genotyping were tested by F2-R2 primers and C. Sequencing results for F1-R1 (left) and F2-R2 (right) PCR products from all three positive clones. ESCs; Embryonic stem cells and PCR; Polymerase chain reaction.

    Techniques Used: Knock-In, Plasmid Preparation, Polymerase Chain Reaction, Transfection, Expressing, Sequencing, Clone Assay

    18) Product Images from "A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids"

    Article Title: A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

    Journal: eLife

    doi: 10.7554/eLife.52904

    AMOs allow HTS-compatible toxicity evaluation in whole organoids or specific cellular subpopulations. ( a ) Higher toxin concentrations resulted in increased cCasp3+ apoptotic signal in AMOs. Panel shows representative single plane confocal micrographs from our high-content analysis pipeline after adding G418 for 4 days starting at day 50 of culture. Scale bars = 100 μm. ( b ) Increase of cCasp3+ cells follows a typical sigmoidal dose-response kinetic in AMOs with escalating concentrations of G418. Depicted is the total number of cCasp3 cells per organoid normalized by the organoid area on the y-axis against the logarithmic concentration of G418 on the x-axis. n ≥ 3, error bars = SEM. ( c ) The cCasp3 signal shows little colocalization with Sox2, indicating that not neuronal precursors but other, more mature cell types are primarily affected by the treatment. The percentage of Sox2+ neural precursors among the apoptotic cells increased with the inhibitor concentration but remained relatively low with a maximum of approximately 15%. n ≥ 3, error bars = SEM ( d/e ) The dose-response curve ( d ) and colocalization analysis ( e ) based on a single plane display almost identical results to the ones based on entire organoids (b/c). This potentially allows a substantial reduction of imaging time and costs by acquiring only single medial slices instead of entire organoid volumes. n ≥ 3, error bars = SEM. ( f ) Bar graph showing the number of apoptotic cCasp3+ cells (normalized by area) for single confocal planes. Each bar represents one plane; bars are grouped by the concentration of the G418 treatment. n ≥ 3, error bars = SEM. ( g/h ) Followup high-content imaging-based evaluation of G418 toxicity with two different AMO lines and hiPSC organoids using a wider range of G418 concentrations than first tested in ( a–f ) illustrating the drawbacks of cCasp3-based toxicity screening. As cCasp3 labels cells only for a transient time during cell death, the peak signal can be missed and results can be misleading. Here, higher concentrations of G418 resulted in lower levels of cCasp3 positive cells after 96 hr ( g ). The size (area of the cross section) of the organoids from ( g ) did not change with increased concentrations of G418 ( h ). n ≥ 3, Error bars: SEM. ( i ) ATPglo-based cell viability measurements of G418 treated AMOs and hiPSC organoids (each from the same batch and treated together with the samples shown in ( g )), confirmed that higher concentrations of G418 are indeed more toxic and the cCasp3-based readout failed to detect this effect. In all three assays ( g-i ) AMOs showed lower variance than hiPSC organoids. n AMO line 1 2 = 6, n hiPSC organoids ≥ 5, Error bars: SD. All samples in ( g-i ) were at day 35 at the time of analysis.
    Figure Legend Snippet: AMOs allow HTS-compatible toxicity evaluation in whole organoids or specific cellular subpopulations. ( a ) Higher toxin concentrations resulted in increased cCasp3+ apoptotic signal in AMOs. Panel shows representative single plane confocal micrographs from our high-content analysis pipeline after adding G418 for 4 days starting at day 50 of culture. Scale bars = 100 μm. ( b ) Increase of cCasp3+ cells follows a typical sigmoidal dose-response kinetic in AMOs with escalating concentrations of G418. Depicted is the total number of cCasp3 cells per organoid normalized by the organoid area on the y-axis against the logarithmic concentration of G418 on the x-axis. n ≥ 3, error bars = SEM. ( c ) The cCasp3 signal shows little colocalization with Sox2, indicating that not neuronal precursors but other, more mature cell types are primarily affected by the treatment. The percentage of Sox2+ neural precursors among the apoptotic cells increased with the inhibitor concentration but remained relatively low with a maximum of approximately 15%. n ≥ 3, error bars = SEM ( d/e ) The dose-response curve ( d ) and colocalization analysis ( e ) based on a single plane display almost identical results to the ones based on entire organoids (b/c). This potentially allows a substantial reduction of imaging time and costs by acquiring only single medial slices instead of entire organoid volumes. n ≥ 3, error bars = SEM. ( f ) Bar graph showing the number of apoptotic cCasp3+ cells (normalized by area) for single confocal planes. Each bar represents one plane; bars are grouped by the concentration of the G418 treatment. n ≥ 3, error bars = SEM. ( g/h ) Followup high-content imaging-based evaluation of G418 toxicity with two different AMO lines and hiPSC organoids using a wider range of G418 concentrations than first tested in ( a–f ) illustrating the drawbacks of cCasp3-based toxicity screening. As cCasp3 labels cells only for a transient time during cell death, the peak signal can be missed and results can be misleading. Here, higher concentrations of G418 resulted in lower levels of cCasp3 positive cells after 96 hr ( g ). The size (area of the cross section) of the organoids from ( g ) did not change with increased concentrations of G418 ( h ). n ≥ 3, Error bars: SEM. ( i ) ATPglo-based cell viability measurements of G418 treated AMOs and hiPSC organoids (each from the same batch and treated together with the samples shown in ( g )), confirmed that higher concentrations of G418 are indeed more toxic and the cCasp3-based readout failed to detect this effect. In all three assays ( g-i ) AMOs showed lower variance than hiPSC organoids. n AMO line 1 2 = 6, n hiPSC organoids ≥ 5, Error bars: SD. All samples in ( g-i ) were at day 35 at the time of analysis.

    Techniques Used: High Content Screening, Concentration Assay, Imaging

    19) Product Images from "Distinct SoxB1 networks are required for naïve and primed pluripotency"

    Article Title: Distinct SoxB1 networks are required for naïve and primed pluripotency

    Journal: eLife

    doi: 10.7554/eLife.27746

    ( A ) Targeting T2A-H2b-tdTomato to the Sox2 locus stop codon yields the Sox2::HT fluorescent reporter allele. The Sox2 ORF is represented by a blue box and the Sox2 UTRs by beige boxes; homology arms are shown (red line). The Sox2::HT ORF is followed by GtxIRES-Neo and an FRT-flanked PGK-Neo-pA (PNpA)/MC1-TK-pA (MTpA) selection cassette. The FRT sites (cyan triangles), EcoRI sites (E), HindIII sites (H) and probes (black boxes) used for Southern analysis are shown. Sizes of relevant fragments produced before and after targeting and excision of the FRT-flanked cassette are indicated. ( B ) Southern analysis of TST cell lines expanded after G418 selection. FNF denotes lines prior to excision of the FRT-flanked cassette.
    Figure Legend Snippet: ( A ) Targeting T2A-H2b-tdTomato to the Sox2 locus stop codon yields the Sox2::HT fluorescent reporter allele. The Sox2 ORF is represented by a blue box and the Sox2 UTRs by beige boxes; homology arms are shown (red line). The Sox2::HT ORF is followed by GtxIRES-Neo and an FRT-flanked PGK-Neo-pA (PNpA)/MC1-TK-pA (MTpA) selection cassette. The FRT sites (cyan triangles), EcoRI sites (E), HindIII sites (H) and probes (black boxes) used for Southern analysis are shown. Sizes of relevant fragments produced before and after targeting and excision of the FRT-flanked cassette are indicated. ( B ) Southern analysis of TST cell lines expanded after G418 selection. FNF denotes lines prior to excision of the FRT-flanked cassette.

    Techniques Used: Selection, Produced

    20) Product Images from "Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7"

    Article Title: Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7

    Journal: Journal of Fungi

    doi: 10.3390/jof6030156

    Overexpression of mrpigG in M. ruber M7. ( a ) Schematic representation of the homologous recombination strategy yielding mrpigG overexpression strains. ( b ) Construction of mrpigG overexpression construct by double-joint PCR. Lane 1, 5′ flanking region of mrpigG ; lane 2, G418 resistance cassette; lane 3, the trpC promoter; lane 4, mrpigG ORF regions plus 3′ flanking region of mrpigG ; lane 5, double-joint PCR product. ( c ) Confirmation of mrpigG homologous recombination events. Three primer pairs were used, and PCR amplifications showed distinct bands in different strains. Lane 1, the M7:: PtrpC-mrpigG strain; lane 2, the wild-type strain.
    Figure Legend Snippet: Overexpression of mrpigG in M. ruber M7. ( a ) Schematic representation of the homologous recombination strategy yielding mrpigG overexpression strains. ( b ) Construction of mrpigG overexpression construct by double-joint PCR. Lane 1, 5′ flanking region of mrpigG ; lane 2, G418 resistance cassette; lane 3, the trpC promoter; lane 4, mrpigG ORF regions plus 3′ flanking region of mrpigG ; lane 5, double-joint PCR product. ( c ) Confirmation of mrpigG homologous recombination events. Three primer pairs were used, and PCR amplifications showed distinct bands in different strains. Lane 1, the M7:: PtrpC-mrpigG strain; lane 2, the wild-type strain.

    Techniques Used: Over Expression, Homologous Recombination, Construct, Polymerase Chain Reaction

    Complementation of mrpigG in M. ruber M7. ( a ) Schematic representation of the homologous recombination strategy to construct mrpigG complementation strains ( b ) Construction of mrpigG complementation cassette by double-joint PCR. Lane 1, 5′ flanking region of mrpigG plus mrpigG ORF regions; lane 2, G418 resistance cassette; lane 3, 3′ flanking region of mrpigG ; lane 4, double-joint PCR product. ( c ) Confirmation of mrpigG homologous recombination events. Four primer pairs were used, and PCR amplifications showed distinct bands in different strains. Lane 1, the Δ mrpigG :: mrpigG strain; lane 2, the Δ mrpigG strain.
    Figure Legend Snippet: Complementation of mrpigG in M. ruber M7. ( a ) Schematic representation of the homologous recombination strategy to construct mrpigG complementation strains ( b ) Construction of mrpigG complementation cassette by double-joint PCR. Lane 1, 5′ flanking region of mrpigG plus mrpigG ORF regions; lane 2, G418 resistance cassette; lane 3, 3′ flanking region of mrpigG ; lane 4, double-joint PCR product. ( c ) Confirmation of mrpigG homologous recombination events. Four primer pairs were used, and PCR amplifications showed distinct bands in different strains. Lane 1, the Δ mrpigG :: mrpigG strain; lane 2, the Δ mrpigG strain.

    Techniques Used: Homologous Recombination, Construct, Polymerase Chain Reaction

    21) Product Images from "The fusion proteins TEL-PDGFR? and FIP1L1-PDGFR? escape ubiquitination and degradation"

    Article Title: The fusion proteins TEL-PDGFR? and FIP1L1-PDGFR? escape ubiquitination and degradation

    Journal:

    doi: 10.3324/haematol.2008.001149

    The hybrid ZNF198-FGFR1 is also a stable protein. Ba/F3 cells were electroporated with wild-type human FGFR1 or ZNF198-FGFR1 and selected in the presence of G418. Cells stably expressing the receptors were treated with cycloheximide (50 μg/mL)
    Figure Legend Snippet: The hybrid ZNF198-FGFR1 is also a stable protein. Ba/F3 cells were electroporated with wild-type human FGFR1 or ZNF198-FGFR1 and selected in the presence of G418. Cells stably expressing the receptors were treated with cycloheximide (50 μg/mL)

    Techniques Used: Stable Transfection, Expressing

    22) Product Images from "Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells"

    Article Title: Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.104

    c-FLIP L was responsible for the synergistic effect of Mapa and hyperthermia. ( a ) CX-1 cells were transfected with pCR3.V64-Met-Flag-FLIPL and stable clones were selected with G418 (500 μ g/ml). ( b ) Control plasmid or pCR3.V64-Met-Flag-FLIPL stably transfected cells from a pool of clone 1#, 8# and 11# were heated at 42 °C for 1 h in the presence or absence of 500 ng/ml Mapa, and then incubated at 37 °C for 3 h. The level of c-FLIP L and the cleavage of caspase 8, caspase 9 and PARP were detected by immunoblotting. Actin was used as loading control. ( c ) CX-1 cells were transfected with nonsense sequence (control) or FLIP siRNA-targeting FLIP mRNA. After 48 h, cells were heated at 42 °C for 1 h in the presence or absence of 100 ng/ml Mapa, and then incubated for 3 h. The levels of c-FLIP L and PARP were detected by immunoblotting. Actin was used as a loading control
    Figure Legend Snippet: c-FLIP L was responsible for the synergistic effect of Mapa and hyperthermia. ( a ) CX-1 cells were transfected with pCR3.V64-Met-Flag-FLIPL and stable clones were selected with G418 (500 μ g/ml). ( b ) Control plasmid or pCR3.V64-Met-Flag-FLIPL stably transfected cells from a pool of clone 1#, 8# and 11# were heated at 42 °C for 1 h in the presence or absence of 500 ng/ml Mapa, and then incubated at 37 °C for 3 h. The level of c-FLIP L and the cleavage of caspase 8, caspase 9 and PARP were detected by immunoblotting. Actin was used as loading control. ( c ) CX-1 cells were transfected with nonsense sequence (control) or FLIP siRNA-targeting FLIP mRNA. After 48 h, cells were heated at 42 °C for 1 h in the presence or absence of 100 ng/ml Mapa, and then incubated for 3 h. The levels of c-FLIP L and PARP were detected by immunoblotting. Actin was used as a loading control

    Techniques Used: Transfection, Clone Assay, Plasmid Preparation, Stable Transfection, Incubation, Sequencing

    23) Product Images from "Manipulations in HIWI level exerts influence on the proliferation of human non-small cell lung cancer cells"

    Article Title: Manipulations in HIWI level exerts influence on the proliferation of human non-small cell lung cancer cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3106

    HIWI overexpression was stabilized in the HIWI and EGFP co-expressing A549 HIWI (+) cells. (A) A sketch map of the HIWI and EGFP genes joined with a self-cleaving 2A linkage sequence. (B) The EGFP-positive A549 HIWI (+) cells were selected under G418
    Figure Legend Snippet: HIWI overexpression was stabilized in the HIWI and EGFP co-expressing A549 HIWI (+) cells. (A) A sketch map of the HIWI and EGFP genes joined with a self-cleaving 2A linkage sequence. (B) The EGFP-positive A549 HIWI (+) cells were selected under G418

    Techniques Used: Over Expression, Expressing, Sequencing

    24) Product Images from "UBE2N Regulates Paclitaxel Sensitivity of Ovarian Cancer via Fos/P53 Axis"

    Article Title: UBE2N Regulates Paclitaxel Sensitivity of Ovarian Cancer via Fos/P53 Axis

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S271164

    Fos regulated paclitaxel sensitivity in vitro and participated in UBE2N regulation of the paclitaxel sensitivity via P53. ( A and B ) Western blotting of UBE2N, Fos and P53 in A2780 and SKOV3 cells with UBE2N-knockdown. ( C and D ) Western blotting of Fos, P53 in A2780 and SKOV3 cells with Fos-knockdown. ( E and F ) Western blotting of Fos, P53 in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. ( G and H ) Cell viability assays in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. ( I and J ) IC50 of paclitaxel in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days. Results are shown as means±SEM for at least 3 separate experiments. The level of significance is indicated by * P
    Figure Legend Snippet: Fos regulated paclitaxel sensitivity in vitro and participated in UBE2N regulation of the paclitaxel sensitivity via P53. ( A and B ) Western blotting of UBE2N, Fos and P53 in A2780 and SKOV3 cells with UBE2N-knockdown. ( C and D ) Western blotting of Fos, P53 in A2780 and SKOV3 cells with Fos-knockdown. ( E and F ) Western blotting of Fos, P53 in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. ( G and H ) Cell viability assays in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. ( I and J ) IC50 of paclitaxel in A2780 and SKOV3 cells with Fos-knockdown, which were transfected in advance with UBE2N-specific shRNA and selected with G418 (400 μg/mL) for 14 days. Results are shown as means±SEM for at least 3 separate experiments. The level of significance is indicated by * P

    Techniques Used: In Vitro, Western Blot, Transfection, shRNA

    25) Product Images from "Partial Antiviral Activities Detection of Chicken Mx Jointing with Neuraminidase Gene (NA) against Newcastle Disease Virus"

    Article Title: Partial Antiviral Activities Detection of Chicken Mx Jointing with Neuraminidase Gene (NA) against Newcastle Disease Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071688

    HA of antibody group infected NDV at different time. After G418 selection for two weeks, the antibody incubated CEF cells were trypsinized and seeded into 24-well plates. After overnight cultivation, 100 TCID50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24, 48 and 72 h infection, supernatants of the cell cultures were collected for NDV titration on CEF cells.
    Figure Legend Snippet: HA of antibody group infected NDV at different time. After G418 selection for two weeks, the antibody incubated CEF cells were trypsinized and seeded into 24-well plates. After overnight cultivation, 100 TCID50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24, 48 and 72 h infection, supernatants of the cell cultures were collected for NDV titration on CEF cells.

    Techniques Used: Infection, Selection, Incubation, Titration

    HA of transfection group infected NDV at different time. After G418 selection for two weeks, the transfected CEF cells were trypsinized and seeded into 24-well plates. After overnight cultivation, 100 TCID50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24, 48 and 72 h infection, supernatants of the cell cultures were collected for NDV titration on CEF cells.
    Figure Legend Snippet: HA of transfection group infected NDV at different time. After G418 selection for two weeks, the transfected CEF cells were trypsinized and seeded into 24-well plates. After overnight cultivation, 100 TCID50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24, 48 and 72 h infection, supernatants of the cell cultures were collected for NDV titration on CEF cells.

    Techniques Used: Transfection, Infection, Selection, Incubation, Titration

    The cell morphology of CEF inoculated NDV for each transfection group at different time (200×). CEF cells were transfected with pVITRO 2 -Mx-NA, pVITRO 2 -Mx, pVITRO 2 -NA and pVITRO 2 . After G418 selection for two weeks, the transfected CEF cells were trypsinized and seeded into 6-well plates. After overnight cultivation, 100 TCID 50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24 h, 48 h, 72 h, 96 h and 120 h infection, cell cultures were observed under microscope for cytopathic effect (CPE) and 50% CPE inhibition was recorded as described. Normal CEF cells infected with NDV was used as a positive control. pVITRO 2 -Mx-NA: pVITRO 2 -MN; pVITRO 2 -Mx: pVITRO 2 -M; pVITRO 2 -NA: pVITRO 2 -N.
    Figure Legend Snippet: The cell morphology of CEF inoculated NDV for each transfection group at different time (200×). CEF cells were transfected with pVITRO 2 -Mx-NA, pVITRO 2 -Mx, pVITRO 2 -NA and pVITRO 2 . After G418 selection for two weeks, the transfected CEF cells were trypsinized and seeded into 6-well plates. After overnight cultivation, 100 TCID 50 NDV was added to each well (in triplicates) and incubation was continued for 1 h at 37°C. After 24 h, 48 h, 72 h, 96 h and 120 h infection, cell cultures were observed under microscope for cytopathic effect (CPE) and 50% CPE inhibition was recorded as described. Normal CEF cells infected with NDV was used as a positive control. pVITRO 2 -Mx-NA: pVITRO 2 -MN; pVITRO 2 -Mx: pVITRO 2 -M; pVITRO 2 -NA: pVITRO 2 -N.

    Techniques Used: Transfection, Selection, Incubation, Infection, Microscopy, Inhibition, Positive Control

    26) Product Images from "Hiwi overexpression does not affect proliferation, migration or apoptosis of liver cancer cells in vitro or in vivo"

    Article Title: Hiwi overexpression does not affect proliferation, migration or apoptosis of liver cancer cells in vitro or in vivo

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8585

    Generation of human Hiwi-expressing plasmid. (A) PcDNA3.1-myc-Hiwi plasmids were cut using Eco RI and Xho I restriction enzymes and separated by agarose gel electrophoresis. The arrow indicates inserts of ~2.5 kb. (B) 293 cells were transfected with PcDNA3.1-myc-Hiwi plasmid, and the cell lysates were subjected to western blotting using a Hiwi-directed antibody. (C) At 24 h after plating, primary mouse hepatocytes were incubated for 4 h with recombinant adenoviruses expressing Hiwi at a total multiplicity of infection of 10 pfu per cell. The cells were then cultured for an additional 24 h in fresh medium, and the cell lysates were subjected to western blotting using a myc-directed antibody. The arrow indicates the Hiwi protein band. (D) SMMC7721 cells were transfected with pcDNA3.1-myc-Hiwi plasmid and were selected in 400 µg/ml G418 for 2 weeks. The cell lines that stably express Hiwi at high (lanes 3 and 6) or low (lanes 1 and 4) levels were identified by western blot analysis using a Hiwi-directed antibody. The cell lines that do not express Hiwi were used as controls. Hiwi, piwi like RNA-mediated gene silencing 1.
    Figure Legend Snippet: Generation of human Hiwi-expressing plasmid. (A) PcDNA3.1-myc-Hiwi plasmids were cut using Eco RI and Xho I restriction enzymes and separated by agarose gel electrophoresis. The arrow indicates inserts of ~2.5 kb. (B) 293 cells were transfected with PcDNA3.1-myc-Hiwi plasmid, and the cell lysates were subjected to western blotting using a Hiwi-directed antibody. (C) At 24 h after plating, primary mouse hepatocytes were incubated for 4 h with recombinant adenoviruses expressing Hiwi at a total multiplicity of infection of 10 pfu per cell. The cells were then cultured for an additional 24 h in fresh medium, and the cell lysates were subjected to western blotting using a myc-directed antibody. The arrow indicates the Hiwi protein band. (D) SMMC7721 cells were transfected with pcDNA3.1-myc-Hiwi plasmid and were selected in 400 µg/ml G418 for 2 weeks. The cell lines that stably express Hiwi at high (lanes 3 and 6) or low (lanes 1 and 4) levels were identified by western blot analysis using a Hiwi-directed antibody. The cell lines that do not express Hiwi were used as controls. Hiwi, piwi like RNA-mediated gene silencing 1.

    Techniques Used: Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Transfection, Western Blot, Incubation, Recombinant, Infection, Cell Culture, Stable Transfection

    27) Product Images from "N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro"

    Article Title: N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro

    Journal:

    doi: 10.3748/wjg.15.697

    EGFP expression in PT67 cells and EC9706 cells after pMSCVneo/N-cadherin transfection. A: Selected by 1000 mg/L G418 for 15 d and 300 mg/L G418 for 15 d, the PT-67 cells transfected with pMSCVneo/N-cadherin plasmid expressed EGFP stably. The figure was
    Figure Legend Snippet: EGFP expression in PT67 cells and EC9706 cells after pMSCVneo/N-cadherin transfection. A: Selected by 1000 mg/L G418 for 15 d and 300 mg/L G418 for 15 d, the PT-67 cells transfected with pMSCVneo/N-cadherin plasmid expressed EGFP stably. The figure was

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

    28) Product Images from "LGR5 promotes cancer stem cell traits and chemoresistance in cervical cancer"

    Article Title: LGR5 promotes cancer stem cell traits and chemoresistance in cervical cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.393

    LGR5 + cervical cancer cells are capable of differentiating in vitro and in vivo . ( a – d ) LGR5 + and LGR5 − cells were isolated from LGR5-overexpressing SiHa and HeLa or control cells and cultured in DMEM medium supplemented with 10% FBS and 1000 μ g/ml G418 for 2 weeks in vitro . The xenograft tumor cells from LGR5 + and LGR5 − cells in vivo were digested by collagenase IV overnight before detection. The expression of LGR5 was analyzed by flow cytometry
    Figure Legend Snippet: LGR5 + cervical cancer cells are capable of differentiating in vitro and in vivo . ( a – d ) LGR5 + and LGR5 − cells were isolated from LGR5-overexpressing SiHa and HeLa or control cells and cultured in DMEM medium supplemented with 10% FBS and 1000 μ g/ml G418 for 2 weeks in vitro . The xenograft tumor cells from LGR5 + and LGR5 − cells in vivo were digested by collagenase IV overnight before detection. The expression of LGR5 was analyzed by flow cytometry

    Techniques Used: In Vitro, In Vivo, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry

    29) Product Images from "Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology"

    Article Title: Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108311

    Transformation of pZLY1 into Candida shehatae 20335 and detection of the dominant selective marker (G418 resistance) and reporter genes (GFP gene). A. 1: Colonies were not observed on YEPX solid medium with G418 when Candida shehatae 20335 was not transformed by pZLY1; 2: several colonies grew on YEPX solid medium with G418 when Candida shehatae 20335 was transformed by pZLY1. B. Candida shehatae 20335 cells were observed under light microscopy (1600×). C. Candida shehatae 20335 transformants were observed under light microscopy (1600×). No morphological differences can be seen between the cells. D. Candida shehatae 20335 cells observed under fluorescence microscopy (1000×), showing GFP is not expressed in untransformed cells. E. Candida shehatae 20335 cells observed under fluorescence microscopy (1000×), showing GFP is expressed in transformants.
    Figure Legend Snippet: Transformation of pZLY1 into Candida shehatae 20335 and detection of the dominant selective marker (G418 resistance) and reporter genes (GFP gene). A. 1: Colonies were not observed on YEPX solid medium with G418 when Candida shehatae 20335 was not transformed by pZLY1; 2: several colonies grew on YEPX solid medium with G418 when Candida shehatae 20335 was transformed by pZLY1. B. Candida shehatae 20335 cells were observed under light microscopy (1600×). C. Candida shehatae 20335 transformants were observed under light microscopy (1600×). No morphological differences can be seen between the cells. D. Candida shehatae 20335 cells observed under fluorescence microscopy (1000×), showing GFP is not expressed in untransformed cells. E. Candida shehatae 20335 cells observed under fluorescence microscopy (1000×), showing GFP is expressed in transformants.

    Techniques Used: Transformation Assay, Marker, Light Microscopy, Fluorescence, Microscopy

    Screening of protoplast fusants. A. Many colonies are found to grow on YEPDS regenerated solid medium when protoplast fusants are spread on it. B and C. Several colonies grow on YEPDS regenerated solid medium with blasticidin and G418.
    Figure Legend Snippet: Screening of protoplast fusants. A. Many colonies are found to grow on YEPDS regenerated solid medium when protoplast fusants are spread on it. B and C. Several colonies grow on YEPDS regenerated solid medium with blasticidin and G418.

    Techniques Used:

    Structures of the pZLY1 plasmid (left) with the G418 resistance gene ( Kan R) and adh 1P– GFP – adh 1t reporter gene, and the pZLY2 plasmid (right) with the blasticidin resistance gene ( bsd ) and adh 1P– gus A– adh 1t reporter gene. adh 1P and adh 1t represent, respectively, the promoter and terminator of the adh 1 gene of Saccharomyces cerevisiae . Kan R represents the G418 resistance gene, gus A represents the gus A gene and GFP represent the GFP gene.
    Figure Legend Snippet: Structures of the pZLY1 plasmid (left) with the G418 resistance gene ( Kan R) and adh 1P– GFP – adh 1t reporter gene, and the pZLY2 plasmid (right) with the blasticidin resistance gene ( bsd ) and adh 1P– gus A– adh 1t reporter gene. adh 1P and adh 1t represent, respectively, the promoter and terminator of the adh 1 gene of Saccharomyces cerevisiae . Kan R represents the G418 resistance gene, gus A represents the gus A gene and GFP represent the GFP gene.

    Techniques Used: Plasmid Preparation

    30) Product Images from "Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity"

    Article Title: Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02788-14

    HCV replicon elimination with DCV. Huh-7 cells harboring GT-1 to -6 replicons were treated with the indicated concentrations of DCV for 14 days in the absence of G418. Cell cultures were maintained at subconfluency by splitting as described in Materials
    Figure Legend Snippet: HCV replicon elimination with DCV. Huh-7 cells harboring GT-1 to -6 replicons were treated with the indicated concentrations of DCV for 14 days in the absence of G418. Cell cultures were maintained at subconfluency by splitting as described in Materials

    Techniques Used:

    GT-2a M31 replicon cells were treated for 3 or 7 days with the indicated concentration of a PI, MK-5172, in the absence or presence of 250 nM DCV and without G418. After either 3 or 7 days of treatment (D3 and D7, respectively), inhibitors were removed
    Figure Legend Snippet: GT-2a M31 replicon cells were treated for 3 or 7 days with the indicated concentration of a PI, MK-5172, in the absence or presence of 250 nM DCV and without G418. After either 3 or 7 days of treatment (D3 and D7, respectively), inhibitors were removed

    Techniques Used: Concentration Assay

    31) Product Images from "Elevated expression of solute carrier family 22 member 18 increases the sensitivity of U251 glioma cells to BCNU"

    Article Title: Elevated expression of solute carrier family 22 member 18 increases the sensitivity of U251 glioma cells to BCNU

    Journal: Oncology Letters

    doi: 10.3892/ol.2011.371

    Microscopic images of different groups cells in the selection. (A) Normal U251; (B) Normal U251 cells cultured in the presence of G418 for 2 weeks; (C and D) U251-SLC22A18 cells cultured in the presence of G418 for 3 weeks.
    Figure Legend Snippet: Microscopic images of different groups cells in the selection. (A) Normal U251; (B) Normal U251 cells cultured in the presence of G418 for 2 weeks; (C and D) U251-SLC22A18 cells cultured in the presence of G418 for 3 weeks.

    Techniques Used: Selection, Cell Culture

    32) Product Images from "New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism"

    Article Title: New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism

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

    doi: 10.1073/pnas.1602049113

    Screening of the lead compounds that mediate nonsense suppression of nsCre transgene in vivo. Fluorescence image shows tdTomato expression profile (red) in the ARC region of Agrp nsCre/+ ::Rosa26 tdTomato mice 4 d after injection of either G418 ( A and E
    Figure Legend Snippet: Screening of the lead compounds that mediate nonsense suppression of nsCre transgene in vivo. Fluorescence image shows tdTomato expression profile (red) in the ARC region of Agrp nsCre/+ ::Rosa26 tdTomato mice 4 d after injection of either G418 ( A and E

    Techniques Used: In Vivo, Fluorescence, Expressing, Mouse Assay, Injection

    Chemical structures of the aminoglycoside G418 and its synthetic derivatives NB124, NB127, and NB128 that were investigated in this study. The common part of all of the structures is highlighted in blue color. The ring numbers are in capital roman numerals
    Figure Legend Snippet: Chemical structures of the aminoglycoside G418 and its synthetic derivatives NB124, NB127, and NB128 that were investigated in this study. The common part of all of the structures is highlighted in blue color. The ring numbers are in capital roman numerals

    Techniques Used:

    33) Product Images from "SETMAR functions in illegitimate DNA recombination and non-homologous end joining"

    Article Title: SETMAR functions in illegitimate DNA recombination and non-homologous end joining

    Journal: bioRxiv

    doi: 10.1101/465138

    The SET and MAR domains increase the frequency of illegitimate DNA integration. A , Representation of the integration assay. Cells are transfected with a circular plasmid encoding a neomycin resistance gene. For integration to occur through the NHEJ pathway, the plasmid needs to be linearized by a DSB and a plasmid free end has to be in close vicinity of a genomic DSB. The linearized plasmid can also be repaired, which re-circularized the plasmid, or be degraded. Following G418 treatment for two weeks, surviving cells form foci which can be detected by methylene blue staining. B , Number of illegitimate integration events in the genome of a circular plasmid encoding a neomycin resistance gene. Average ± S.E.M. of 3 biological replicates. Statistical test: paired t-test, ∗ p-value
    Figure Legend Snippet: The SET and MAR domains increase the frequency of illegitimate DNA integration. A , Representation of the integration assay. Cells are transfected with a circular plasmid encoding a neomycin resistance gene. For integration to occur through the NHEJ pathway, the plasmid needs to be linearized by a DSB and a plasmid free end has to be in close vicinity of a genomic DSB. The linearized plasmid can also be repaired, which re-circularized the plasmid, or be degraded. Following G418 treatment for two weeks, surviving cells form foci which can be detected by methylene blue staining. B , Number of illegitimate integration events in the genome of a circular plasmid encoding a neomycin resistance gene. Average ± S.E.M. of 3 biological replicates. Statistical test: paired t-test, ∗ p-value

    Techniques Used: Transfection, Plasmid Preparation, Non-Homologous End Joining, Staining

    34) Product Images from "Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells"

    Article Title: Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.202810

    The enhancement of nuclear envelope anchorage inhibits APB formation. ( A ) Schematic diagrams of cells overexpressing SUN1, RAP1-RCT-domain-deleted-SUN1 (RAP1ΔC-SUN1), or RAP1-SUN1 fusion chimera protein are shown. NE, the nuclear envelope. ( B ) U2OS and VA13 cells were infected with lentivirus expressing the empty vector control (EV), SUN1, RAP1ΔC-SUN1, or RAP1-SUN1 fusion and then selected in medium containing G418 for 5 days. Cell lysates were analyzed by immunoblotting with anti-RAP1, anti-SUN1, and anti-GAPDH antibodies. The arrowhead indicates the RAP1-SUN1 fusion protein. The arrow indicates endogenous SUN1. The asterisk indicates endogenous RAP1. The ladders under the major protein band show possible products of protein degradation. GAPDH was used as the loading control. ( C ) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel), as shown in Figure 1 . Scale bar, 20 μm. ( D ) Quantification of APBs (%) in the U2OS and VA13 cells shown in ( C ). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); * P
    Figure Legend Snippet: The enhancement of nuclear envelope anchorage inhibits APB formation. ( A ) Schematic diagrams of cells overexpressing SUN1, RAP1-RCT-domain-deleted-SUN1 (RAP1ΔC-SUN1), or RAP1-SUN1 fusion chimera protein are shown. NE, the nuclear envelope. ( B ) U2OS and VA13 cells were infected with lentivirus expressing the empty vector control (EV), SUN1, RAP1ΔC-SUN1, or RAP1-SUN1 fusion and then selected in medium containing G418 for 5 days. Cell lysates were analyzed by immunoblotting with anti-RAP1, anti-SUN1, and anti-GAPDH antibodies. The arrowhead indicates the RAP1-SUN1 fusion protein. The arrow indicates endogenous SUN1. The asterisk indicates endogenous RAP1. The ladders under the major protein band show possible products of protein degradation. GAPDH was used as the loading control. ( C ) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel), as shown in Figure 1 . Scale bar, 20 μm. ( D ) Quantification of APBs (%) in the U2OS and VA13 cells shown in ( C ). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); * P

    Techniques Used: Infection, Expressing, Plasmid Preparation

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    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Expressing:

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer
    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1
    Article Snippet: All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere. .. NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen). ..

    Construct:

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer
    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Generated:

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer
    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1
    Article Snippet: All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere. .. NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen). ..

    Selection:

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer
    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Article Title: Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells
    Article Snippet: After 16 h, transfection supernatant was used to infect xenotropic PG13 packaging cells , which produce the gibbon ape leukemia virus (GALV) env protein. .. Subsequent selection in G418 (Sigma; 400 μg/ml) resulted in 33 clones, which were screened using a modified viral RNA dot blot assay ( ) to identify potential high titre candidates. ..

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1
    Article Snippet: All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere. .. NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen). ..

    Cell Culture:

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer
    Article Snippet: Where indicated, 24 hours before transfection, cells were plated at optimal density and the following day transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). .. Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418. ..

    Article Title: Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection
    Article Snippet: .. During short- or long-term ARB treatment, FL-Neo cells were cultured in medium without G418. ..

    Transfection:

    Article Title: Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism
    Article Snippet: Site-directed mutagenesis (Stratagene, Quickchange) was used to introduce the K42A mutation in the RAD50 ORF and generate the Psp72-α-NEO -α-RAD50 K42A using primers Q and R ( ). .. Both Psp72-α-NEO -α-RAD50 WT and Psp72-α-NEO -α-RAD50 K42A plasmids were then transfected by electroporation in the L . infantum BLAST RAD50 -/+ mutants and cells were selected with 40μg/ml of G418 (Geneticin; Sigma-Aldrich). .. After inactivation of the second RAD50 genomic allele with the PURO cassette, attempts to lose the Psp72-α-NEO -α-RAD50 construct were performed by removing the G418 drug pressure up to 55 passages.

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation
    Article Snippet: Protein band intensities were quantified using Image Quant 3.0 software. .. Colony formation assay 10 million HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C, Myc-Bcl6 and GFP vector by electroporation and allowed to grow in DMEM supplemented with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). .. After two weeks selection, GFP fluorescence of every plate was scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA).

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1
    Article Snippet: All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere. .. NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen). ..

    Electroporation:

    Article Title: Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism
    Article Snippet: Site-directed mutagenesis (Stratagene, Quickchange) was used to introduce the K42A mutation in the RAD50 ORF and generate the Psp72-α-NEO -α-RAD50 K42A using primers Q and R ( ). .. Both Psp72-α-NEO -α-RAD50 WT and Psp72-α-NEO -α-RAD50 K42A plasmids were then transfected by electroporation in the L . infantum BLAST RAD50 -/+ mutants and cells were selected with 40μg/ml of G418 (Geneticin; Sigma-Aldrich). .. After inactivation of the second RAD50 genomic allele with the PURO cassette, attempts to lose the Psp72-α-NEO -α-RAD50 construct were performed by removing the G418 drug pressure up to 55 passages.

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation
    Article Snippet: Protein band intensities were quantified using Image Quant 3.0 software. .. Colony formation assay 10 million HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C, Myc-Bcl6 and GFP vector by electroporation and allowed to grow in DMEM supplemented with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). .. After two weeks selection, GFP fluorescence of every plate was scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA).

    Colony Assay:

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation
    Article Snippet: Protein band intensities were quantified using Image Quant 3.0 software. .. Colony formation assay 10 million HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C, Myc-Bcl6 and GFP vector by electroporation and allowed to grow in DMEM supplemented with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). .. After two weeks selection, GFP fluorescence of every plate was scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA).

    Plasmid Preparation:

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation
    Article Snippet: Protein band intensities were quantified using Image Quant 3.0 software. .. Colony formation assay 10 million HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C, Myc-Bcl6 and GFP vector by electroporation and allowed to grow in DMEM supplemented with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). .. After two weeks selection, GFP fluorescence of every plate was scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA).

    Clone Assay:

    Article Title: Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells
    Article Snippet: After 16 h, transfection supernatant was used to infect xenotropic PG13 packaging cells , which produce the gibbon ape leukemia virus (GALV) env protein. .. Subsequent selection in G418 (Sigma; 400 μg/ml) resulted in 33 clones, which were screened using a modified viral RNA dot blot assay ( ) to identify potential high titre candidates. ..

    Modification:

    Article Title: Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells
    Article Snippet: After 16 h, transfection supernatant was used to infect xenotropic PG13 packaging cells , which produce the gibbon ape leukemia virus (GALV) env protein. .. Subsequent selection in G418 (Sigma; 400 μg/ml) resulted in 33 clones, which were screened using a modified viral RNA dot blot assay ( ) to identify potential high titre candidates. ..

    Dot Blot:

    Article Title: Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells
    Article Snippet: After 16 h, transfection supernatant was used to infect xenotropic PG13 packaging cells , which produce the gibbon ape leukemia virus (GALV) env protein. .. Subsequent selection in G418 (Sigma; 400 μg/ml) resulted in 33 clones, which were screened using a modified viral RNA dot blot assay ( ) to identify potential high titre candidates. ..

    Activity Assay:

    Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb
    Article Snippet: .. To assess telomerase activity in the immortalized (early, ≤ 20 subpassages) versus primary (L-OB hTERT- ) OB cultures as well as in late subpassage OBGF400 cultures that had or had not undergone re-exposure to the neomycin analog, G418, a TRAP assay was conducted using the TRAPeze Detection Kit (Chemicon International, Inc, Temecula, CA). ..

    TRAP Assay:

    Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb
    Article Snippet: .. To assess telomerase activity in the immortalized (early, ≤ 20 subpassages) versus primary (L-OB hTERT- ) OB cultures as well as in late subpassage OBGF400 cultures that had or had not undergone re-exposure to the neomycin analog, G418, a TRAP assay was conducted using the TRAPeze Detection Kit (Chemicon International, Inc, Temecula, CA). ..

    Marker:

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1
    Article Snippet: All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere. .. NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen). ..

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  • 97
    Millipore g418
    Fbx4 serine 12 is required for Cyclin D1 ubiquitination and proteolysis A. 293T cells were transfected with plasmids encoding Flag-tagged Fbx4 mutants and wild type Myc-tagged Fbx4. Complexes were isolated by affinity chromatography using the M2 conjugated agarose and individual components detected by immunoblot with Fbx4 and Skp1 antibodies. B. NIH-3T3 cells, wherein Fbx4 had previously been knocked down by shRNA, were transfected with wt, S12A or S12E Fbx4 pcDNA3 followed by <t>G418</t> selection of stably expressing clones. Asynchronous cells were harvested and subjected to Western blotting with cyclin D1, TRF1 and Fbx4 antibodies. C. Quantification of B. Error bars indicate +/−SD. D. Ligase complexes (purified protein shown in bottom panel) were purified from stable cell lines expressing wt, S12E and S12A Fbx4 (described in B). In vitro ubiquitination reactions were performed using GST-tagged purified cyclin D1. Ubiquitin-conjugated cyclin D1 was detected by immunoblot. Non-specific complexes are denoted by asterisk. E. NIH3T3 cells were serum starved for 48 hrs and released into 10% FBS containing media for the indicated intervals. Immunoblot analysis was performed using pS11/12-Fbx4 and a total Fbx4 antibody. F. Cells synchronized via a nocodazole block were harvested at the indicated intervals following nocodazole release. Fbx4 phosphorylation was detected by precipitation with the pS11/12 antibody and immunoblot with the total Fbx4 antibody. Total cyclin D1 and Fbx4 levels were assessed by direct western.
    G418, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/g418/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    g418 - by Bioz Stars, 2021-06
    97/100 stars
      Buy from Supplier

    95
    Millipore g 418 disulfate salt
    Sex selection and fitness of the flies carrying two copy of sex-sorter cassette. The sex-sorter cassette includes NeoR traF and PuroR dsxM genes that confer resistance to puromycin and <t>geneticin</t> antibiotics, respectively, in the sex-specific manner ( Figure 1a ). The PuroR dsxM gene is properly spliced and results in expression of the functional PuroR protein only in males, while NeoR traF expresses the NeoR protein only in females. To estimate the lowest concentration of an antibiotic at which male or female selection is enforced at 100%, the homozygous sex-sorter flies were raised on various concentrations of puromycin or geneticin. (a) Only male flies emerged from the food supplemented with 0.4 mg/mL or more of puromycin. (b) Raising the same flies on the food containing 0.2 mg/mL or more of geneticin resulted in the emergence of only female flies. To compare the fitness of homozygous sex-sorter flies to that of wild type ( wt ) flies, the embryo-to-adult survival of both fly types were compared under normal and selective conditions. (c) Embryos of both sex-sorter (gray bars) and wt flies (white bars) survived to the adulthood equally well on food without any antibiotics and died on the food supplemented with both puromycin and geneticin to 0.4 and 1.2 mg/mL. (d) The survival of male or female sex-sorter flies under selection treatments was statistically identical to that of the corresponding gender from wt flies raised under normal conditions. Bar plots show the average ± SD over at least three biological replicates. Statistical significance was estimated using a t test with equal variance. ( P ≥ 0.05 ns , P
    G 418 Disulfate Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/g 418 disulfate salt/product/Millipore
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    g 418 disulfate salt - by Bioz Stars, 2021-06
    95/100 stars
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    This vector allows the expression of two genes from one vector where the second gene produced from the mRNA is the neomycin G418 G418 antibiotic resistance gene Neo under the
      Buy from Supplier

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    This vector contains a Neomycin Neo G418 resistance expression cassette under control of the RSV promoter The entire RSV Neo G418 cassette is flanked by AscI sites The RSV promoter
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    Fbx4 serine 12 is required for Cyclin D1 ubiquitination and proteolysis A. 293T cells were transfected with plasmids encoding Flag-tagged Fbx4 mutants and wild type Myc-tagged Fbx4. Complexes were isolated by affinity chromatography using the M2 conjugated agarose and individual components detected by immunoblot with Fbx4 and Skp1 antibodies. B. NIH-3T3 cells, wherein Fbx4 had previously been knocked down by shRNA, were transfected with wt, S12A or S12E Fbx4 pcDNA3 followed by G418 selection of stably expressing clones. Asynchronous cells were harvested and subjected to Western blotting with cyclin D1, TRF1 and Fbx4 antibodies. C. Quantification of B. Error bars indicate +/−SD. D. Ligase complexes (purified protein shown in bottom panel) were purified from stable cell lines expressing wt, S12E and S12A Fbx4 (described in B). In vitro ubiquitination reactions were performed using GST-tagged purified cyclin D1. Ubiquitin-conjugated cyclin D1 was detected by immunoblot. Non-specific complexes are denoted by asterisk. E. NIH3T3 cells were serum starved for 48 hrs and released into 10% FBS containing media for the indicated intervals. Immunoblot analysis was performed using pS11/12-Fbx4 and a total Fbx4 antibody. F. Cells synchronized via a nocodazole block were harvested at the indicated intervals following nocodazole release. Fbx4 phosphorylation was detected by precipitation with the pS11/12 antibody and immunoblot with the total Fbx4 antibody. Total cyclin D1 and Fbx4 levels were assessed by direct western.

    Journal: Cancer cell

    Article Title: Mutations in Fbx4 inhibit phosphorylation-dependent dimerization of the SCFFbx4 ligase and contribute to cyclin D1 overexpression in human cancer

    doi: 10.1016/j.ccr.2008.05.017

    Figure Lengend Snippet: Fbx4 serine 12 is required for Cyclin D1 ubiquitination and proteolysis A. 293T cells were transfected with plasmids encoding Flag-tagged Fbx4 mutants and wild type Myc-tagged Fbx4. Complexes were isolated by affinity chromatography using the M2 conjugated agarose and individual components detected by immunoblot with Fbx4 and Skp1 antibodies. B. NIH-3T3 cells, wherein Fbx4 had previously been knocked down by shRNA, were transfected with wt, S12A or S12E Fbx4 pcDNA3 followed by G418 selection of stably expressing clones. Asynchronous cells were harvested and subjected to Western blotting with cyclin D1, TRF1 and Fbx4 antibodies. C. Quantification of B. Error bars indicate +/−SD. D. Ligase complexes (purified protein shown in bottom panel) were purified from stable cell lines expressing wt, S12E and S12A Fbx4 (described in B). In vitro ubiquitination reactions were performed using GST-tagged purified cyclin D1. Ubiquitin-conjugated cyclin D1 was detected by immunoblot. Non-specific complexes are denoted by asterisk. E. NIH3T3 cells were serum starved for 48 hrs and released into 10% FBS containing media for the indicated intervals. Immunoblot analysis was performed using pS11/12-Fbx4 and a total Fbx4 antibody. F. Cells synchronized via a nocodazole block were harvested at the indicated intervals following nocodazole release. Fbx4 phosphorylation was detected by precipitation with the pS11/12 antibody and immunoblot with the total Fbx4 antibody. Total cyclin D1 and Fbx4 levels were assessed by direct western.

    Article Snippet: Stable cell lines expressing Fbx4 constructs were generated by G418 (Calbiochem, San Diego, CA) selection (1mg/ml) for 21 days and subsequently cultured in medium containing 500µg/ml G418.

    Techniques: Transfection, Isolation, Affinity Chromatography, shRNA, Selection, Stable Transfection, Expressing, Clone Assay, Western Blot, Purification, In Vitro, Blocking Assay

    RAD50 gene conditional inactivation in L . infantum . (A) Schematic representation of the RAD50 locus in L . infantum before and after integration of the inactivation cassettes blasticidin-S deaminase (5’- BLAST-3’ ), puromycin acetyltransferase (5’- PURO-3’ ) and transfection construct Psp- NEO - RAD50 . S, SacI restriction sites. (B, C) Southern blot analysis with genomic DNAs digested with SacI were hybridized with probes covering either the 5’ flanking region of RAD50 (B) or the RAD50 ORF (C). (D) PCR analysis with primers set aa’ and bb’ the chromosomal copies of the MRE11 and RAD50 genes respectively. Lanes: 1, L . infantum WT; 2, WT Psp- NEO-RAD50 ; 3, WT Rev Psp- NEO - RAD50 ; 4, RAD50 -/- Psp- NEO - RAD50 ; 5, RAD50 -/- Rev Psp- NEO - RAD50 grown for 55 passages in absence of G418; 6, MRE11 -/- and 7, MRE11 -/- RAD50 -/- .

    Journal: PLoS Genetics

    Article Title: Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

    doi: 10.1371/journal.pgen.1006117

    Figure Lengend Snippet: RAD50 gene conditional inactivation in L . infantum . (A) Schematic representation of the RAD50 locus in L . infantum before and after integration of the inactivation cassettes blasticidin-S deaminase (5’- BLAST-3’ ), puromycin acetyltransferase (5’- PURO-3’ ) and transfection construct Psp- NEO - RAD50 . S, SacI restriction sites. (B, C) Southern blot analysis with genomic DNAs digested with SacI were hybridized with probes covering either the 5’ flanking region of RAD50 (B) or the RAD50 ORF (C). (D) PCR analysis with primers set aa’ and bb’ the chromosomal copies of the MRE11 and RAD50 genes respectively. Lanes: 1, L . infantum WT; 2, WT Psp- NEO-RAD50 ; 3, WT Rev Psp- NEO - RAD50 ; 4, RAD50 -/- Psp- NEO - RAD50 ; 5, RAD50 -/- Rev Psp- NEO - RAD50 grown for 55 passages in absence of G418; 6, MRE11 -/- and 7, MRE11 -/- RAD50 -/- .

    Article Snippet: Both Psp72-α-NEO -α-RAD50 WT and Psp72-α-NEO -α-RAD50 K42A plasmids were then transfected by electroporation in the L . infantum BLAST RAD50 -/+ mutants and cells were selected with 40μg/ml of G418 (Geneticin; Sigma-Aldrich).

    Techniques: Transfection, Construct, Southern Blot, Polymerase Chain Reaction

    Ectopic expression of p120 E4F reduces the growth rate of NIH 3T3 fibroblasts. (A) NIH 3T3 cell lines that express E4F cDNA or an amino-terminal E4F fragment were plated at 5 × 10 4 cells per 35-mm-diameter well, and cell counts were determined after the indicated times of growth. Cell lines included the parental line (NIH 3T3), a G418-resistant control (3T3/neo 23-1), three lines that express p120 E4F from E4F cDNA (E4F2.5K/3T3-4, -5, and -7), and two lines that express the first 262 amino acids from E4F cDNA (E4F252/3T3 25-2 and 26-2). Growth rates of the indicated lines were measured in parallel in three independent experiments. Growth curves shown are from a single representative experiment, with each point being the average from duplicate wells; the standard error between duplicates was less than 4% at all points. (B) Expression levels of ectopically expressed p120 E4F in E4F2.5K/3T3 and control cell lines were determined by precipitation with S-protein–agarose and Western blotting using α-E4F-Nterm antiserum after separation of proteins by SDS–10% PAGE. The position of p120 E4F is indicated by the arrow. The positions of molecular mass markers (in kilodaltons) are also shown. (C) Expression levels of the ectopically expressed p50 E4F -like E4F amino-terminal fragment in E4F262 cell lines were determined by Western blotting as described for panel B. The position of the E4F262 protein is indicated by the arrow.

    Journal: Molecular and Cellular Biology

    Article Title: Adenovirus E1A-Regulated Transcription Factor p120E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21WAF1

    doi:

    Figure Lengend Snippet: Ectopic expression of p120 E4F reduces the growth rate of NIH 3T3 fibroblasts. (A) NIH 3T3 cell lines that express E4F cDNA or an amino-terminal E4F fragment were plated at 5 × 10 4 cells per 35-mm-diameter well, and cell counts were determined after the indicated times of growth. Cell lines included the parental line (NIH 3T3), a G418-resistant control (3T3/neo 23-1), three lines that express p120 E4F from E4F cDNA (E4F2.5K/3T3-4, -5, and -7), and two lines that express the first 262 amino acids from E4F cDNA (E4F252/3T3 25-2 and 26-2). Growth rates of the indicated lines were measured in parallel in three independent experiments. Growth curves shown are from a single representative experiment, with each point being the average from duplicate wells; the standard error between duplicates was less than 4% at all points. (B) Expression levels of ectopically expressed p120 E4F in E4F2.5K/3T3 and control cell lines were determined by precipitation with S-protein–agarose and Western blotting using α-E4F-Nterm antiserum after separation of proteins by SDS–10% PAGE. The position of p120 E4F is indicated by the arrow. The positions of molecular mass markers (in kilodaltons) are also shown. (C) Expression levels of the ectopically expressed p50 E4F -like E4F amino-terminal fragment in E4F262 cell lines were determined by Western blotting as described for panel B. The position of the E4F262 protein is indicated by the arrow.

    Article Snippet: NIH 3T3 clone 7 mouse fibroblast cells were a gift from M. Roussel (St. Jude Children’s Research Hospital, Memphis, Tenn.). p120E4F -expressing cell lines (E4F2.5K/3T3) were generated by Lipofectamine- (Life Technologies) or calcium phosphate-mediated transfection with pCMVs-E4F2.5K ( ) and pβA-Pr-neo ( ) as a selection marker and selected in media containing 400 μg of G418 per ml. pCMVs-E4F2.5K expresses the 783-amino-acid E4F open reading frame tagged at the amino terminus with the S-peptide (Novagen).

    Techniques: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis

    Sex selection and fitness of the flies carrying two copy of sex-sorter cassette. The sex-sorter cassette includes NeoR traF and PuroR dsxM genes that confer resistance to puromycin and geneticin antibiotics, respectively, in the sex-specific manner ( Figure 1a ). The PuroR dsxM gene is properly spliced and results in expression of the functional PuroR protein only in males, while NeoR traF expresses the NeoR protein only in females. To estimate the lowest concentration of an antibiotic at which male or female selection is enforced at 100%, the homozygous sex-sorter flies were raised on various concentrations of puromycin or geneticin. (a) Only male flies emerged from the food supplemented with 0.4 mg/mL or more of puromycin. (b) Raising the same flies on the food containing 0.2 mg/mL or more of geneticin resulted in the emergence of only female flies. To compare the fitness of homozygous sex-sorter flies to that of wild type ( wt ) flies, the embryo-to-adult survival of both fly types were compared under normal and selective conditions. (c) Embryos of both sex-sorter (gray bars) and wt flies (white bars) survived to the adulthood equally well on food without any antibiotics and died on the food supplemented with both puromycin and geneticin to 0.4 and 1.2 mg/mL. (d) The survival of male or female sex-sorter flies under selection treatments was statistically identical to that of the corresponding gender from wt flies raised under normal conditions. Bar plots show the average ± SD over at least three biological replicates. Statistical significance was estimated using a t test with equal variance. ( P ≥ 0.05 ns , P

    Journal: bioRxiv

    Article Title: A novel drug-inducible sex-separation technique for insects

    doi: 10.1101/2019.12.13.875716

    Figure Lengend Snippet: Sex selection and fitness of the flies carrying two copy of sex-sorter cassette. The sex-sorter cassette includes NeoR traF and PuroR dsxM genes that confer resistance to puromycin and geneticin antibiotics, respectively, in the sex-specific manner ( Figure 1a ). The PuroR dsxM gene is properly spliced and results in expression of the functional PuroR protein only in males, while NeoR traF expresses the NeoR protein only in females. To estimate the lowest concentration of an antibiotic at which male or female selection is enforced at 100%, the homozygous sex-sorter flies were raised on various concentrations of puromycin or geneticin. (a) Only male flies emerged from the food supplemented with 0.4 mg/mL or more of puromycin. (b) Raising the same flies on the food containing 0.2 mg/mL or more of geneticin resulted in the emergence of only female flies. To compare the fitness of homozygous sex-sorter flies to that of wild type ( wt ) flies, the embryo-to-adult survival of both fly types were compared under normal and selective conditions. (c) Embryos of both sex-sorter (gray bars) and wt flies (white bars) survived to the adulthood equally well on food without any antibiotics and died on the food supplemented with both puromycin and geneticin to 0.4 and 1.2 mg/mL. (d) The survival of male or female sex-sorter flies under selection treatments was statistically identical to that of the corresponding gender from wt flies raised under normal conditions. Bar plots show the average ± SD over at least three biological replicates. Statistical significance was estimated using a t test with equal variance. ( P ≥ 0.05 ns , P

    Article Snippet: A 1.1 g of the dry food was aliquoted per fly vial (FlyStaff.com) and mixed with 5 mL of distilled water supplemented with puromycin (Sigma #P8833) or geneticin (G418, Sigma #A1720), in varying concentrations from 0 to 1.2 mg/mL.

    Techniques: Selection, Expressing, Functional Assay, Concentration Assay

    Development of sex-sorter cassette in Drosophila . (a) Schematic maps of genetic constructs engineered and tested in the study. (b) Expression of antibiotic resistance genes ( PuroR and NeoR ) throughout Drosophila development confers resistance to puromycin and geneticin, respectively, supplemented on fly food. To insure that functional antibiotic-resistance proteins will be produced only in one or the other gender, sex-specific introns from two sex-determination genes ( tra and dsx ) were inserted into coding sequences of PuroR and NeoR . The entire sequences of female-specific traF and male-specific dsxM introns (highlighted in pink) are spliced out in the corresponding sex, but some sequences carrying a stop codon (TGA) are retained in the opposite sex ( Figure S2 ). The transgenic flies harboring one copy of a genetic construct were identified by a strong ubiquitous expression of dsRed (highlighted in purple). (c) Expression of Opie2-PuroR dsxM or Opie2-PuroR traF transgene rescues only transgenic males or females (red fluorescence) raised on the food supplemented with puromycin, while all wild type flies (no red fluorescence) and the transgenic flies of selected-out sex die during early development.

    Journal: bioRxiv

    Article Title: A novel drug-inducible sex-separation technique for insects

    doi: 10.1101/2019.12.13.875716

    Figure Lengend Snippet: Development of sex-sorter cassette in Drosophila . (a) Schematic maps of genetic constructs engineered and tested in the study. (b) Expression of antibiotic resistance genes ( PuroR and NeoR ) throughout Drosophila development confers resistance to puromycin and geneticin, respectively, supplemented on fly food. To insure that functional antibiotic-resistance proteins will be produced only in one or the other gender, sex-specific introns from two sex-determination genes ( tra and dsx ) were inserted into coding sequences of PuroR and NeoR . The entire sequences of female-specific traF and male-specific dsxM introns (highlighted in pink) are spliced out in the corresponding sex, but some sequences carrying a stop codon (TGA) are retained in the opposite sex ( Figure S2 ). The transgenic flies harboring one copy of a genetic construct were identified by a strong ubiquitous expression of dsRed (highlighted in purple). (c) Expression of Opie2-PuroR dsxM or Opie2-PuroR traF transgene rescues only transgenic males or females (red fluorescence) raised on the food supplemented with puromycin, while all wild type flies (no red fluorescence) and the transgenic flies of selected-out sex die during early development.

    Article Snippet: A 1.1 g of the dry food was aliquoted per fly vial (FlyStaff.com) and mixed with 5 mL of distilled water supplemented with puromycin (Sigma #P8833) or geneticin (G418, Sigma #A1720), in varying concentrations from 0 to 1.2 mg/mL.

    Techniques: Construct, Expressing, Functional Assay, Produced, Transgenic Assay, Fluorescence