Structured Review

Bio-Rad electroporation cuvettes
Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after <t>electroporation</t> with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value
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Images

1) Product Images from "Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction"

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction

Journal: Scientific Reports

doi: 10.1038/s41598-018-30035-2

Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value
Figure Legend Snippet: Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value

Techniques Used: Functional Assay, In Situ Hybridization, Expressing, Transgenic Assay, Electroporation, ALP Assay

2) Product Images from "The role of DNA polymerase activity in human non-homologous end joining"

Article Title: The role of DNA polymerase activity in human non-homologous end joining

Journal: Nucleic Acids Research

doi:

NHEJ of a DSB model substrate in HeLa cells in vivo . Substrate XM was introduced into HeLa cells by electroporation. After transfection, cells were cultured for the indicated times after which substrate DNA was recovered and transfected into E.coli to assess formation of circular monomeric end joining products as described in Materials and Methods. NHEJ activity after 2 h in culture was taken as 100%. Error bars represent the standard deviation of two independent experiments.
Figure Legend Snippet: NHEJ of a DSB model substrate in HeLa cells in vivo . Substrate XM was introduced into HeLa cells by electroporation. After transfection, cells were cultured for the indicated times after which substrate DNA was recovered and transfected into E.coli to assess formation of circular monomeric end joining products as described in Materials and Methods. NHEJ activity after 2 h in culture was taken as 100%. Error bars represent the standard deviation of two independent experiments.

Techniques Used: Non-Homologous End Joining, In Vivo, Electroporation, Transfection, Cell Culture, Activity Assay, Standard Deviation

3) Product Images from "Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation"

Article Title: Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-8-90

Electroporation of 3d mDC and 7d mDC with long MART-1/Melan-A peptide and MART-1/Melan-A-encoding ivt RNA . (A) 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 1 μg, 5 μg and 10 μg long MART-1/Melan-A peptide. After 24 h incubation at 37°C and 5% CO 2 , the DC were cocultured with A42 CTL for 24 h. (B) 2d iDC, 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 24 μg MART-1/Melan-A ivt RNA, incubated at 37°C for 24 h and cocultured with A42 CTL for 24 h (n = 3). (C) 3d mDC and 7d mDC were electroporated with 12 μg MART-1/Melan-A ivt RNA at different electroporation conditions (250 V, 150 μF and 300 V, 300 μF), respectively. 3 h after electroporation, mDC were stained intracellularly with a MART-1/Melan-A-specific antibody and analyzed by flow cytometry (n = 2). (D) 24 h after electroporation with MART-1/Melan-A ivt RNA, DC were cocultured with A42 CTL for 24 h (n = 2). The IFN-γ release of the A42 CTL was measured by IFN-γ-ELISA. The bars in A, B and D show mean values of triplicates with standard deviations (Rec.: recovery; n.d.: not detected).
Figure Legend Snippet: Electroporation of 3d mDC and 7d mDC with long MART-1/Melan-A peptide and MART-1/Melan-A-encoding ivt RNA . (A) 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 1 μg, 5 μg and 10 μg long MART-1/Melan-A peptide. After 24 h incubation at 37°C and 5% CO 2 , the DC were cocultured with A42 CTL for 24 h. (B) 2d iDC, 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 24 μg MART-1/Melan-A ivt RNA, incubated at 37°C for 24 h and cocultured with A42 CTL for 24 h (n = 3). (C) 3d mDC and 7d mDC were electroporated with 12 μg MART-1/Melan-A ivt RNA at different electroporation conditions (250 V, 150 μF and 300 V, 300 μF), respectively. 3 h after electroporation, mDC were stained intracellularly with a MART-1/Melan-A-specific antibody and analyzed by flow cytometry (n = 2). (D) 24 h after electroporation with MART-1/Melan-A ivt RNA, DC were cocultured with A42 CTL for 24 h (n = 2). The IFN-γ release of the A42 CTL was measured by IFN-γ-ELISA. The bars in A, B and D show mean values of triplicates with standard deviations (Rec.: recovery; n.d.: not detected).

Techniques Used: Electroporation, Incubation, CTL Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

4) Product Images from "Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli"

Article Title: Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-4-11

Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan R transformants per 10 8 survivor and total number of Kan R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).
Figure Legend Snippet: Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan R transformants per 10 8 survivor and total number of Kan R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).

Techniques Used: Concentration Assay, Electroporation, Polymerase Chain Reaction

5) Product Images from "Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum"

Article Title: Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum

Journal: Malaria Journal

doi: 10.1186/1475-2875-11-210

Quantitative analysis of the relative efficiency of electroporation-based transfection techniques (A) Schematic representing the four protocols employed in this study. Protocol 1, direct electroporation of 40 μg of plasmids pΔ1 and pINT into ring stage IE on Day 0; Protocol 2, preloading of 40 μg of plasmids into erythrocytes which are mixed with mature stage IE on Day 1; Protocols 3 (40 μg of plasmids) and 4 (20 μg of plasmids) use a combination of both Protocols 1 and 2 and is termed here the “double-tap” technique. ( B ) The timecourse of log 10 RLU (Mean ± StDev, n = 3) over time for three independent transfections using each protocol. The key indicates the transfection tracking number (#), the days (D) post-transfection that an at least 1% asexual stage parasitaemia was directly observed microscopically and the mean transformation efficiency (parasites estimated to survive transfection N o /input parasites). *Transfection #7 was lost on day 3 with evidence of bacterial contamination of culture. **In transfection #12, the lid of the culture flask split between days 34–36, resulting in loss of low oxygen environment. No determination (n.d) of transfection efficiency was made for this experiment for this reason.
Figure Legend Snippet: Quantitative analysis of the relative efficiency of electroporation-based transfection techniques (A) Schematic representing the four protocols employed in this study. Protocol 1, direct electroporation of 40 μg of plasmids pΔ1 and pINT into ring stage IE on Day 0; Protocol 2, preloading of 40 μg of plasmids into erythrocytes which are mixed with mature stage IE on Day 1; Protocols 3 (40 μg of plasmids) and 4 (20 μg of plasmids) use a combination of both Protocols 1 and 2 and is termed here the “double-tap” technique. ( B ) The timecourse of log 10 RLU (Mean ± StDev, n = 3) over time for three independent transfections using each protocol. The key indicates the transfection tracking number (#), the days (D) post-transfection that an at least 1% asexual stage parasitaemia was directly observed microscopically and the mean transformation efficiency (parasites estimated to survive transfection N o /input parasites). *Transfection #7 was lost on day 3 with evidence of bacterial contamination of culture. **In transfection #12, the lid of the culture flask split between days 34–36, resulting in loss of low oxygen environment. No determination (n.d) of transfection efficiency was made for this experiment for this reason.

Techniques Used: Electroporation, Transfection, Transformation Assay

6) Product Images from "Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes"

Article Title: Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.01745

Electroporation of MRSA with antimicrobial Peptides (AMPs). Electrocompetent WBG 8287 cells suspended in (A) 2.5 μg ml -1 melittin, (B) 50 μg ml -1 mel12-26, and (C) 2 μg ml -1 bac8c and electroporated immediately (EP). Melittin-resistant WBG 8287 suspended in (D) 10 μg ml -1 melittin or (E) 50 μg ml -1 mel12-26 and electroporated. Survival was measured as a percentage of zero peptide, unelectroporated controls. Data represent the mean of six samples and error bars represent standard deviation. ∗ P ≤ 0.05; ∗∗ P ≤ 0.001.
Figure Legend Snippet: Electroporation of MRSA with antimicrobial Peptides (AMPs). Electrocompetent WBG 8287 cells suspended in (A) 2.5 μg ml -1 melittin, (B) 50 μg ml -1 mel12-26, and (C) 2 μg ml -1 bac8c and electroporated immediately (EP). Melittin-resistant WBG 8287 suspended in (D) 10 μg ml -1 melittin or (E) 50 μg ml -1 mel12-26 and electroporated. Survival was measured as a percentage of zero peptide, unelectroporated controls. Data represent the mean of six samples and error bars represent standard deviation. ∗ P ≤ 0.05; ∗∗ P ≤ 0.001.

Techniques Used: Electroporation, Standard Deviation

7) Product Images from "The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress"

Article Title: The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162065

Disruption of the TspC and TspD genes. (A) Genomic 5´ and 3´ fragments of TspC and TspD were amplified by PCR, ligated into a blasticidin-resistance cassette and each construct was transferred into AX2 wildtype cells by electroporation. The adjacent genes of TspC ( lipocalin , DDB_G0269882; pseudogene samkB , DDB_G0270988) and TspD ( TspA ; DDB_G0269884) are shown. (B) For each gene, two independent blasticidin resistant clones were compared with the wildtype (wt) for correct genomic 5´ integration (primers 1/2), insertion of the blasticidin-resistance gene (primers 3/4), and 3´ integration (primers 5/6).
Figure Legend Snippet: Disruption of the TspC and TspD genes. (A) Genomic 5´ and 3´ fragments of TspC and TspD were amplified by PCR, ligated into a blasticidin-resistance cassette and each construct was transferred into AX2 wildtype cells by electroporation. The adjacent genes of TspC ( lipocalin , DDB_G0269882; pseudogene samkB , DDB_G0270988) and TspD ( TspA ; DDB_G0269884) are shown. (B) For each gene, two independent blasticidin resistant clones were compared with the wildtype (wt) for correct genomic 5´ integration (primers 1/2), insertion of the blasticidin-resistance gene (primers 3/4), and 3´ integration (primers 5/6).

Techniques Used: Amplification, Polymerase Chain Reaction, Construct, Electroporation, Clone Assay

8) Product Images from "Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells"

Article Title: Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells

Journal: Leukemia research

doi: 10.1016/j.leukres.2008.11.024

Effects of irradiation on cytolytic properties of electroporated NK-92 cells. Cytotoxicity, expressed in percentage of killed target cells, of NK-92 against cell lines K562, REH, and SUP-B15 for an effector to target ratio of 5:1. NK-92 cells were electroporated without nucleic acid (control, full box color ) or with αCD19-CAR mRNA ( stripes ), and irradiated either 4 h prior to electroporation (■) or 20 h after electroporation ( ).
Figure Legend Snippet: Effects of irradiation on cytolytic properties of electroporated NK-92 cells. Cytotoxicity, expressed in percentage of killed target cells, of NK-92 against cell lines K562, REH, and SUP-B15 for an effector to target ratio of 5:1. NK-92 cells were electroporated without nucleic acid (control, full box color ) or with αCD19-CAR mRNA ( stripes ), and irradiated either 4 h prior to electroporation (■) or 20 h after electroporation ( ).

Techniques Used: Irradiation, Electroporation

Viability of cells after electroporation. NK-92 cells were stained with PI 24 h after electroporation with GFP (■) or αCD19-CAR ( ), either in the form of DNA or mRNA, or without any nucleic acid (□, control).
Figure Legend Snippet: Viability of cells after electroporation. NK-92 cells were stained with PI 24 h after electroporation with GFP (■) or αCD19-CAR ( ), either in the form of DNA or mRNA, or without any nucleic acid (□, control).

Techniques Used: Electroporation, Staining

Expression and stability of the exogenous proteins. (A) Right: FACS plots of a representative experiment showing GFP expression (FITC), αCD19-CAR expression (APC), and PI staining (PE) following mRNA electroporation. Left: Expression levels of GFP (■) or αCD19-CAR ( ) proteins at 24 h post-electroporation in NK-92 cells electroporated with DNA or mRNA, shown in percentage of the whole cell population. (B) Expression of GFP (●) or αCD19-CAR ( ) proteins over time in hours in NK-92 cells electroporated with mRNA.
Figure Legend Snippet: Expression and stability of the exogenous proteins. (A) Right: FACS plots of a representative experiment showing GFP expression (FITC), αCD19-CAR expression (APC), and PI staining (PE) following mRNA electroporation. Left: Expression levels of GFP (■) or αCD19-CAR ( ) proteins at 24 h post-electroporation in NK-92 cells electroporated with DNA or mRNA, shown in percentage of the whole cell population. (B) Expression of GFP (●) or αCD19-CAR ( ) proteins over time in hours in NK-92 cells electroporated with mRNA.

Techniques Used: Expressing, FACS, Staining, Electroporation

9) Product Images from "Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction"

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction

Journal: Scientific Reports

doi: 10.1038/s41598-018-30035-2

β-Catenin-dependent regulation of HmTSP expression. ( a ) Topography of the HmTSP promoter including -2191 bp of 5′ untranslated region. Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA (accession no. XM_012702849), and ATG the translation start site. Positions of canonical TCF binding motifs are indicated by red (5′-CTTTGTT-3′) or blue (5′-AACAAAG-3′) bars, respectively. The localization and size of DNA segments flanked by specific ChIP primer pairs are indicated by horizontal grey bars. ( b ) ChIP analysis of the HmTSP promoter region using chromatin from whole Hydra animals. A polyclonal antibody directed against Hydra TCF was used for immunoprecipitation, followed by PCR amplification of the indicated fragments from the HmTSP regulatory region including a region without a TCF binding site (control). Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as further controls. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. One representative of two replicates using independent chromatin preparations is shown (see Supplementary Fig. S3a ). ( c , d ) ISH analysis of HmTSP expression (red) in the head regions of transgenic animals with ectodermal GFP expression (green) electroporated with control GFP siRNA ( c ) or GFP and β-Catenin siRNAs ( d ). HmTSP transcript was absent in electroporated areas with impaired β-Catenin expression. Note that GFP-positive areas indicate patches of tissue not affected by the electroporation. ISH was performed 8 days after electroporation with siRNAs. Scale bars = 50 µm. Representative of 10 hydras examined. E . Quantitative rt PCR analysis of β-Catenin and HmTSP expression upon siRNA mediated β-Catenin knockdown. Reduced expression of β-Catenin in siRNA-treated animals correlated with a major decrease in expression of HmTSP . Columns represent the mean and error bars the standard deviation from three independent experiments.
Figure Legend Snippet: β-Catenin-dependent regulation of HmTSP expression. ( a ) Topography of the HmTSP promoter including -2191 bp of 5′ untranslated region. Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA (accession no. XM_012702849), and ATG the translation start site. Positions of canonical TCF binding motifs are indicated by red (5′-CTTTGTT-3′) or blue (5′-AACAAAG-3′) bars, respectively. The localization and size of DNA segments flanked by specific ChIP primer pairs are indicated by horizontal grey bars. ( b ) ChIP analysis of the HmTSP promoter region using chromatin from whole Hydra animals. A polyclonal antibody directed against Hydra TCF was used for immunoprecipitation, followed by PCR amplification of the indicated fragments from the HmTSP regulatory region including a region without a TCF binding site (control). Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as further controls. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. One representative of two replicates using independent chromatin preparations is shown (see Supplementary Fig. S3a ). ( c , d ) ISH analysis of HmTSP expression (red) in the head regions of transgenic animals with ectodermal GFP expression (green) electroporated with control GFP siRNA ( c ) or GFP and β-Catenin siRNAs ( d ). HmTSP transcript was absent in electroporated areas with impaired β-Catenin expression. Note that GFP-positive areas indicate patches of tissue not affected by the electroporation. ISH was performed 8 days after electroporation with siRNAs. Scale bars = 50 µm. Representative of 10 hydras examined. E . Quantitative rt PCR analysis of β-Catenin and HmTSP expression upon siRNA mediated β-Catenin knockdown. Reduced expression of β-Catenin in siRNA-treated animals correlated with a major decrease in expression of HmTSP . Columns represent the mean and error bars the standard deviation from three independent experiments.

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, In Situ Hybridization, Transgenic Assay, Electroporation, Quantitative RT-PCR, Standard Deviation

Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value
Figure Legend Snippet: Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value

Techniques Used: Functional Assay, In Situ Hybridization, Expressing, Transgenic Assay, Electroporation, ALP Assay

10) Product Images from "Mutational Analysis of DBD*--a Unique Antileukemic Gene Sequence 1"

Article Title: Mutational Analysis of DBD*--a Unique Antileukemic Gene Sequence 1

Journal: Neoplasia (New York, N.Y.)

doi:

Comparison of lethality of DBD* and loss-of-function mutant F444P. Twenty-four hours after electroporation with various pHook2 plasmids, cells were evaluated for PI staining and light scattering by flow cytometry. The threshold was set to gate out cell debris and aggregates. Only the singlet cell population was analyzed; 10,000 to 20,000 events were accumulated. G1 represents viable cells; G2 represents the population of cells undergoing apoptosis. (A) Electroporation with pHook2, (B) with pHook2/DBD*, (C) with the loss-of-function mutant pHook2/F444P. (D) shows the ratios of cells in the dead (G2)/viable (G1) populations after each treatment. The G2/G1 ratio for DBD* (B) is significantly higher than pHook2 vector (A) or the mutant F444P (C); n =5. **Indicates significant difference of DBD* from (A), P=.002 or (C), P=.007.
Figure Legend Snippet: Comparison of lethality of DBD* and loss-of-function mutant F444P. Twenty-four hours after electroporation with various pHook2 plasmids, cells were evaluated for PI staining and light scattering by flow cytometry. The threshold was set to gate out cell debris and aggregates. Only the singlet cell population was analyzed; 10,000 to 20,000 events were accumulated. G1 represents viable cells; G2 represents the population of cells undergoing apoptosis. (A) Electroporation with pHook2, (B) with pHook2/DBD*, (C) with the loss-of-function mutant pHook2/F444P. (D) shows the ratios of cells in the dead (G2)/viable (G1) populations after each treatment. The G2/G1 ratio for DBD* (B) is significantly higher than pHook2 vector (A) or the mutant F444P (C); n =5. **Indicates significant difference of DBD* from (A), P=.002 or (C), P=.007.

Techniques Used: Mutagenesis, Electroporation, Staining, Flow Cytometry, Cytometry, Plasmid Preparation

11) Product Images from "MR Imaging of Transplanted Stem Cells in Myocardial Infarction"

Article Title: MR Imaging of Transplanted Stem Cells in Myocardial Infarction

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-60761-901-7_10

Instant MR labeling of cells using magnetoelectroporation (MEP). a Cells are suspended in a sterile electroporation cuvette ( arrow ), mixed with Feridex ® , and placed in a cuvette holder. b Using the connected electroporator, cells are mildly permeabilized
Figure Legend Snippet: Instant MR labeling of cells using magnetoelectroporation (MEP). a Cells are suspended in a sterile electroporation cuvette ( arrow ), mixed with Feridex ® , and placed in a cuvette holder. b Using the connected electroporator, cells are mildly permeabilized

Techniques Used: Labeling, Electroporation

12) Product Images from "Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication"

Article Title: Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication

Journal: Antiviral research

doi: 10.1016/j.antiviral.2011.04.009

A SARS-CoV replicon expressing a luciferase reporter. (A) The Feo gene fused or non-fused to TRS9 was inserted into pBAC-SARS-CoV-REP (REP) to construct pSARS-CoV-REP-Feo (Feo) or pSARS-CoV-REP-ΔTRSFeo (ΔTRS), respectively. The pSARS-CoV-REP-Feo-MluIrev (MluIrev), which is defective in synthesis of functional replicase proteins, was used as a negative control plasmid. TRSs are indicated by a black box and leader sequences by a box with deviant crease lines. Black arrows indicate primers used for detection of N gene-specific sg-mRNAs and gray arrows for detection of sg-mRNAs containing the Feo gene. (B and C) BHK-21 cells were co-transfected with replicon plasmids and pRL-TK used for normalization of transfection efficiency, by electroporation. At 30 h post-transfection, cells were harvested and analyzed for sg-mRNA level, luciferase activity, and intracellular SARS-CoV nucleocapsid N protein level. (B) The N gene-specific sg-mRNA level was quantified by real-time qRT-PCR using a TaqMan probe. Subgenomic RNA copy numbers per μg total RNA are shown. ND, not detected. (C) Firefiy luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. Normalized luciferase activity of cells transfected with pSARS-CoV-REP was defined as 100. Endogenous sg-mRNAs containing Feo gene was amplified by RT-PCR and resulting PCR products were resolved by agarose gel electrophoresis (low panel). (D) BHK-21 or HEK293 cells were left untransfected (Mock) or transfected with the plasmid indicated above the blots. N protein and α-tubulin were detected by Western blot analysis. (E) Kinetics of SARS-CoV replicon replication in transiently transfected cells. BHK-21 cells were transfected with pSARS-REP-Feo (●) or pSARS-REP-ΔTRSFeo (▲) by electroporation. Cells were harvested at each given time point and store at −80 °C until analysis. Luciferase activity was measured with the same amount of cell lysate. Data from one representative experiment from two independent experiments with similar results are shown.
Figure Legend Snippet: A SARS-CoV replicon expressing a luciferase reporter. (A) The Feo gene fused or non-fused to TRS9 was inserted into pBAC-SARS-CoV-REP (REP) to construct pSARS-CoV-REP-Feo (Feo) or pSARS-CoV-REP-ΔTRSFeo (ΔTRS), respectively. The pSARS-CoV-REP-Feo-MluIrev (MluIrev), which is defective in synthesis of functional replicase proteins, was used as a negative control plasmid. TRSs are indicated by a black box and leader sequences by a box with deviant crease lines. Black arrows indicate primers used for detection of N gene-specific sg-mRNAs and gray arrows for detection of sg-mRNAs containing the Feo gene. (B and C) BHK-21 cells were co-transfected with replicon plasmids and pRL-TK used for normalization of transfection efficiency, by electroporation. At 30 h post-transfection, cells were harvested and analyzed for sg-mRNA level, luciferase activity, and intracellular SARS-CoV nucleocapsid N protein level. (B) The N gene-specific sg-mRNA level was quantified by real-time qRT-PCR using a TaqMan probe. Subgenomic RNA copy numbers per μg total RNA are shown. ND, not detected. (C) Firefiy luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. Normalized luciferase activity of cells transfected with pSARS-CoV-REP was defined as 100. Endogenous sg-mRNAs containing Feo gene was amplified by RT-PCR and resulting PCR products were resolved by agarose gel electrophoresis (low panel). (D) BHK-21 or HEK293 cells were left untransfected (Mock) or transfected with the plasmid indicated above the blots. N protein and α-tubulin were detected by Western blot analysis. (E) Kinetics of SARS-CoV replicon replication in transiently transfected cells. BHK-21 cells were transfected with pSARS-REP-Feo (●) or pSARS-REP-ΔTRSFeo (▲) by electroporation. Cells were harvested at each given time point and store at −80 °C until analysis. Luciferase activity was measured with the same amount of cell lysate. Data from one representative experiment from two independent experiments with similar results are shown.

Techniques Used: Expressing, Luciferase, Construct, Functional Assay, Negative Control, Plasmid Preparation, Transfection, Electroporation, Activity Assay, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

13) Product Images from "The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway"

Article Title: The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0703534

OA enhances NF-κB transcriptional activity. (A) Schematic representation of the two IL-6 promoter constructs and the pGAL4p65, pGAL4dbd, and pGAL4tkluc plasmids applied in the transfections of TF-1 cell line. Fragments of 122 (pIL6(-122)luc) and 60 (pIL6(-60)luc) basepairs, respectively, of the human IL-6 promoter are fused to the luciferase gene. Binding site for the transcription factors NF-κB is denoted above the largest construct. The Gal4-transactivator fusion proteins, pGal4p65 and pGal4dbd, are exclusively nuclear and are regulated independently of IκB. The reporter gene is under the control of five Gal4-binding sites. (B) Schematic representation of NF-κB- and p65-mediated promoter activity in the transiently transfected TF-1 cells. TF-1 cells were cultured as described in ‘Methods' and subsequently transiently transfected by means of electroporation with the respective plasmids. Six hours after transfection cells were stimulated for 24 h with medium or OA (30 ng ml −1 ). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Basal promoter activity for each construct when treated with medium alone is set at 1. Mean fold induction and standard error of the mean represent six or more identical experiments. * P
Figure Legend Snippet: OA enhances NF-κB transcriptional activity. (A) Schematic representation of the two IL-6 promoter constructs and the pGAL4p65, pGAL4dbd, and pGAL4tkluc plasmids applied in the transfections of TF-1 cell line. Fragments of 122 (pIL6(-122)luc) and 60 (pIL6(-60)luc) basepairs, respectively, of the human IL-6 promoter are fused to the luciferase gene. Binding site for the transcription factors NF-κB is denoted above the largest construct. The Gal4-transactivator fusion proteins, pGal4p65 and pGal4dbd, are exclusively nuclear and are regulated independently of IκB. The reporter gene is under the control of five Gal4-binding sites. (B) Schematic representation of NF-κB- and p65-mediated promoter activity in the transiently transfected TF-1 cells. TF-1 cells were cultured as described in ‘Methods' and subsequently transiently transfected by means of electroporation with the respective plasmids. Six hours after transfection cells were stimulated for 24 h with medium or OA (30 ng ml −1 ). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Basal promoter activity for each construct when treated with medium alone is set at 1. Mean fold induction and standard error of the mean represent six or more identical experiments. * P

Techniques Used: Activity Assay, Construct, Transfection, Luciferase, Binding Assay, Cell Culture, Electroporation, Produced

14) Product Images from "Superparamagnetic Iron Oxide Labeling of Stem Cells for MRI Tracking and Delivery in Cardiovascular Disease"

Article Title: Superparamagnetic Iron Oxide Labeling of Stem Cells for MRI Tracking and Delivery in Cardiovascular Disease

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-60761-705-1_11

BTX electroporation system ( a ) that can be used for magnetoelectroporation. This system may be operated by a switch ( b ) or foot pedal (not shown).
Figure Legend Snippet: BTX electroporation system ( a ) that can be used for magnetoelectroporation. This system may be operated by a switch ( b ) or foot pedal (not shown).

Techniques Used: Electroporation

15) Product Images from "A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-β1 and IL-35"

Article Title: A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-β1 and IL-35

Journal: Scientific Reports

doi: 10.1038/s41598-017-04322-3

TolDC and C5-DC successfully express porcine antigen following electroporation of porcine-specific ivtRNA. MHC-I expression was used as surrogate marker for porcine antigen expression. ( a ) Flow cytometry analysis at different time points after electroporation using a porcine MHC-I-specific monoclonal antibody. Mock-electroporated tolDC and C5-DC were used as controls (grey peaks). ( b ) in % of cells, ( c ) as MFI.
Figure Legend Snippet: TolDC and C5-DC successfully express porcine antigen following electroporation of porcine-specific ivtRNA. MHC-I expression was used as surrogate marker for porcine antigen expression. ( a ) Flow cytometry analysis at different time points after electroporation using a porcine MHC-I-specific monoclonal antibody. Mock-electroporated tolDC and C5-DC were used as controls (grey peaks). ( b ) in % of cells, ( c ) as MFI.

Techniques Used: Electroporation, Expressing, Marker, Flow Cytometry, Cytometry

16) Product Images from "The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia"

Article Title: The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia

Journal:

doi: 10.1182/blood-2007-09-111872

siRNA knockdown of SET and SET-NUP214 in the LOUCY T-ALL cell line. (A) SET expression using Western blot analysis 4 days after electroporation with the following conditions: no siRNA, control siRNA, siRNA SET exon 8, or siRNA SET exon 5. (B) Relative
Figure Legend Snippet: siRNA knockdown of SET and SET-NUP214 in the LOUCY T-ALL cell line. (A) SET expression using Western blot analysis 4 days after electroporation with the following conditions: no siRNA, control siRNA, siRNA SET exon 8, or siRNA SET exon 5. (B) Relative

Techniques Used: Expressing, Western Blot, Electroporation

ChIP and coIP analysis of T-ALL cell lines LOUCY and SKW3. (A) ChIP analysis of SKW3, LOUCY, and LOUCY 4 days after electroporation with siRNA SET exon 5, for promoter sequences of HOXA1 , HOXA3 , HOXA9 , HOXA10 , and HOXA11 . The amount of bound DNA was calculated
Figure Legend Snippet: ChIP and coIP analysis of T-ALL cell lines LOUCY and SKW3. (A) ChIP analysis of SKW3, LOUCY, and LOUCY 4 days after electroporation with siRNA SET exon 5, for promoter sequences of HOXA1 , HOXA3 , HOXA9 , HOXA10 , and HOXA11 . The amount of bound DNA was calculated

Techniques Used: Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay, Electroporation

17) Product Images from "Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone 1"

Article Title: Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone 1

Journal: Plant Physiology

doi:

The effect of electroporation of protein kinase substrate peptides on GA-induced amylase secretion. Freshly isolated protoplasts were loaded with 25 μm syntide-2, malantide, or BSA by electroporation and treated for 48 h with or without 5 μm GA. Secreted amylase activity was then assessed. A set of nonelectroporated control protoplasts was treated identically. Results represent the mean ± se of three separate experiments.
Figure Legend Snippet: The effect of electroporation of protein kinase substrate peptides on GA-induced amylase secretion. Freshly isolated protoplasts were loaded with 25 μm syntide-2, malantide, or BSA by electroporation and treated for 48 h with or without 5 μm GA. Secreted amylase activity was then assessed. A set of nonelectroporated control protoplasts was treated identically. Results represent the mean ± se of three separate experiments.

Techniques Used: Electroporation, Isolation, Activity Assay

18) Product Images from "A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways"

Article Title: A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01509

TAARD increases the luciferase reporter activities of NF-κB and STAT3 via TLR1 and TLR3, respectively. (A) NKL cells were electroporated with either pGL3-κB-Luc (1 µg) or pGL3-Basic (1 µg) and a pRL-TK renilla-luciferase control plasmid (50 ng). After electroporation, cells were immediately transferred into fresh medium and cultured for an additional 6 h. Then, cells were treated with different concentrations of TAARD. (B) NKL cells were electroporated as described in (A) but with 4×M67 pTATA TK-Luc plasmid (1 µg). Cells were treated and measured as described in (A) . (C) 293T cells were co-transfected with pGL3-κB-luc (1 µg) or pGL3-Basic (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR1 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. (D) 293T cells were co-transfected with 4×M67 pTATA TK-Luc (1 µg) or pGL Basic plasmid (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR3 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. The ratio of firefly to renilla luciferase activities was used to show the relative luciferase activity, which corresponded to NF-κB or STAT activation. Data shown represent one of three independent experiments with similar results. * p
Figure Legend Snippet: TAARD increases the luciferase reporter activities of NF-κB and STAT3 via TLR1 and TLR3, respectively. (A) NKL cells were electroporated with either pGL3-κB-Luc (1 µg) or pGL3-Basic (1 µg) and a pRL-TK renilla-luciferase control plasmid (50 ng). After electroporation, cells were immediately transferred into fresh medium and cultured for an additional 6 h. Then, cells were treated with different concentrations of TAARD. (B) NKL cells were electroporated as described in (A) but with 4×M67 pTATA TK-Luc plasmid (1 µg). Cells were treated and measured as described in (A) . (C) 293T cells were co-transfected with pGL3-κB-luc (1 µg) or pGL3-Basic (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR1 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. (D) 293T cells were co-transfected with 4×M67 pTATA TK-Luc (1 µg) or pGL Basic plasmid (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR3 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. The ratio of firefly to renilla luciferase activities was used to show the relative luciferase activity, which corresponded to NF-κB or STAT activation. Data shown represent one of three independent experiments with similar results. * p

Techniques Used: Luciferase, Plasmid Preparation, Electroporation, Cell Culture, Transfection, Expressing, Activity Assay, Activation Assay

19) Product Images from "Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance"

Article Title: Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance

Journal: Oncotarget

doi:

Translocation of TFEB-FLAG from the cytoplasm to the nucleus after exposure of malignant and non-malignant human cells to doxorubicin, mitoxantrone and sunitinib Stably transfected MCF-7 TFEB-3XFLAG cells were exposed to 0.5 μM doxorubicin, 0.5 μM mitoxantrone or 100 μM chloroquine for 24 hr. In an independent set of experiments, 24 hr after transient transfection of HEK293 and HeLa cells with FLAG-tagged TFEB using electroporation, The cells were exposed to 0.5 μM doxorubicin, mitoxantrone, or sunitinib for 24 hr. Cells were then fixed, stained with the DNA dye DAPI (blue fluorescence), incubated with an anti-FLAG antibody (green fluorescence) and analyzed by a fluorescence microscope. The first row represents the drug-free control cells.
Figure Legend Snippet: Translocation of TFEB-FLAG from the cytoplasm to the nucleus after exposure of malignant and non-malignant human cells to doxorubicin, mitoxantrone and sunitinib Stably transfected MCF-7 TFEB-3XFLAG cells were exposed to 0.5 μM doxorubicin, 0.5 μM mitoxantrone or 100 μM chloroquine for 24 hr. In an independent set of experiments, 24 hr after transient transfection of HEK293 and HeLa cells with FLAG-tagged TFEB using electroporation, The cells were exposed to 0.5 μM doxorubicin, mitoxantrone, or sunitinib for 24 hr. Cells were then fixed, stained with the DNA dye DAPI (blue fluorescence), incubated with an anti-FLAG antibody (green fluorescence) and analyzed by a fluorescence microscope. The first row represents the drug-free control cells.

Techniques Used: Translocation Assay, Stable Transfection, Transfection, Electroporation, Staining, Fluorescence, Incubation, Microscopy

20) Product Images from "Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer"

Article Title: Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer

Journal: Theranostics

doi: 10.7150/thno.16532

Characterization of AS1411-EVs for breast cancer targeting: A. Zeta potential distribution of EVs (blue), AS1411-EVs (red) and AS1411-EVs digested by DNase I (green) measured by nanoparticle tracking analysis. B. Fluorescence microscopy analysis of the T-AS1411 stable binding with EVs membrane outer surface. T-AS1411 was modified by Cy3 and bound to EVs via cholesterol affinity. The Cy3-AS1411-EVs were digested by DNase I and then captured by Dynabeads ® with CD63 antibody (down). The resulting fluorescence signal was very weak. However, control group Cy3-AS1411-EVs not digested by DNase I (up) had a very strong fluorescence signal. (BL: bright light, FL: fluorescence light, Scale bars: 100 μm). C. Size distribution of AS1411-EVs measured by nanoparticle tracking analysis. The peak diameter was at 77 nm for AS1411-EVs. D. AS1411-EVs-let-7-Cy3 was captured by beads and the fluorescence signal detected by fluorescence microscopy. The red fluorescence signal showed successful electroporation of let-7-Cy3 into EVs (Scale bars: 100 μm). E. Transmission electron microscopy of AS1411-EVs-let-7. The morphology of AS1411-EVs-let-7 was intact and the size was approximately 30-100 nm (Arrows indicate AS1411-EVs-let-7, Scale bars: 100 nm). F. Release kinetics of encapsulated cel-miRNA-67 from the AS1411-EVs as determined by incubation for up to 24 hours in PBS or mouse serum at 37°C (n=3) (Each bar represents the mean ±SD of three replicates).
Figure Legend Snippet: Characterization of AS1411-EVs for breast cancer targeting: A. Zeta potential distribution of EVs (blue), AS1411-EVs (red) and AS1411-EVs digested by DNase I (green) measured by nanoparticle tracking analysis. B. Fluorescence microscopy analysis of the T-AS1411 stable binding with EVs membrane outer surface. T-AS1411 was modified by Cy3 and bound to EVs via cholesterol affinity. The Cy3-AS1411-EVs were digested by DNase I and then captured by Dynabeads ® with CD63 antibody (down). The resulting fluorescence signal was very weak. However, control group Cy3-AS1411-EVs not digested by DNase I (up) had a very strong fluorescence signal. (BL: bright light, FL: fluorescence light, Scale bars: 100 μm). C. Size distribution of AS1411-EVs measured by nanoparticle tracking analysis. The peak diameter was at 77 nm for AS1411-EVs. D. AS1411-EVs-let-7-Cy3 was captured by beads and the fluorescence signal detected by fluorescence microscopy. The red fluorescence signal showed successful electroporation of let-7-Cy3 into EVs (Scale bars: 100 μm). E. Transmission electron microscopy of AS1411-EVs-let-7. The morphology of AS1411-EVs-let-7 was intact and the size was approximately 30-100 nm (Arrows indicate AS1411-EVs-let-7, Scale bars: 100 nm). F. Release kinetics of encapsulated cel-miRNA-67 from the AS1411-EVs as determined by incubation for up to 24 hours in PBS or mouse serum at 37°C (n=3) (Each bar represents the mean ±SD of three replicates).

Techniques Used: Fluorescence, Microscopy, Binding Assay, Modification, Electroporation, Transmission Assay, Electron Microscopy, Incubation

21) Product Images from "The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation"

Article Title: The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

Journal: Toxins

doi: 10.3390/toxins8040090

Effect of TcsL-cat or TcsL mutants treatment on cell viability. Cells were electroporated with TcsL-cat or mutants and proliferation tests were performed 72 h after electroporation. Data are expressed as mean ± SEM of at least three independent experiments. Statistically significant differences between results were evaluated by t test with Welch correction. A p value of
Figure Legend Snippet: Effect of TcsL-cat or TcsL mutants treatment on cell viability. Cells were electroporated with TcsL-cat or mutants and proliferation tests were performed 72 h after electroporation. Data are expressed as mean ± SEM of at least three independent experiments. Statistically significant differences between results were evaluated by t test with Welch correction. A p value of

Techniques Used: Electroporation

22) Product Images from "Potential Benefits of Sequential Inhibitor-Mutagen Treatments of RNA Virus Infections"

Article Title: Potential Benefits of Sequential Inhibitor-Mutagen Treatments of RNA Virus Infections

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000658

Effects of ribavirin (R) and guanidinium (GU) on the interference on FMDV replication exerted by capsid and 3D polymerase mutants. (A) Cells were mock-electroporated (control), or electroporated with 2 µg of pMT28 transcript (expresing FMDV C-S8c1), or co-electroporated with a mixture of transcripts obtained from pMT28 and from capsid mutant Q2027A and 3D polymerase mutant MD [63] . +R and +GU indicate the presence of R (1 mM) or GU (2 mM), respectively, as indicated in the box on the right. The ratio of pMT28 RNA to the total amount of mutant RNA was 1∶10, and the total amount of FMDV RNA was the same in each electroporation assay. Infectivity values were determined in triplicate at the indicated hours post-electroporation. Virus titers are expressed as percentage of the titers produced, relative to those produced by pMT28 RNA at the corresponding hours post-electroporation, taken as 100%. (B) Same as in (A) but using in the coelectroporations RNA from non-interfering polymerase mutants DMD and D3 [63] . In (A) and (B), no infectivity was obtained from the mock-electroporated samples (control box in dark grey, not visible). The origin of FMDV mutants, and procedures for titration of virus are detailed in Materials and Methods .
Figure Legend Snippet: Effects of ribavirin (R) and guanidinium (GU) on the interference on FMDV replication exerted by capsid and 3D polymerase mutants. (A) Cells were mock-electroporated (control), or electroporated with 2 µg of pMT28 transcript (expresing FMDV C-S8c1), or co-electroporated with a mixture of transcripts obtained from pMT28 and from capsid mutant Q2027A and 3D polymerase mutant MD [63] . +R and +GU indicate the presence of R (1 mM) or GU (2 mM), respectively, as indicated in the box on the right. The ratio of pMT28 RNA to the total amount of mutant RNA was 1∶10, and the total amount of FMDV RNA was the same in each electroporation assay. Infectivity values were determined in triplicate at the indicated hours post-electroporation. Virus titers are expressed as percentage of the titers produced, relative to those produced by pMT28 RNA at the corresponding hours post-electroporation, taken as 100%. (B) Same as in (A) but using in the coelectroporations RNA from non-interfering polymerase mutants DMD and D3 [63] . In (A) and (B), no infectivity was obtained from the mock-electroporated samples (control box in dark grey, not visible). The origin of FMDV mutants, and procedures for titration of virus are detailed in Materials and Methods .

Techniques Used: Mutagenesis, Electroporation, Infection, Produced, Titration

23) Product Images from "Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion"

Article Title: Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion

Journal: AIDS Research and Human Retroviruses

doi: 10.1089/aid.2009.0068

( A ). Truncation mutagenesis to determine Nef secretion sequences. The relative fluorescence of carboxy-terminal deletion mutants of Nef compared to the wtNef-GFP is shown. Media were collected and assayed from the 48-h-old cultures of HEK293 cells transfected with the wt, NefΔ31–206 (1–30), NefΔ51–206 (1–50), NefΔ66–206 (1–65), NefΔ71–206 (1–70), NefΔ91–206 (1–90), NefΔ151–206 (1–150), NefΔ201–206 (1–200), NefΔ1–12 (13–206), NefΔ1–12 and Δ71–206 (13–70), NefΔ1–40 and Δ71–206 (41–70), and untransfected HEK293 cells (bar 12). ( B R/4A, and untransfected HEK293 cells. ( C , and untransfected HEK293 cells. ( D – F ) The Nef-induced secretion domains function similarly in multiple cell types. Jurkats (1 × 10 6 (SMR, bar 7), GFP (bar 8), and untransfected cells (bar 9) ( D ), THP-1 ( E ), and U937 ( F ) monocytes by Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, Inc., CA). Cells were incubated in RPMI 1640 medium for 48 h at 37°C and removed from the culture supernatant by centrifugation at 2000 × g for 5 min. In all experiments, the error bars show the standard errors of the measurements. Transfection efficiencies for Jurkat cells (80–86.67%), for THP-1 cells (60–65%), and for U937 cells (55–60%). These results are a compilation of at least three independent experiments.
Figure Legend Snippet: ( A ). Truncation mutagenesis to determine Nef secretion sequences. The relative fluorescence of carboxy-terminal deletion mutants of Nef compared to the wtNef-GFP is shown. Media were collected and assayed from the 48-h-old cultures of HEK293 cells transfected with the wt, NefΔ31–206 (1–30), NefΔ51–206 (1–50), NefΔ66–206 (1–65), NefΔ71–206 (1–70), NefΔ91–206 (1–90), NefΔ151–206 (1–150), NefΔ201–206 (1–200), NefΔ1–12 (13–206), NefΔ1–12 and Δ71–206 (13–70), NefΔ1–40 and Δ71–206 (41–70), and untransfected HEK293 cells (bar 12). ( B R/4A, and untransfected HEK293 cells. ( C , and untransfected HEK293 cells. ( D – F ) The Nef-induced secretion domains function similarly in multiple cell types. Jurkats (1 × 10 6 (SMR, bar 7), GFP (bar 8), and untransfected cells (bar 9) ( D ), THP-1 ( E ), and U937 ( F ) monocytes by Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, Inc., CA). Cells were incubated in RPMI 1640 medium for 48 h at 37°C and removed from the culture supernatant by centrifugation at 2000 × g for 5 min. In all experiments, the error bars show the standard errors of the measurements. Transfection efficiencies for Jurkat cells (80–86.67%), for THP-1 cells (60–65%), and for U937 cells (55–60%). These results are a compilation of at least three independent experiments.

Techniques Used: Mutagenesis, Fluorescence, Transfection, Electroporation, Incubation, Centrifugation

24) Product Images from "Magnetic Resonance Imaging of Monocytes Labeled with Ultrasmall Superparamagnetic Particles of Iron Oxide Using Magnetoelectroporation in an Animal Model of Multiple Sclerosis"

Article Title: Magnetic Resonance Imaging of Monocytes Labeled with Ultrasmall Superparamagnetic Particles of Iron Oxide Using Magnetoelectroporation in an Animal Model of Multiple Sclerosis

Journal: Molecular imaging

doi:

A , Iron incorporation as quantified by the ferrozin assay. No increase in intracellular iron was detected following electroporation with Sinerem. MEP with Supravist at a concentration of 3 mg Fe/mL resulted in a 400% increase in intracellular iron concentration.
Figure Legend Snippet: A , Iron incorporation as quantified by the ferrozin assay. No increase in intracellular iron was detected following electroporation with Sinerem. MEP with Supravist at a concentration of 3 mg Fe/mL resulted in a 400% increase in intracellular iron concentration.

Techniques Used: Electroporation, Concentration Assay

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Electroporation:

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Article Title: Optimal Electroporation Condition for Small Interfering RNA Transfection into MDA-MB-468 Cell Line
Article Snippet: .. The RNA-cells mixtures were transferred into 4 mm Biorad Gene Pulser cuvettes, and electroporated at different conditions ( ) by Gene Pulser X cell Electroporation system (BioRad, US). ..

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Transfection:

Article Title: A Novel Self-Replicating Chimeric Lentivirus-Like Particle
Article Snippet: .. Fifty micrograms of in vitro synthesized replicon RNA was transfected into Vero or BHK cells by electroporation of 800 μl of resuspended cells using five electrical pulses of 450 V (Vero) or three electrical pulses of 850 V (BHK) at 25 μF with a Bio-Rad gene pulser II electroporator and 0.4-cm-gap gene pulser cuvettes (Bio-Rad). .. Transfected BHK cells were placed into 75-cm2 flasks containing complete medium, and transfected Vero cells were placed into 175-cm2 flasks containing either complete medium or virus production serum-free medium (VP-SFM; Invitrogen) supplemented with 0.29 μg/ml of l -glutamine.

Article Title: A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids
Article Snippet: .. For transfections with the BTX ECM 830 square wave electroporation system, 107 cells were pulsed three times with 1.5 kV (unipolar, 100 µs, 10s interval) in 0.4 cm gap Gene Pulser electroporation cuvettes (BioRad). .. For gene tagging with pPLOT constructs, 3 × 106 cells were transfected with the PCR reactions for the sgRNA and donor DNA (combined volume approx.

In Vitro:

Article Title: A Novel Self-Replicating Chimeric Lentivirus-Like Particle
Article Snippet: .. Fifty micrograms of in vitro synthesized replicon RNA was transfected into Vero or BHK cells by electroporation of 800 μl of resuspended cells using five electrical pulses of 450 V (Vero) or three electrical pulses of 850 V (BHK) at 25 μF with a Bio-Rad gene pulser II electroporator and 0.4-cm-gap gene pulser cuvettes (Bio-Rad). .. Transfected BHK cells were placed into 75-cm2 flasks containing complete medium, and transfected Vero cells were placed into 175-cm2 flasks containing either complete medium or virus production serum-free medium (VP-SFM; Invitrogen) supplemented with 0.29 μg/ml of l -glutamine.

Transformation Assay:

Article Title: Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion
Article Snippet: .. M1 5448 cells were transformed with 10 μg of pHY304-ess ::cat by electroporation using BioRad Gene Pulser II and Gene Pulser/MicroPulser Electroporation Cuvettes, 0.2 cm gap (BioRad) (cooled on ice before application) at the following settings: voltage 1.75 kV, resistance 400 Ω; capacitance: 25 μF. .. Electroporated cells were mixed with 500 μL of THY broth and 0.25 M Sucrose, and were transferred to an Eppendorf tube.

Synthesized:

Article Title: A Novel Self-Replicating Chimeric Lentivirus-Like Particle
Article Snippet: .. Fifty micrograms of in vitro synthesized replicon RNA was transfected into Vero or BHK cells by electroporation of 800 μl of resuspended cells using five electrical pulses of 450 V (Vero) or three electrical pulses of 850 V (BHK) at 25 μF with a Bio-Rad gene pulser II electroporator and 0.4-cm-gap gene pulser cuvettes (Bio-Rad). .. Transfected BHK cells were placed into 75-cm2 flasks containing complete medium, and transfected Vero cells were placed into 175-cm2 flasks containing either complete medium or virus production serum-free medium (VP-SFM; Invitrogen) supplemented with 0.29 μg/ml of l -glutamine.

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