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    Structured Review

    Millipore rt qpcr analysis total rna
    Co-purification of HPat (A) or AGO1 (B) with GW182 in NOT1 knockdown cells. A, B: Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD) or NOT1 (NOT1 KD). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–5 and 14–18, in B: lanes 1–6 and 15–21. IPs in A lanes 6 and 19, in B lanes 7 and 22), anti-c-myc (Input in A: lanes 7–12 and 20–23. IPs in A lanes 13 and 24) or anti-AGO1 antibody (Input in B: lanes 8–13 and 23–29. IPs in B lanes 14 and 30). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. C, D: The amount of Myc-HPat/HA-GW182 ( C ) or AGO1/HA-GW182 ( D ) in immunoprecipitates (IP) from lysates of control and NOT1 knockdown cells. The IP was normalized (Supporting Figure S4 ) and the value of the control IP set to 1. E: Analysis of NOT1 mRNA levels in knockdown cells compared to control cells treated with dsYFP <t>RNA.</t> The levels of NOT1 mRNA in total RNA of input samples were analyzed by <t>RT-qPCR</t> and normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. F: Upregulation of endogenous miRNA targets in knockdown cells. Total RNA of input samples were analyzed by RT-qPCR for changes of CG5123 mRNA levels in NOT1 knockdown cells. mRNA levels were normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s t test and significance values are as follows: ns, not significant; *, p
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    1) Product Images from "HPat a Decapping Activator Interacting with the miRNA Effector Complex"

    Article Title: HPat a Decapping Activator Interacting with the miRNA Effector Complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071860

    Co-purification of HPat (A) or AGO1 (B) with GW182 in NOT1 knockdown cells. A, B: Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD) or NOT1 (NOT1 KD). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–5 and 14–18, in B: lanes 1–6 and 15–21. IPs in A lanes 6 and 19, in B lanes 7 and 22), anti-c-myc (Input in A: lanes 7–12 and 20–23. IPs in A lanes 13 and 24) or anti-AGO1 antibody (Input in B: lanes 8–13 and 23–29. IPs in B lanes 14 and 30). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. C, D: The amount of Myc-HPat/HA-GW182 ( C ) or AGO1/HA-GW182 ( D ) in immunoprecipitates (IP) from lysates of control and NOT1 knockdown cells. The IP was normalized (Supporting Figure S4 ) and the value of the control IP set to 1. E: Analysis of NOT1 mRNA levels in knockdown cells compared to control cells treated with dsYFP RNA. The levels of NOT1 mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. F: Upregulation of endogenous miRNA targets in knockdown cells. Total RNA of input samples were analyzed by RT-qPCR for changes of CG5123 mRNA levels in NOT1 knockdown cells. mRNA levels were normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s t test and significance values are as follows: ns, not significant; *, p
    Figure Legend Snippet: Co-purification of HPat (A) or AGO1 (B) with GW182 in NOT1 knockdown cells. A, B: Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD) or NOT1 (NOT1 KD). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–5 and 14–18, in B: lanes 1–6 and 15–21. IPs in A lanes 6 and 19, in B lanes 7 and 22), anti-c-myc (Input in A: lanes 7–12 and 20–23. IPs in A lanes 13 and 24) or anti-AGO1 antibody (Input in B: lanes 8–13 and 23–29. IPs in B lanes 14 and 30). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. C, D: The amount of Myc-HPat/HA-GW182 ( C ) or AGO1/HA-GW182 ( D ) in immunoprecipitates (IP) from lysates of control and NOT1 knockdown cells. The IP was normalized (Supporting Figure S4 ) and the value of the control IP set to 1. E: Analysis of NOT1 mRNA levels in knockdown cells compared to control cells treated with dsYFP RNA. The levels of NOT1 mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. F: Upregulation of endogenous miRNA targets in knockdown cells. Total RNA of input samples were analyzed by RT-qPCR for changes of CG5123 mRNA levels in NOT1 knockdown cells. mRNA levels were normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s t test and significance values are as follows: ns, not significant; *, p

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

    Co-purification of HPat with GW182 in AGO1 knockdown cells. A: Cells stably expressing HA-GW182 and Myc-HPat were treated for four days with dsRNA against YFP (control KD) or AGO1 (AGO1 KD). Protein complexes were immunoprecipiated from cell lysates using anti-HA antibody. Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Inputs lanes 1–4, 12–15, and IPs lanes 5 and 16) or anti-c-myc antibody (Inputs lanes 6–10, 17–21 and IPs lanes 11 and 22). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. B: Quantitative analysis of the western blot in (A). The amount of Myc-HPat/HA-GW182 in the IP was normalized and the value for the control IP set to 1. C: Knockdown efficiency of AGO1. Cell lysate of AGO1 knockdown cells and various amounts of control cell lysate were analyzed by western blot analysis. Tubulin was used as a loading control. D: Upregulation of endogenous miRNA targets CG5123 and CG6770 in AGO1 knockdown cells. Total RNA from input samples of (A) were analyzed by RT-qPCR and normalized to rp49 levels. The values of dsYFP treated cells were set to 1. In all figures bars represent mean values and error bars standard deviations of at least three biological replicates. Statistical analysis was performed using the Student’s t test and significance values are as follows: *, p
    Figure Legend Snippet: Co-purification of HPat with GW182 in AGO1 knockdown cells. A: Cells stably expressing HA-GW182 and Myc-HPat were treated for four days with dsRNA against YFP (control KD) or AGO1 (AGO1 KD). Protein complexes were immunoprecipiated from cell lysates using anti-HA antibody. Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Inputs lanes 1–4, 12–15, and IPs lanes 5 and 16) or anti-c-myc antibody (Inputs lanes 6–10, 17–21 and IPs lanes 11 and 22). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. B: Quantitative analysis of the western blot in (A). The amount of Myc-HPat/HA-GW182 in the IP was normalized and the value for the control IP set to 1. C: Knockdown efficiency of AGO1. Cell lysate of AGO1 knockdown cells and various amounts of control cell lysate were analyzed by western blot analysis. Tubulin was used as a loading control. D: Upregulation of endogenous miRNA targets CG5123 and CG6770 in AGO1 knockdown cells. Total RNA from input samples of (A) were analyzed by RT-qPCR and normalized to rp49 levels. The values of dsYFP treated cells were set to 1. In all figures bars represent mean values and error bars standard deviations of at least three biological replicates. Statistical analysis was performed using the Student’s t test and significance values are as follows: *, p

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

    Co-purification of HPat with GW182 in EDC4 and Dcp1 (A), or XRN1 (B) knockdown cells. A, B: Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD), EDC4 and Dcp1 (EDC4/Dcp1 KD, A ) or XRN1 (XRN1 KD, B ). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–3 and 11–14, in B: lanes 1–5 and 14–18. IPs in A: lanes 4 and 16, in B: lanes 6 and 19) or anti-c-myc antibody (Input in A: lanes 5–9 and 17–21, in B: lanes 7–12 and 20–25. IPs in A: lanes 10 and 22, in B: lanes 13 and 26). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. C: The amount of Myc-HPat/HA-GW182 in immunoprecipitates (IP) from lysates of control, EDC4 and Dcp1, or XRN1 knockdown cells. The IP was normalized (Supporting Figure S5 ) and the value of the control IP set to 1. D: Analysis of EDC4, Dcp1, and XRN1 mRNA levels. The levels of EDC4, Dcp1, and XRN1 mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. E: Upregulation of endogenous miRNA targets in knockdown cells. CG6770 mRNA levels in total RNA of EDC4/Dcp1, XRN1, and YFP knockdown cells were analyzed by RT-qPCR. mRNA levels were normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s t test and significance values are as follows: ns, not significant; *, p
    Figure Legend Snippet: Co-purification of HPat with GW182 in EDC4 and Dcp1 (A), or XRN1 (B) knockdown cells. A, B: Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD), EDC4 and Dcp1 (EDC4/Dcp1 KD, A ) or XRN1 (XRN1 KD, B ). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–3 and 11–14, in B: lanes 1–5 and 14–18. IPs in A: lanes 4 and 16, in B: lanes 6 and 19) or anti-c-myc antibody (Input in A: lanes 5–9 and 17–21, in B: lanes 7–12 and 20–25. IPs in A: lanes 10 and 22, in B: lanes 13 and 26). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. C: The amount of Myc-HPat/HA-GW182 in immunoprecipitates (IP) from lysates of control, EDC4 and Dcp1, or XRN1 knockdown cells. The IP was normalized (Supporting Figure S5 ) and the value of the control IP set to 1. D: Analysis of EDC4, Dcp1, and XRN1 mRNA levels. The levels of EDC4, Dcp1, and XRN1 mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. E: Upregulation of endogenous miRNA targets in knockdown cells. CG6770 mRNA levels in total RNA of EDC4/Dcp1, XRN1, and YFP knockdown cells were analyzed by RT-qPCR. mRNA levels were normalized to rp49 mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s t test and significance values are as follows: ns, not significant; *, p

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

    2) Product Images from "TGF-? Signaling Is Often Attenuated during Hepatotumorigenesis, but Is Retained for the Malignancy of Hepatocellular Carcinoma Cells"

    Article Title: TGF-? Signaling Is Often Attenuated during Hepatotumorigenesis, but Is Retained for the Malignancy of Hepatocellular Carcinoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063436

    TβRII expression and its role in supporting HCC cell viability. (A) TβRII expression was compared in different stages of HCCs from ONCOMINE (left panel) and in 38 HCC tissue specimens by quantitative real-time RT-PCR (right panel). **, p
    Figure Legend Snippet: TβRII expression and its role in supporting HCC cell viability. (A) TβRII expression was compared in different stages of HCCs from ONCOMINE (left panel) and in 38 HCC tissue specimens by quantitative real-time RT-PCR (right panel). **, p

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "A Long ncRNA Links Copy Number Variation to a Polycomb/Trithorax Epigenetic Switch in FSHD Muscular Dystrophy"

    Article Title: A Long ncRNA Links Copy Number Variation to a Polycomb/Trithorax Epigenetic Switch in FSHD Muscular Dystrophy

    Journal: Cell

    doi: 10.1016/j.cell.2012.03.035

    DBE-T Directly Binds the TrxG Protein Ash1L and Recruits It to the FSHD Locus (A) RNA immunoprecipitation (IP) following UV crosslinking for Ash1L or IgG on AZA+TSA treated chr4/CHO cells. DBE-T or, as control, pre-miR19A and U1 snRNA enrichments were measured by qRT-PCR. The error bars represent SEM. (B) In vitro RNA-GST pull-down assay showing the interaction between recombinant GST-fused Ash1L SET domain or GST and in vitro transcribed DBE-T . On the right, Coomassie staining of purified recombinant proteins. After RNA recovery, samples were analyzed by qRT-PCR. The error bars represent SEM. (C) Following AZA+TSA treatment, chr4/CHO cells stably expressing a nonsilencing control shRNA or sh DBE-T were analyzed by ChIP for Ash1L or IgG. Enrichment for NDE was analyzed by qPCR and displayed as enrichment relative to input. The error bars represent SEM. (D) Upon AZA+TSA treatment, control shRNA, and sh Ash1L cells were collected to analyze DBE-T expression by qRT-PCR. Results are expressed over β-actin . The error bars represent SEM. See also Figure S8 .
    Figure Legend Snippet: DBE-T Directly Binds the TrxG Protein Ash1L and Recruits It to the FSHD Locus (A) RNA immunoprecipitation (IP) following UV crosslinking for Ash1L or IgG on AZA+TSA treated chr4/CHO cells. DBE-T or, as control, pre-miR19A and U1 snRNA enrichments were measured by qRT-PCR. The error bars represent SEM. (B) In vitro RNA-GST pull-down assay showing the interaction between recombinant GST-fused Ash1L SET domain or GST and in vitro transcribed DBE-T . On the right, Coomassie staining of purified recombinant proteins. After RNA recovery, samples were analyzed by qRT-PCR. The error bars represent SEM. (C) Following AZA+TSA treatment, chr4/CHO cells stably expressing a nonsilencing control shRNA or sh DBE-T were analyzed by ChIP for Ash1L or IgG. Enrichment for NDE was analyzed by qPCR and displayed as enrichment relative to input. The error bars represent SEM. (D) Upon AZA+TSA treatment, control shRNA, and sh Ash1L cells were collected to analyze DBE-T expression by qRT-PCR. Results are expressed over β-actin . The error bars represent SEM. See also Figure S8 .

    Techniques Used: Immunoprecipitation, Quantitative RT-PCR, In Vitro, Pull Down Assay, Recombinant, Staining, Purification, Stable Transfection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    4) Product Images from "Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis"

    Article Title: Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098048

    Oral vaccination with IV activates innate immune response in wild boar. (A) Comparison of PBMC mRNA levels between vaccinated (N = 7) and control (N = 8) animals. (B) Comparison of PBMC mRNA levels between control animals with lower (N = 5) and higher (N = 3) tuberculous lesion score. The mRNA levels of selected genes were analyzed by real-time RT-PCR in PBMC of vaccinated and control wild boar collected at T0 (day 1, before vaccination), T1 (day 126, before challenge) and T2 (day 255 at necropsy). The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD and normalized Ct values were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05). (C) Serum C3 protein levels (µg/ml) were determined by ELISA in vaccinated and control wild boar at T0, T1 and T2 and compared between groups by Student’s t-test with unequal variance (P > 0.05). (D) The difference in serum C3 protein levels (µg/ml) determined by ELISA in vaccinated and control wild boar was calculated between T2 and T1 and represented as Ave±S.D. (E) Serum cytokine protein levels were determined using the Quantibody porcine cytokine array in vaccinated (N = 7) and control (N = 8) wild boar at T0, T1 and T2, represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).
    Figure Legend Snippet: Oral vaccination with IV activates innate immune response in wild boar. (A) Comparison of PBMC mRNA levels between vaccinated (N = 7) and control (N = 8) animals. (B) Comparison of PBMC mRNA levels between control animals with lower (N = 5) and higher (N = 3) tuberculous lesion score. The mRNA levels of selected genes were analyzed by real-time RT-PCR in PBMC of vaccinated and control wild boar collected at T0 (day 1, before vaccination), T1 (day 126, before challenge) and T2 (day 255 at necropsy). The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD and normalized Ct values were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05). (C) Serum C3 protein levels (µg/ml) were determined by ELISA in vaccinated and control wild boar at T0, T1 and T2 and compared between groups by Student’s t-test with unequal variance (P > 0.05). (D) The difference in serum C3 protein levels (µg/ml) determined by ELISA in vaccinated and control wild boar was calculated between T2 and T1 and represented as Ave±S.D. (E) Serum cytokine protein levels were determined using the Quantibody porcine cytokine array in vaccinated (N = 7) and control (N = 8) wild boar at T0, T1 and T2, represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).

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

    Mycobacterial DNA and RNA in the IV increase C3 expression levels. (A) Total RNA (4 µg) extracted from the IV or IV (2 µl) were subjected to different treatments and analyzed by agarose gel electrophoresis after RT-PCR for the amplification of Mycobacterium spp. 16S rRNA. (B) Total RNA extracted from the IV was treated with DNase I and/or RNase and used for cDNA synthesis using random primers 16S rRNA PCR. (C) Radial un-rooted tree of the 16S rRNA phylogenetic analysis using Neighbor Joining. Mycobacterium species and sequence Genbank accession numbers are shown. (D) Alignment of 16S rRNA sequences from the same mycobacteria used in the phylogenetic analysis to show characteristic single nucleotide polymorphisms in the vaccination isolate ( M. bovis (IV)). (E) C3 mRNA levels in the oral mucosa and PBMC at T2. (F) Serum C3 protein levels in pigs vaccinated with the IV and IV-DNA/RNA determined by ELISA at T0, T1 and T2. The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD. Normalized Ct values and protein levels (µg/ml) were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).
    Figure Legend Snippet: Mycobacterial DNA and RNA in the IV increase C3 expression levels. (A) Total RNA (4 µg) extracted from the IV or IV (2 µl) were subjected to different treatments and analyzed by agarose gel electrophoresis after RT-PCR for the amplification of Mycobacterium spp. 16S rRNA. (B) Total RNA extracted from the IV was treated with DNase I and/or RNase and used for cDNA synthesis using random primers 16S rRNA PCR. (C) Radial un-rooted tree of the 16S rRNA phylogenetic analysis using Neighbor Joining. Mycobacterium species and sequence Genbank accession numbers are shown. (D) Alignment of 16S rRNA sequences from the same mycobacteria used in the phylogenetic analysis to show characteristic single nucleotide polymorphisms in the vaccination isolate ( M. bovis (IV)). (E) C3 mRNA levels in the oral mucosa and PBMC at T2. (F) Serum C3 protein levels in pigs vaccinated with the IV and IV-DNA/RNA determined by ELISA at T0, T1 and T2. The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD. Normalized Ct values and protein levels (µg/ml) were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Sequencing, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Transcription of Satellite III non-coding RNAs is a general stress response in human cells"

    Article Title: Transcription of Satellite III non-coding RNAs is a general stress response in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1056

    Expression of Satellite III RNAs. ( A ) RT-PCR analysis of SatIII transcripts in total RNA prepared from unstressed (37°C) and heat-shocked (1 h at 42°C followed by 2 h at 37°C) (42°C) HeLa cells. G-rich transcripts are reverse transcribed with the RSM13 primer and PCR amplified with Hur98R and M13 primers for 25 or 40 cycles. C-rich RNAs are reverse transcribed with FSM13 and amplified for 40 cycles with Hur98F and M13. The same amount of RNA (1 µg) was reverse transcribed with an oligo dT and amplified with oligos specific for GAPDH and hsp70.1 transcripts (see Supplementary Table 1). M: GeneRuler 100 bp DNA ladder plus (Fermentas) ( B ) qRT-PCR analysis of G-rich (white columns) and C-rich (gray columns) transcripts in total RNAs prepared from unstressed HeLa cells, from heat-shocked cells and from cells allowed to recover for the indicated times at 37°C. Primers used are the same described in panel A. Columns: mean of three independent experiments. Error bars are indicated. (C) qRT-PCR analysis of G-rich transcripts in total RNA prepared from HeLa cells subjected to Osmotic stress (0.8 M sorbitol) for the indicated times. Results were normalized against the housekeeping hP0 mRNA level and expressed as a function of the SatIII RNA level observed in unstressed control (C) cells. (HS): RNAs from HeLa cells kept 1 h at 42°C and allowed to recover 1 h at 37°C. Columns: mean of three independent experiments. Error bars are indicated. ( D ) Sequence of the amplified G-rich RNA in panel A (25 cy). Arrows: primers used in RT-PCR. GGAAT pentamers are underlined. The terminator sequence is boxed. ( E ) RT-PCR analysis of SatIII transcripts in total RNA prepared from HeLa, B14-150, GM-106 and GM-114 cell lines. For each cell lines we have analyzed RNA extracted from cells subjected to heat shock (H; 1h at 42°C followed by 1 h at 37°C) or to osmotic stress (8 h in 0.8 M sorbitol). G-rich transcripts were PCR amplified for 40 cycles.
    Figure Legend Snippet: Expression of Satellite III RNAs. ( A ) RT-PCR analysis of SatIII transcripts in total RNA prepared from unstressed (37°C) and heat-shocked (1 h at 42°C followed by 2 h at 37°C) (42°C) HeLa cells. G-rich transcripts are reverse transcribed with the RSM13 primer and PCR amplified with Hur98R and M13 primers for 25 or 40 cycles. C-rich RNAs are reverse transcribed with FSM13 and amplified for 40 cycles with Hur98F and M13. The same amount of RNA (1 µg) was reverse transcribed with an oligo dT and amplified with oligos specific for GAPDH and hsp70.1 transcripts (see Supplementary Table 1). M: GeneRuler 100 bp DNA ladder plus (Fermentas) ( B ) qRT-PCR analysis of G-rich (white columns) and C-rich (gray columns) transcripts in total RNAs prepared from unstressed HeLa cells, from heat-shocked cells and from cells allowed to recover for the indicated times at 37°C. Primers used are the same described in panel A. Columns: mean of three independent experiments. Error bars are indicated. (C) qRT-PCR analysis of G-rich transcripts in total RNA prepared from HeLa cells subjected to Osmotic stress (0.8 M sorbitol) for the indicated times. Results were normalized against the housekeeping hP0 mRNA level and expressed as a function of the SatIII RNA level observed in unstressed control (C) cells. (HS): RNAs from HeLa cells kept 1 h at 42°C and allowed to recover 1 h at 37°C. Columns: mean of three independent experiments. Error bars are indicated. ( D ) Sequence of the amplified G-rich RNA in panel A (25 cy). Arrows: primers used in RT-PCR. GGAAT pentamers are underlined. The terminator sequence is boxed. ( E ) RT-PCR analysis of SatIII transcripts in total RNA prepared from HeLa, B14-150, GM-106 and GM-114 cell lines. For each cell lines we have analyzed RNA extracted from cells subjected to heat shock (H; 1h at 42°C followed by 1 h at 37°C) or to osmotic stress (8 h in 0.8 M sorbitol). G-rich transcripts were PCR amplified for 40 cycles.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Sequencing

    Quantitative RT-PCR analysis of hsp70.1 mRNA level in HeLa cells subjected to different stress treatments. ( A ) Total RNA (1 µg) from HeLa cells irradiated with UV-C at the indicated doses was reverse transcribed with oligo dT. An aliquot (1/10th) was tested in qPCR to assess the level of hsp70 A1A mRNA. Black bars: no recovery. Dark gray bars: 4 h of recovery. Light gray bars: 8 h of recovery. White bars: 15 h of recovery after irradiation. ( B ) HeLa cells were treated with 5 µM cadmium sulfate for 1 h and allowed to recover for the indicated times (white bars). Gray bars: cell treated for the indicated times with 5 µM cadmium sulfate. ( C ) HeLa cells were grown for the indicated time periods in 0.8 M sorbitol. White columns: hsp70.1. Gray columns: hsp70.2. ( D ) HeLa cells heat shocked 1 h at 42°C and the allowed to recover 1 h at 37°C (HS). White columns: hsp70.1. Gray columns: hsp70.2. C represents unstressed cells.
    Figure Legend Snippet: Quantitative RT-PCR analysis of hsp70.1 mRNA level in HeLa cells subjected to different stress treatments. ( A ) Total RNA (1 µg) from HeLa cells irradiated with UV-C at the indicated doses was reverse transcribed with oligo dT. An aliquot (1/10th) was tested in qPCR to assess the level of hsp70 A1A mRNA. Black bars: no recovery. Dark gray bars: 4 h of recovery. Light gray bars: 8 h of recovery. White bars: 15 h of recovery after irradiation. ( B ) HeLa cells were treated with 5 µM cadmium sulfate for 1 h and allowed to recover for the indicated times (white bars). Gray bars: cell treated for the indicated times with 5 µM cadmium sulfate. ( C ) HeLa cells were grown for the indicated time periods in 0.8 M sorbitol. White columns: hsp70.1. Gray columns: hsp70.2. ( D ) HeLa cells heat shocked 1 h at 42°C and the allowed to recover 1 h at 37°C (HS). White columns: hsp70.1. Gray columns: hsp70.2. C represents unstressed cells.

    Techniques Used: Quantitative RT-PCR, Irradiation, Real-time Polymerase Chain Reaction

    Different SatIII chromosomal domains are activated by osmotic stress. ( A ) The number of nSBs per cell has been measured. Cells have been grouped in four classes containing the indicated number of nSBs. Percentages have been calculated on 300 cells with nSBs in three different experiments. White columns: Cells grown for 8 h in 0.8 M Sorbitol. Gray columns: 1 h at 42°C followed by 1 h at 37°C. ( B ) Parental B14-150 cells, and somatic human > hamster cell hybrids GM-106, GM-114 and YXY-95S grown 8 h in 0.8 M sorbitol, heat shocked (1 h at 42°C followed by 1 h at 37°C) or untreated (NT). Cells were analyzed by RNA in situ hybridization (FISH) and co-stained with DAPI. Confocal images of the same cells are shown. ( C ) qRT-PCR analysis of G-rich SatIII RNAs extracted from the indicated cells lines untreated (C), heat shocked (HS) or grown for the indicated hours in 0.8 M sorbitol. RNAs were first standardized by measuring by qRT-PCR the level of the GAPDH transcripts. The induction fold has been calculated using the level of SatIII RNAs in unstressed cells as reference. Dots represent the results of a second experiment. ND: non detectable.
    Figure Legend Snippet: Different SatIII chromosomal domains are activated by osmotic stress. ( A ) The number of nSBs per cell has been measured. Cells have been grouped in four classes containing the indicated number of nSBs. Percentages have been calculated on 300 cells with nSBs in three different experiments. White columns: Cells grown for 8 h in 0.8 M Sorbitol. Gray columns: 1 h at 42°C followed by 1 h at 37°C. ( B ) Parental B14-150 cells, and somatic human > hamster cell hybrids GM-106, GM-114 and YXY-95S grown 8 h in 0.8 M sorbitol, heat shocked (1 h at 42°C followed by 1 h at 37°C) or untreated (NT). Cells were analyzed by RNA in situ hybridization (FISH) and co-stained with DAPI. Confocal images of the same cells are shown. ( C ) qRT-PCR analysis of G-rich SatIII RNAs extracted from the indicated cells lines untreated (C), heat shocked (HS) or grown for the indicated hours in 0.8 M sorbitol. RNAs were first standardized by measuring by qRT-PCR the level of the GAPDH transcripts. The induction fold has been calculated using the level of SatIII RNAs in unstressed cells as reference. Dots represent the results of a second experiment. ND: non detectable.

    Techniques Used: RNA In Situ Hybridization, Fluorescence In Situ Hybridization, Staining, Quantitative RT-PCR

    6) Product Images from "The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1"

    Article Title: The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks912

    Expression of human miRNAs suppress HIV-1 replication in cell culture. ( A ) Effects of transfection of the indicated individual miRNAs on HIV-1 replication in cultured cells. The 42CD4 cells were transfected with the indicated miRNAs, and cells were infected with HIV-1 NL4-3 24 h later. Viral replication was monitored by measuring RT production in culture supernatants, 3 days after infection. Values are expressed as percent of a control irrelevant RNA oligonucleotide (oligo fluo). Arrows indicate miRNA chosen for follow-up experiments. ( B ) Time course replication curves spanning 2, 3, 4 and 6 days post-infection in cells transfected with miR-138, miR-149, miR-326, miR-195 and miR-29b (indicated by an arrow in panel A) and irrelevant control RNA oligonucleotide (oligo fluo); miR-29b is a positive control; oligo fluo is a negative control. Results are representative of three independent experiments. Values are the mean of three separate experiments with standard deviations.
    Figure Legend Snippet: Expression of human miRNAs suppress HIV-1 replication in cell culture. ( A ) Effects of transfection of the indicated individual miRNAs on HIV-1 replication in cultured cells. The 42CD4 cells were transfected with the indicated miRNAs, and cells were infected with HIV-1 NL4-3 24 h later. Viral replication was monitored by measuring RT production in culture supernatants, 3 days after infection. Values are expressed as percent of a control irrelevant RNA oligonucleotide (oligo fluo). Arrows indicate miRNA chosen for follow-up experiments. ( B ) Time course replication curves spanning 2, 3, 4 and 6 days post-infection in cells transfected with miR-138, miR-149, miR-326, miR-195 and miR-29b (indicated by an arrow in panel A) and irrelevant control RNA oligonucleotide (oligo fluo); miR-29b is a positive control; oligo fluo is a negative control. Results are representative of three independent experiments. Values are the mean of three separate experiments with standard deviations.

    Techniques Used: Expressing, Cell Culture, Transfection, Infection, Positive Control, Negative Control

    7) Product Images from "Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors"

    Article Title: Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00937.x

    CD24 + cells express markers of embryonic renal progenitors. FACS analysis exhibits the co-expression of surface markers of embryonic renal progenitors in CD24 + cells, CD24 and Cadherin-11(95.5 ± 2.1%), isotype control (A). The cells showed expression of CXCR-4 (78.7 ± 1.6%) (B). qRT-PCR was performed on CD24 + cells for the expression of genes specific to metanephric progenitors (n = 3) (C). RNA was obtained from single cell colony of CD24 + cells at passages 1 and 5. All mRNA expression levels were normalized to the house keeping gene GAPDH expression. Graphs are represented as fold difference in Ct value of passage 5 over passage 1. Data are represented as mean ± S.E.M. (* P
    Figure Legend Snippet: CD24 + cells express markers of embryonic renal progenitors. FACS analysis exhibits the co-expression of surface markers of embryonic renal progenitors in CD24 + cells, CD24 and Cadherin-11(95.5 ± 2.1%), isotype control (A). The cells showed expression of CXCR-4 (78.7 ± 1.6%) (B). qRT-PCR was performed on CD24 + cells for the expression of genes specific to metanephric progenitors (n = 3) (C). RNA was obtained from single cell colony of CD24 + cells at passages 1 and 5. All mRNA expression levels were normalized to the house keeping gene GAPDH expression. Graphs are represented as fold difference in Ct value of passage 5 over passage 1. Data are represented as mean ± S.E.M. (* P

    Techniques Used: FACS, Expressing, Quantitative RT-PCR

    GPECs undergo epithelial EMT in vitro . qRT-PCR analysis for the expression of epithelial and mesenchymal markers as well as transcript levels of major EMT regulators in CD24 + cells at passage 5 over P1( n = 3). Graphs are represented as fold difference in Ct value of passage 5 over passage 1. RNA was obtained from single cell colony of CD24 + cells at passages 1 and 5. All mRNA expression levels were normalized to the house keeping gene GAPDH expression. Data are represented as mean ± S.E.M., (*P
    Figure Legend Snippet: GPECs undergo epithelial EMT in vitro . qRT-PCR analysis for the expression of epithelial and mesenchymal markers as well as transcript levels of major EMT regulators in CD24 + cells at passage 5 over P1( n = 3). Graphs are represented as fold difference in Ct value of passage 5 over passage 1. RNA was obtained from single cell colony of CD24 + cells at passages 1 and 5. All mRNA expression levels were normalized to the house keeping gene GAPDH expression. Data are represented as mean ± S.E.M., (*P

    Techniques Used: In Vitro, Quantitative RT-PCR, Expressing

    8) Product Images from "An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis"

    Article Title: An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010993

    Xenograft tumors established with parent and hBD-3 overexpressing HEK293 cells in nude mice. (A) RT-PCR of hBD-3 on total RNA samples extracted from parent HEK293 and hBD-3 overexpressed HEK293 cells, respectively. (B and C) ELISA of hBD-3 using culture supernatants (B) or cell lysates (C) derived from parent HEK293 and hBD-3 overexpressed cells. HEK293 and hBD-3 overexpressing cells were cultured in serum-free medium for 3 days, followed by ELISA of collected media and cell lysates, respectively. *, p = 0.00. (D) Representative mice bearing tumors after 10 days post inoculation. Yellow arrows, inoculation sites. (E) Representative tumors isolated from mice inoculated with parent HEK293 cells and hBD-3 overexpressing cells. (F) The incidence and sizes of xenograft tumors generated using parent HEK293 and hBD-3 overexpressed HEK293 cells. The mean volume for each group of tumors is represented as black lines; in HEK293 tumors, the value is 27.9 mm 3 , while in hBD-3 overexpressed tumors, the value is 66.9 mm 3 . *, p
    Figure Legend Snippet: Xenograft tumors established with parent and hBD-3 overexpressing HEK293 cells in nude mice. (A) RT-PCR of hBD-3 on total RNA samples extracted from parent HEK293 and hBD-3 overexpressed HEK293 cells, respectively. (B and C) ELISA of hBD-3 using culture supernatants (B) or cell lysates (C) derived from parent HEK293 and hBD-3 overexpressed cells. HEK293 and hBD-3 overexpressing cells were cultured in serum-free medium for 3 days, followed by ELISA of collected media and cell lysates, respectively. *, p = 0.00. (D) Representative mice bearing tumors after 10 days post inoculation. Yellow arrows, inoculation sites. (E) Representative tumors isolated from mice inoculated with parent HEK293 cells and hBD-3 overexpressing cells. (F) The incidence and sizes of xenograft tumors generated using parent HEK293 and hBD-3 overexpressed HEK293 cells. The mean volume for each group of tumors is represented as black lines; in HEK293 tumors, the value is 27.9 mm 3 , while in hBD-3 overexpressed tumors, the value is 66.9 mm 3 . *, p

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Isolation, Generated

    9) Product Images from "Phosphatidylserine Increases IKBKAP Levels in Familial Dysautonomia Cells"

    Article Title: Phosphatidylserine Increases IKBKAP Levels in Familial Dysautonomia Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015884

    PS raises IKAP mRNA levels in FD cell lines. ( A ) Chemical structure of PS. ( B ) FDA cells or ( C ) FDB cells were treated with 0, 5, 10, 100, 200 and 300 µg/ml PS. RNA was extracted after 24 hr for FDA cell line and after 48 hr for FDB cell line. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP mRNA in FD cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.
    Figure Legend Snippet: PS raises IKAP mRNA levels in FD cell lines. ( A ) Chemical structure of PS. ( B ) FDA cells or ( C ) FDB cells were treated with 0, 5, 10, 100, 200 and 300 µg/ml PS. RNA was extracted after 24 hr for FDA cell line and after 48 hr for FDB cell line. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP mRNA in FD cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.

    Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    Expression of IKAP mRNA and protein in FD cells. ( A ) Schematic diagram illustrating the area in IKAP responsible for FD. The FD mutation at position six of exon 20 splice donor site is shown by an arrow. In FD patients, two mRNA isoforms, one containing exon 20 and one without, can be present. ( B ) RT-PCR analysis of IKAP mRNA. RNA was extracted from control, heterozygous and FD cells and the endogenous splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exon 19 and 21. Isoforms were quantified using ImageJ. GAPDH was used as control for cDNA amounts. ( C ) QPCR analysis of the IKAP mRNA. Left side: Level of exon 20 inclusion isoform (wt). Relative quantity represents normalization to control cells. Right side: Level of exon 20 skipped isoform (mut). Relative quantity represents normalization to heterozygous (Htz) cells. All values were normalized to HPRT mRNA. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. ( D ) Analysis of IKAP protein levels. Western blotting of extracts from the indicated cell lines using an anti-IKAP antibody (Santa Cruz Biotechnology, D-17). Band intensities were quantified using ImageJ.
    Figure Legend Snippet: Expression of IKAP mRNA and protein in FD cells. ( A ) Schematic diagram illustrating the area in IKAP responsible for FD. The FD mutation at position six of exon 20 splice donor site is shown by an arrow. In FD patients, two mRNA isoforms, one containing exon 20 and one without, can be present. ( B ) RT-PCR analysis of IKAP mRNA. RNA was extracted from control, heterozygous and FD cells and the endogenous splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exon 19 and 21. Isoforms were quantified using ImageJ. GAPDH was used as control for cDNA amounts. ( C ) QPCR analysis of the IKAP mRNA. Left side: Level of exon 20 inclusion isoform (wt). Relative quantity represents normalization to control cells. Right side: Level of exon 20 skipped isoform (mut). Relative quantity represents normalization to heterozygous (Htz) cells. All values were normalized to HPRT mRNA. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. ( D ) Analysis of IKAP protein levels. Western blotting of extracts from the indicated cell lines using an anti-IKAP antibody (Santa Cruz Biotechnology, D-17). Band intensities were quantified using ImageJ.

    Techniques Used: Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Western Blot

    Other supplements also increase IKAP mRNA levels in an FD cell line. ( A ) Sharp·PS GOLD4508P (Gold), ( B ) SharpGPC 85F (GPC), or ( C ) Krill oil+4225F (Krill oil) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the supplement. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.
    Figure Legend Snippet: Other supplements also increase IKAP mRNA levels in an FD cell line. ( A ) Sharp·PS GOLD4508P (Gold), ( B ) SharpGPC 85F (GPC), or ( C ) Krill oil+4225F (Krill oil) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the supplement. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.

    Techniques Used: Gel Permeation Chromatography, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    The effect of substances already tested in FD models or patients on IKAP mRNA levels. ( A ) Kinetin or ( B ) tocotrienol (Toco) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the substance. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP . All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.
    Figure Legend Snippet: The effect of substances already tested in FD models or patients on IKAP mRNA levels. ( A ) Kinetin or ( B ) tocotrienol (Toco) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the substance. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP . All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.

    Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    PS raises IKAP mRNA and protein levels following long-term treatment. PS was added to FDB cells at a concentration of 100 µg/ml. Every two days the medium was replaced and fresh PS was added. RNA was extracted 3, 7 and 14 days following the initial addition of PS. All experiments were repeated independently three times, and the results shown are representative of an average experiment. ( A ) QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells harvested on the same day. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. ( B ) RT-PCR analysis of the splicing of the endogenous IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. ( C ) Western blotting using an anti-IKAP antibody (BD-Bioscience) of FDB cells treated with PS for two weeks. The exposure of the analysis of 293T cells was reduced to one fourth of its original amount in order to avoid intense background and to better visualize the effect of PS. Band intensities were quantified using ImageJ.
    Figure Legend Snippet: PS raises IKAP mRNA and protein levels following long-term treatment. PS was added to FDB cells at a concentration of 100 µg/ml. Every two days the medium was replaced and fresh PS was added. RNA was extracted 3, 7 and 14 days following the initial addition of PS. All experiments were repeated independently three times, and the results shown are representative of an average experiment. ( A ) QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells harvested on the same day. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. ( B ) RT-PCR analysis of the splicing of the endogenous IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. ( C ) Western blotting using an anti-IKAP antibody (BD-Bioscience) of FDB cells treated with PS for two weeks. The exposure of the analysis of 293T cells was reduced to one fourth of its original amount in order to avoid intense background and to better visualize the effect of PS. Band intensities were quantified using ImageJ.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Western Blot

    10) Product Images from "Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action"

    Article Title: Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action

    Journal: Scientific Reports

    doi: 10.1038/srep25689

    IL-3 dictates the content of miR-126-3p in EC-derived EVs. (a) miR‐126-3p, miR-221 and miR-222 expression was evaluated by quantitative real-time PCR (qRT-PCR) on ECs, untreated ( c ) or treated with IL-3 (IL-3) in the presence or in the absence of the anti-IL3Ralpha blocking antibody (anti-IL-3R). Data normalized to RNU6B are representative of four different experiments performed in triplicate (n = 4) ( p = 0.056 IL-3 vs none and p = 0.075 IL-3 vs anti-IL3R+ IL-3 for miR-126-3p; p = 0.0003 IL-3 vs none and p = 0.0001 IL-3 vs anti-IL3R+ IL-3 for miR-221: p = 0.04 IL-3 vs none and p = 0.008 IL-3 vs anti-IL3R+ IL-3 for miR-222) (b) miR‐126-3p, miR-221 and miR-222 expression was evaluated as above on EVs recovered from ECs, treated as above ( n = 5) ( p = 0.01 IL-3-EVs vs EVs and p = 0.04 IL-3-EVs vs anti-IL3R-EVs for miR-126-3p; p = 0.7 IL-3-EVs vs EVs and p = 0.9 IL-3-EVs vs anti-IL3R-EVs for miR-221: p = 0.2 IL-3-EVs vs EVs and p = 0.5 IL-3-EVs vs anti-IL3R-EVs for miR-222). (c) miR‐126-3p expression evaluated on EC-derived EVs was normalized to EC miR‐126-3p content. Data are representative of five experiments performed in triplicate ( n = 5) ( p = 0.03 IL-3-EVs vs EVs and p = 0.02 IL-3-EVs vs anti-IL-3R-EVs). (d) miR‐126-3p expression was evaluated by qRT-PCR on ECs untreated or treated with EVs derived from ECs stimulated as above. Data normalized to RNU6B are representative of four different experiments performed in triplicate ( n = 4) ( p = 0.004 EC+ IL-3-EVs vs EC+ EVs and p = 0.02 EC+ IL-3-EVs vs EC+ anti-IL-3R-EVs). (e) ECs incubated in the presence of 50 μ g/ml of α -amanitin to inhibit EC transcription were stimulated or not stimulated with EVs or IL-3-EVs. EV-miR-126 transfer was evaluated by q-RT-PCR. The difference in Ct values (Δ Ct) between α -amanitin-treated ECs alone or with the indicated EVs is reported ( p = 0.001 ) (mean ± SD). (f) Representative photomicrographs of an in vitro angiogenesis assay performed on ECs treated as indicated. The quantitative analysis of the number and length of branches and percentage of area of in vitro formed vessel-like structures is reported as mean ± SD. The results are representative of four different experiments performed in triplicate ( n = 4) (for length, *** p
    Figure Legend Snippet: IL-3 dictates the content of miR-126-3p in EC-derived EVs. (a) miR‐126-3p, miR-221 and miR-222 expression was evaluated by quantitative real-time PCR (qRT-PCR) on ECs, untreated ( c ) or treated with IL-3 (IL-3) in the presence or in the absence of the anti-IL3Ralpha blocking antibody (anti-IL-3R). Data normalized to RNU6B are representative of four different experiments performed in triplicate (n = 4) ( p = 0.056 IL-3 vs none and p = 0.075 IL-3 vs anti-IL3R+ IL-3 for miR-126-3p; p = 0.0003 IL-3 vs none and p = 0.0001 IL-3 vs anti-IL3R+ IL-3 for miR-221: p = 0.04 IL-3 vs none and p = 0.008 IL-3 vs anti-IL3R+ IL-3 for miR-222) (b) miR‐126-3p, miR-221 and miR-222 expression was evaluated as above on EVs recovered from ECs, treated as above ( n = 5) ( p = 0.01 IL-3-EVs vs EVs and p = 0.04 IL-3-EVs vs anti-IL3R-EVs for miR-126-3p; p = 0.7 IL-3-EVs vs EVs and p = 0.9 IL-3-EVs vs anti-IL3R-EVs for miR-221: p = 0.2 IL-3-EVs vs EVs and p = 0.5 IL-3-EVs vs anti-IL3R-EVs for miR-222). (c) miR‐126-3p expression evaluated on EC-derived EVs was normalized to EC miR‐126-3p content. Data are representative of five experiments performed in triplicate ( n = 5) ( p = 0.03 IL-3-EVs vs EVs and p = 0.02 IL-3-EVs vs anti-IL-3R-EVs). (d) miR‐126-3p expression was evaluated by qRT-PCR on ECs untreated or treated with EVs derived from ECs stimulated as above. Data normalized to RNU6B are representative of four different experiments performed in triplicate ( n = 4) ( p = 0.004 EC+ IL-3-EVs vs EC+ EVs and p = 0.02 EC+ IL-3-EVs vs EC+ anti-IL-3R-EVs). (e) ECs incubated in the presence of 50 μ g/ml of α -amanitin to inhibit EC transcription were stimulated or not stimulated with EVs or IL-3-EVs. EV-miR-126 transfer was evaluated by q-RT-PCR. The difference in Ct values (Δ Ct) between α -amanitin-treated ECs alone or with the indicated EVs is reported ( p = 0.001 ) (mean ± SD). (f) Representative photomicrographs of an in vitro angiogenesis assay performed on ECs treated as indicated. The quantitative analysis of the number and length of branches and percentage of area of in vitro formed vessel-like structures is reported as mean ± SD. The results are representative of four different experiments performed in triplicate ( n = 4) (for length, *** p

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Blocking Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, In Vitro, Angiogenesis Assay

    Schematic representation of IL-3 paracrine actions via EC-EVs. IL-3 stimulation leads to Erk1/2 activation. IL-3 stimulation leads to STAT5 activation and cyclin D1 transcription 48 . On the other hand, IL-3-mediated STAT5 activation is involved in the control of EV cargo (at least of pSTAT5, miR-126-3p EV content) and EV release (left panel). The released vesicles are internalized by neighboring ECs. The transfer of EV-miR126-3p and pSTAT5 into recipient ECs independently promotes pro-angiogenic signals (right panel).
    Figure Legend Snippet: Schematic representation of IL-3 paracrine actions via EC-EVs. IL-3 stimulation leads to Erk1/2 activation. IL-3 stimulation leads to STAT5 activation and cyclin D1 transcription 48 . On the other hand, IL-3-mediated STAT5 activation is involved in the control of EV cargo (at least of pSTAT5, miR-126-3p EV content) and EV release (left panel). The released vesicles are internalized by neighboring ECs. The transfer of EV-miR126-3p and pSTAT5 into recipient ECs independently promotes pro-angiogenic signals (right panel).

    Techniques Used: Activation Assay

    IL-3 improves the pro-angiogenic potential of EC-derived EVs. (a) Representative images obtained on a confocal microscope of ECs treated for 1 h with the indicated PKH26-labeled EVs to evaluate up-take of EVs by ECs. The results are representative of four different experiments performed in triplicate (n = 4). Scale bars indicate 10 μ m. (b) Representative photomicrographs of an in vitro angiogenesis assay, showing tube-like structure formation by ECs, either alone or in the presence of IL-3 (10 ng/ml), EVs, IL-3-EVs or anti-IL-3R-EVs. The quantitative analysis of the number and length of branches and percentage of vessel area (% area) of in vitro formed vessel-like structures is reported as mean ± SD. The results are representative of four different experiments performed in triplicate (n = 4) (for length, ** p
    Figure Legend Snippet: IL-3 improves the pro-angiogenic potential of EC-derived EVs. (a) Representative images obtained on a confocal microscope of ECs treated for 1 h with the indicated PKH26-labeled EVs to evaluate up-take of EVs by ECs. The results are representative of four different experiments performed in triplicate (n = 4). Scale bars indicate 10 μ m. (b) Representative photomicrographs of an in vitro angiogenesis assay, showing tube-like structure formation by ECs, either alone or in the presence of IL-3 (10 ng/ml), EVs, IL-3-EVs or anti-IL-3R-EVs. The quantitative analysis of the number and length of branches and percentage of vessel area (% area) of in vitro formed vessel-like structures is reported as mean ± SD. The results are representative of four different experiments performed in triplicate (n = 4) (for length, ** p

    Techniques Used: Derivative Assay, Microscopy, Labeling, In Vitro, Angiogenesis Assay

    EV pSTAT5 content leads to the formation of a STAT5/cyclin D1 transcriptional complex. (a , b) Cell extracts from ECs treated with EC-derived EVs, as indicated, were analyzed for pSTAT5 and STAT5 ( a ) or for cyclin D1 ( b ) and β -actin content. The results are representative of four different experiments performed in triplicate ( n = 4) (*** p
    Figure Legend Snippet: EV pSTAT5 content leads to the formation of a STAT5/cyclin D1 transcriptional complex. (a , b) Cell extracts from ECs treated with EC-derived EVs, as indicated, were analyzed for pSTAT5 and STAT5 ( a ) or for cyclin D1 ( b ) and β -actin content. The results are representative of four different experiments performed in triplicate ( n = 4) (*** p

    Techniques Used: Derivative Assay

    IL-3 treatment drives pSTAT5 EV content. (a) EVs recovered from ECs, untreated (EVs) or treated with IL-3 (IL-3-EVs), in the presence or in the absence of anti-IL3Ralpha blocking antibody (anti-IL-3R-EVs) were analyzed by western blotting for pSTAT5 and STAT5 content and normalized to CD63 content. The results are representative of four different experiments performed in triplicate ( n = 4) ( p
    Figure Legend Snippet: IL-3 treatment drives pSTAT5 EV content. (a) EVs recovered from ECs, untreated (EVs) or treated with IL-3 (IL-3-EVs), in the presence or in the absence of anti-IL3Ralpha blocking antibody (anti-IL-3R-EVs) were analyzed by western blotting for pSTAT5 and STAT5 content and normalized to CD63 content. The results are representative of four different experiments performed in triplicate ( n = 4) ( p

    Techniques Used: Blocking Assay, Western Blot

    EV pSTAT5 content mediates IL-3-EV biological effects. (a , b) Representative photomicrographs of an in vitro angiogenesis assay performed on ECs treated with EC-derived EVs, as indicated, in a or on ECs transfected with Δ NSTAT5 (Δ N) and treated as indicated in ( b ). Quantitative analysis of in vitro formed vessel-like structures (number and length of branches, percentage of area) is reported as mean ± SD. The results are representative of four different experiments performed in triplicate ( n = 4) (for length, *** p
    Figure Legend Snippet: EV pSTAT5 content mediates IL-3-EV biological effects. (a , b) Representative photomicrographs of an in vitro angiogenesis assay performed on ECs treated with EC-derived EVs, as indicated, in a or on ECs transfected with Δ NSTAT5 (Δ N) and treated as indicated in ( b ). Quantitative analysis of in vitro formed vessel-like structures (number and length of branches, percentage of area) is reported as mean ± SD. The results are representative of four different experiments performed in triplicate ( n = 4) (for length, *** p

    Techniques Used: In Vitro, Angiogenesis Assay, Derivative Assay, Transfection

    Characterization of EC-derived EVs. ( a ) Number of EV particles (mean ± SEM) calculated per cell at isolation. Data refer to EVs from ECs (EVs), IL-3-treated ECs (IL-3-EVs) or from ECs pre-treated with the anti-IL3Ralpha blocking antibody (anti-IL-3R) and then stimulated with IL-3 (anti-IL-3R-EVs). The anti-IL-3R alone was also used. The results are representative of four different experiments performed in triplicate ( n = 4) (*** p
    Figure Legend Snippet: Characterization of EC-derived EVs. ( a ) Number of EV particles (mean ± SEM) calculated per cell at isolation. Data refer to EVs from ECs (EVs), IL-3-treated ECs (IL-3-EVs) or from ECs pre-treated with the anti-IL3Ralpha blocking antibody (anti-IL-3R) and then stimulated with IL-3 (anti-IL-3R-EVs). The anti-IL-3R alone was also used. The results are representative of four different experiments performed in triplicate ( n = 4) (*** p

    Techniques Used: Derivative Assay, Isolation, Blocking Assay

    STAT5 dictates the release of EV and miR-126-3p cargo in response to IL-3 stimulation. (a) Number of EV particles (mean ± SEM) calculated per cell at isolation. Data refer to EVs recovered from untreated or IL-3-treated ECs or from ECs transfected with Δ NSTAT5, in the presence of IL-3, or with 1*6STAT5 constructs for 48 h. Results are representative of four different experiments performed in triplicate ( n = 4) (*** p
    Figure Legend Snippet: STAT5 dictates the release of EV and miR-126-3p cargo in response to IL-3 stimulation. (a) Number of EV particles (mean ± SEM) calculated per cell at isolation. Data refer to EVs recovered from untreated or IL-3-treated ECs or from ECs transfected with Δ NSTAT5, in the presence of IL-3, or with 1*6STAT5 constructs for 48 h. Results are representative of four different experiments performed in triplicate ( n = 4) (*** p

    Techniques Used: Isolation, Transfection, Construct

    11) Product Images from "Intrahaplotypic Variants Differentiate Complex Linkage Disequilibrium within Human MHC Haplotypes"

    Article Title: Intrahaplotypic Variants Differentiate Complex Linkage Disequilibrium within Human MHC Haplotypes

    Journal: Scientific Reports

    doi: 10.1038/srep16972

    Association of A2-B46-DR9 intra-CEH variants with ZFP57 expression. (A) qRT-PCR was performed to determine the mRNA level of ZFP57 in COX, QBL, B46BM, B46ZS, B46CM, B58AL, B58SC, and B58CF cell lines. Experiments were carried out in triplicate for each biological replicate. Triangles indicate quantitative expression derived from biological replicate 1, while squares indicate quantitative expression derived from biological replicate 2. The genotype of the eQTL SNPs in each cell line are shown below the plot. (B) Distribution of the 202 A2-B46-DR9 intra-CEH variants across chr6:29.60–29.69(Mb).
    Figure Legend Snippet: Association of A2-B46-DR9 intra-CEH variants with ZFP57 expression. (A) qRT-PCR was performed to determine the mRNA level of ZFP57 in COX, QBL, B46BM, B46ZS, B46CM, B58AL, B58SC, and B58CF cell lines. Experiments were carried out in triplicate for each biological replicate. Triangles indicate quantitative expression derived from biological replicate 1, while squares indicate quantitative expression derived from biological replicate 2. The genotype of the eQTL SNPs in each cell line are shown below the plot. (B) Distribution of the 202 A2-B46-DR9 intra-CEH variants across chr6:29.60–29.69(Mb).

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay

    Mapping of regulatory elements to ZFP57 expression. ( A ) Linkage disequilibrium (LD) plot for region chr6:29.61 – 26.66(Mb) based on the r 2 calculation of 163 A2-B46-DR9 intra-CEH variants/SNPs in 105 unrelated Southern Han Chinese individuals (1000 Genome Project). LD block is defined as a block of SNPs with all pairwise r 2 value > 0.8. (B) Haplotypes found in each LD block. Population haplotype frequency is shown at the side of each haplotype. Connecting lines between blocks indicate crossover to the next block. Block in black indicates allele of variant associated with ZFP57 expression while white indicates allele associated with non ZFP57 expression. (C) Luciferase activity for alleles of each intra-CEH variants in LD blocks 2 to 4. Reporter plasmids carrying the fragment sequence of each unique allele of a certain variant were co-transfected with Renilla control vector into HT1080 cells. The luciferase activity readout was normalized to Renilla luciferase activity and the representative results from three independent experiments are shown. Variants with significant differential allelic luciferase activity (P
    Figure Legend Snippet: Mapping of regulatory elements to ZFP57 expression. ( A ) Linkage disequilibrium (LD) plot for region chr6:29.61 – 26.66(Mb) based on the r 2 calculation of 163 A2-B46-DR9 intra-CEH variants/SNPs in 105 unrelated Southern Han Chinese individuals (1000 Genome Project). LD block is defined as a block of SNPs with all pairwise r 2 value > 0.8. (B) Haplotypes found in each LD block. Population haplotype frequency is shown at the side of each haplotype. Connecting lines between blocks indicate crossover to the next block. Block in black indicates allele of variant associated with ZFP57 expression while white indicates allele associated with non ZFP57 expression. (C) Luciferase activity for alleles of each intra-CEH variants in LD blocks 2 to 4. Reporter plasmids carrying the fragment sequence of each unique allele of a certain variant were co-transfected with Renilla control vector into HT1080 cells. The luciferase activity readout was normalized to Renilla luciferase activity and the representative results from three independent experiments are shown. Variants with significant differential allelic luciferase activity (P

    Techniques Used: Expressing, Blocking Assay, Variant Assay, Luciferase, Activity Assay, Sequencing, Transfection, Plasmid Preparation

    12) Product Images from "Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme"

    Article Title: Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme

    Journal: Nature Communications

    doi: 10.1038/ncomms14758

    Chd7 is required for activation of neuronal genes during GNP differentiation. ( a ) qRT–PCR analysis of Chd7 transcripts in P7 Chd7 WT [ Chd7 f/f ], heterozygous [ Atoh1-Cre::Chd7 f/+ ] and homozygous [ Atoh1-Cre::Chd7 f/f ] mutant GNPs. The level of Gapdh transcripts was used for normalization. Bars represent normalized mean value ± s.d. from three samples for each group. Paired two-tailed t -test with equal variance was performed, P =0.0034 (for Het.); P =0.001 (for Hom.). ( b ) Heatmap shows significantly downregulated genes ( P
    Figure Legend Snippet: Chd7 is required for activation of neuronal genes during GNP differentiation. ( a ) qRT–PCR analysis of Chd7 transcripts in P7 Chd7 WT [ Chd7 f/f ], heterozygous [ Atoh1-Cre::Chd7 f/+ ] and homozygous [ Atoh1-Cre::Chd7 f/f ] mutant GNPs. The level of Gapdh transcripts was used for normalization. Bars represent normalized mean value ± s.d. from three samples for each group. Paired two-tailed t -test with equal variance was performed, P =0.0034 (for Het.); P =0.001 (for Hom.). ( b ) Heatmap shows significantly downregulated genes ( P

    Techniques Used: Activation Assay, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

    13) Product Images from "Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach"

    Article Title: Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004219

    Random RT-PCR amplification of cDNA from clinical specimens and quantitative RT-PCR with virus-specific primers. The samples were nasopharyngeal aspirates and stools (n = 3 and 5, respectively) isolated during 2005–2007 in Osaka, Japan. Influenza A virus and norovirus were detected in the nasopharyngeal aspirates and stool samples, respectively, with other diagnostic methods. (A) RNA extracted from clinical specimens was reverse-transcribed and random-PCR amplified to prepare template DNA for pyrosequencing. One microgram of amplified PCR products in each sample was loaded onto a 1% agarose gel. M indicates 100-bp DNA ladder (NEB). (B) Flu-specific semi-quantitative PCR was performed with 10-fold serial dilutions of the random-PCR products. (C) Norovirus (GII)-specific semi-quantitative PCR was performed with 10-fold serial dilutions of the random-PCR products. Quantitative real-time RT-PCR using a norovirus-specific primer set was also performed, and the estimated copy numbers of norovirus in samples #N1–#N5 are shown in the panel on the right. As a control, cDNA from sample #N3 without random PCR amplification was used (left panel).
    Figure Legend Snippet: Random RT-PCR amplification of cDNA from clinical specimens and quantitative RT-PCR with virus-specific primers. The samples were nasopharyngeal aspirates and stools (n = 3 and 5, respectively) isolated during 2005–2007 in Osaka, Japan. Influenza A virus and norovirus were detected in the nasopharyngeal aspirates and stool samples, respectively, with other diagnostic methods. (A) RNA extracted from clinical specimens was reverse-transcribed and random-PCR amplified to prepare template DNA for pyrosequencing. One microgram of amplified PCR products in each sample was loaded onto a 1% agarose gel. M indicates 100-bp DNA ladder (NEB). (B) Flu-specific semi-quantitative PCR was performed with 10-fold serial dilutions of the random-PCR products. (C) Norovirus (GII)-specific semi-quantitative PCR was performed with 10-fold serial dilutions of the random-PCR products. Quantitative real-time RT-PCR using a norovirus-specific primer set was also performed, and the estimated copy numbers of norovirus in samples #N1–#N5 are shown in the panel on the right. As a control, cDNA from sample #N3 without random PCR amplification was used (left panel).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Isolation, Diagnostic Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    14) Product Images from "Autophagy controls mesenchymal stem cell properties and senescence during bone aging, et al. Autophagy controls mesenchymal stem cell properties and senescence during bone aging"

    Article Title: Autophagy controls mesenchymal stem cell properties and senescence during bone aging, et al. Autophagy controls mesenchymal stem cell properties and senescence during bone aging

    Journal: Aging Cell

    doi: 10.1111/acel.12709

    Comparison of autophagy of young and aged bone marrow and bone marrow‐derived mesenchymal stem cells ( BMMSC s) showed that autophagy was decreased with aging. (a, b) LC 3 of immunohistochemistry ( IHC ) of young and aged bone marrow. Scale bar = 200 μm. (c) Real‐time PCR analysis on whole bone marrow of young and aged femora. BM = bone marrow. (d, e) Western blot was performed to examine expressions of Atg7, Beclin1, P62 and LC 3 in young and aged BMMSC s at protein level. (f) Real‐time PCR was performed to detect mRNA expression of Atg7, Beclin1 and LC 3. (g, h) Immunofluorescence ( IF ) staining of LC 3 in young and aged BMMSC s treated with CQ and PBS . Scale bar = 10 μm. (i) Transmission electron microscopy ( TEM ) was used to detect autophagosomes of young and aged BMMSC s. Scale bar = 500 nm. Results are presented as means ± SD . n = 3. * p
    Figure Legend Snippet: Comparison of autophagy of young and aged bone marrow and bone marrow‐derived mesenchymal stem cells ( BMMSC s) showed that autophagy was decreased with aging. (a, b) LC 3 of immunohistochemistry ( IHC ) of young and aged bone marrow. Scale bar = 200 μm. (c) Real‐time PCR analysis on whole bone marrow of young and aged femora. BM = bone marrow. (d, e) Western blot was performed to examine expressions of Atg7, Beclin1, P62 and LC 3 in young and aged BMMSC s at protein level. (f) Real‐time PCR was performed to detect mRNA expression of Atg7, Beclin1 and LC 3. (g, h) Immunofluorescence ( IF ) staining of LC 3 in young and aged BMMSC s treated with CQ and PBS . Scale bar = 10 μm. (i) Transmission electron microscopy ( TEM ) was used to detect autophagosomes of young and aged BMMSC s. Scale bar = 500 nm. Results are presented as means ± SD . n = 3. * p

    Techniques Used: Derivative Assay, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    15) Product Images from "Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection"

    Article Title: Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00360

    Functional characterization of selected candidate tick protective antigens. (A) Results of the RT-PCR analysis of the expression of ISCW005600 and AAY66632 genes in uninfected and A. phagocytophilum -infected ISE6 tick cells. Results were presented as average + S.D. normalized Ct-values and compared between infected and uninfected cells by Student's t -test with unequal variance ( P ≤ 0.05; N = 4). (B) Comparison of the transcriptomics and RT-PCR results for mRNA levels of ISCW005600 and AAY66632 genes in ISE6 tick cells in response to A. phagocytophilum infection. Transcriptomics results were obtained from Villar et al. ( 2015a ). (C) Representative images of imunofluorescence analysis of uninfected (a,c,e,g,i,k) and A. phagocytophilum -infected (b,d,f,h,j,l) adult female I. scapularis midguts (MG) and salivary glands (SG). Tick tissues were stained with rabbit pre-immune control IgG (a,b) , anti-ISE6 tick cells IgG (c,d) , or anti-tick antigens IgG (e–l) labeled with RFP (red) and DAPI (blue). Yellow arrows illustrate a positive staining for AAY66632 in the SG sections in white squares in infected (l) but not uninfected (k) ticks. (D) The A. phagocytophilum DNA levels were determined after RNAi in infected ISE6 tick cells treated with ISCW005600 and AAY66632 dsRNAs or control Rs86 dsRNA. A. phagocytophilum DNA levels were determined by msp4 real-time PCR normalizing against tick rpS4 . Results are shown as average + S.D. normalized Ct-values and compared between treated and control groups by Student's t -test with unequal variance ( P
    Figure Legend Snippet: Functional characterization of selected candidate tick protective antigens. (A) Results of the RT-PCR analysis of the expression of ISCW005600 and AAY66632 genes in uninfected and A. phagocytophilum -infected ISE6 tick cells. Results were presented as average + S.D. normalized Ct-values and compared between infected and uninfected cells by Student's t -test with unequal variance ( P ≤ 0.05; N = 4). (B) Comparison of the transcriptomics and RT-PCR results for mRNA levels of ISCW005600 and AAY66632 genes in ISE6 tick cells in response to A. phagocytophilum infection. Transcriptomics results were obtained from Villar et al. ( 2015a ). (C) Representative images of imunofluorescence analysis of uninfected (a,c,e,g,i,k) and A. phagocytophilum -infected (b,d,f,h,j,l) adult female I. scapularis midguts (MG) and salivary glands (SG). Tick tissues were stained with rabbit pre-immune control IgG (a,b) , anti-ISE6 tick cells IgG (c,d) , or anti-tick antigens IgG (e–l) labeled with RFP (red) and DAPI (blue). Yellow arrows illustrate a positive staining for AAY66632 in the SG sections in white squares in infected (l) but not uninfected (k) ticks. (D) The A. phagocytophilum DNA levels were determined after RNAi in infected ISE6 tick cells treated with ISCW005600 and AAY66632 dsRNAs or control Rs86 dsRNA. A. phagocytophilum DNA levels were determined by msp4 real-time PCR normalizing against tick rpS4 . Results are shown as average + S.D. normalized Ct-values and compared between treated and control groups by Student's t -test with unequal variance ( P

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Staining, Labeling, Real-time Polymerase Chain Reaction

    16) Product Images from "5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin"

    Article Title: 5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0662-6

    Increased expression of CD105 and selective binding of CD105-saporin in 5-FU resistant cells. a RNA levels (relative quantity) of CD105/ ENG in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines (P
    Figure Legend Snippet: Increased expression of CD105 and selective binding of CD105-saporin in 5-FU resistant cells. a RNA levels (relative quantity) of CD105/ ENG in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines (P

    Techniques Used: Expressing, Binding Assay, Quantitative RT-PCR, Standard Deviation

    5-FU resistant pancreatic cancer cells display increased uptake and accumulation of TPCS 2a , and show increased ROS production following PCT. TPCS 2a accumulation with a increasing TPCS 2a concentration and b increasing incubation time. The graphs show the median TPCS 2a fluorescence intensities in live cells and show the average of three and two individual experiments, respectively (Error bars = S.E.). c Representative epi-fluorescence microscopy images of live 5-FU sensitive and 5-FU resistant cells harvested 1, 2 and 4 h after TPCS 2a incubation. Co-stain of TPCS 2a and Alexa488-Dextran is indicated by yellow fluorescence in the rightmost images. The experiment was performed once but was also verified for the B1Q and the B1L cells (data not shown), confirming the data in ( a ) and ( b ). The scale bar is 20 μm. d Relative ROS generation measured 1 h after light exposure, with increasing PCT (TPCS 2a + light) illumination doses (30–120 s). The graph shows normalized median DCF fluorescence intensities representative of three individual experiments. e RNA levels (relative quantity) of SOD1 and SOD2 in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines ( P
    Figure Legend Snippet: 5-FU resistant pancreatic cancer cells display increased uptake and accumulation of TPCS 2a , and show increased ROS production following PCT. TPCS 2a accumulation with a increasing TPCS 2a concentration and b increasing incubation time. The graphs show the median TPCS 2a fluorescence intensities in live cells and show the average of three and two individual experiments, respectively (Error bars = S.E.). c Representative epi-fluorescence microscopy images of live 5-FU sensitive and 5-FU resistant cells harvested 1, 2 and 4 h after TPCS 2a incubation. Co-stain of TPCS 2a and Alexa488-Dextran is indicated by yellow fluorescence in the rightmost images. The experiment was performed once but was also verified for the B1Q and the B1L cells (data not shown), confirming the data in ( a ) and ( b ). The scale bar is 20 μm. d Relative ROS generation measured 1 h after light exposure, with increasing PCT (TPCS 2a + light) illumination doses (30–120 s). The graph shows normalized median DCF fluorescence intensities representative of three individual experiments. e RNA levels (relative quantity) of SOD1 and SOD2 in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines ( P

    Techniques Used: Concentration Assay, Incubation, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Standard Deviation

    Altered expression and localization of the lysosomal marker LAMP1 in the 5-FUR cell lines. a RNA levels (relative quantity) of LAMP1 in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines ( P
    Figure Legend Snippet: Altered expression and localization of the lysosomal marker LAMP1 in the 5-FUR cell lines. a RNA levels (relative quantity) of LAMP1 in all cell lines, as measured by RT-qPCR. Error bars represent standard deviation. Statistically significant difference between 5-FU sensitive and 5-FU resistant lines ( P

    Techniques Used: Expressing, Marker, Quantitative RT-PCR, Standard Deviation

    17) Product Images from "Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells"

    Article Title: Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177069

    Time course evolution of glutamate transporters and differentiation markers in SVZ NPCs primary culture 1–20 days after differentiation. A-B) Time course evolution of of VGLUTs (E) and EAAT (F) protein expression in NPCs: Quantitative analysis. a-b) Representative images of Western blot experiments of VGLUT1-3 (a) and EAAT1-3 (b). C-D) Time course evolution of of VGLUTs (C) and EAAT (D) mRNA expression in NPCs E-F). Time course evolution of mRNA expression of specific neurogenic markers: Nestin, MAP2, SOX2 and TLX (E) and DCX, B3T, NCAM and GFAP (F). A clear reduction in Nestin mRNA expression is evident along with increased MAP2, SOX2, TLX (E) and DCX, B3T, NCAM, GFAP (F) mRNA expression. Western results were normalized to β-actin and are mean ± SEM of values obtained in four experiments, each one performed by duplicate. Statistics compare the expression at indicated times against control 4 h after seeding.* p
    Figure Legend Snippet: Time course evolution of glutamate transporters and differentiation markers in SVZ NPCs primary culture 1–20 days after differentiation. A-B) Time course evolution of of VGLUTs (E) and EAAT (F) protein expression in NPCs: Quantitative analysis. a-b) Representative images of Western blot experiments of VGLUT1-3 (a) and EAAT1-3 (b). C-D) Time course evolution of of VGLUTs (C) and EAAT (D) mRNA expression in NPCs E-F). Time course evolution of mRNA expression of specific neurogenic markers: Nestin, MAP2, SOX2 and TLX (E) and DCX, B3T, NCAM and GFAP (F). A clear reduction in Nestin mRNA expression is evident along with increased MAP2, SOX2, TLX (E) and DCX, B3T, NCAM, GFAP (F) mRNA expression. Western results were normalized to β-actin and are mean ± SEM of values obtained in four experiments, each one performed by duplicate. Statistics compare the expression at indicated times against control 4 h after seeding.* p

    Techniques Used: Expressing, Western Blot

    Evans Blue (EB) effect on VGLUT and EAAT expression at the protein (A-D) and the mRNA level (E-F) and on mRNA expression of neurogenic markers (G) in SVZ NPCs in primary culture. Cells were acutely (24 h EB + 3 dd) or chronically (4 dd) treated with EB at indicated concentrations and then analyzed by RTqPCR for mRNA expression. A-B) Representative Westerns blots of VGLUTs (A) and EAATs (B) and quantification normalized to β-actin (C-D). (E-G). Graphs show data of VGLUTs (E), EAATs (F) or DCX, GFAP, MAP2 and NCAM (G) mRNA expression. Data are means ± SEM of three experiments each one performed by duplicate in different cultures. Statistical significances against controls were performed by One Way ANOVA followed by Tukey post hoc test, when analysis of variance was significant. (*) P
    Figure Legend Snippet: Evans Blue (EB) effect on VGLUT and EAAT expression at the protein (A-D) and the mRNA level (E-F) and on mRNA expression of neurogenic markers (G) in SVZ NPCs in primary culture. Cells were acutely (24 h EB + 3 dd) or chronically (4 dd) treated with EB at indicated concentrations and then analyzed by RTqPCR for mRNA expression. A-B) Representative Westerns blots of VGLUTs (A) and EAATs (B) and quantification normalized to β-actin (C-D). (E-G). Graphs show data of VGLUTs (E), EAATs (F) or DCX, GFAP, MAP2 and NCAM (G) mRNA expression. Data are means ± SEM of three experiments each one performed by duplicate in different cultures. Statistical significances against controls were performed by One Way ANOVA followed by Tukey post hoc test, when analysis of variance was significant. (*) P

    Techniques Used: Expressing

    18) Product Images from "A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays"

    Article Title: A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6329

    Gene expression analysis using RT-PCR and RT-qPCR. Several identified genes from the FOXA1:ESR1 overlapping cluster were examined following ectopic FOXA1 expression in ESR -positive MCF7 and T47D cell lines at 24 h post-transfection. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (A) Gene expression of FOXA1:ESR1 overlapping genes using RT-PCR. (B) Gene expression of FOXA1:ESR1 overlapping genes using RT-qPCR. (C) FOXA1 overexpression following FOXA1 ectopic expression, as determined by RT-qPCR. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. the control. ns, not significant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCL2 , B-cell lymphoma 2; PGR , progesterone receptor; GATA3 , GATA binding protein 3; FOXA1 , forkhead box protein A1; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; CTRL, control; OE, overexpression; PS 2, trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.
    Figure Legend Snippet: Gene expression analysis using RT-PCR and RT-qPCR. Several identified genes from the FOXA1:ESR1 overlapping cluster were examined following ectopic FOXA1 expression in ESR -positive MCF7 and T47D cell lines at 24 h post-transfection. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (A) Gene expression of FOXA1:ESR1 overlapping genes using RT-PCR. (B) Gene expression of FOXA1:ESR1 overlapping genes using RT-qPCR. (C) FOXA1 overexpression following FOXA1 ectopic expression, as determined by RT-qPCR. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. the control. ns, not significant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCL2 , B-cell lymphoma 2; PGR , progesterone receptor; GATA3 , GATA binding protein 3; FOXA1 , forkhead box protein A1; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; CTRL, control; OE, overexpression; PS 2, trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Electron Paramagnetic Resonance, Transfection, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay

    Quantification of target genes regulated by FOXA1 and FOXA / ESR1 extracted from multi-array analysis. RT-qPCR was performed from FOXA1 and FOXA1 / ESR1 -transfected samples in the T47D cell line. (A) Overexpression of FOXA1 and ESR1 was confirmed by RT-qPCR in FOXA1 and FOXA1 / ESR1 -co-transfected cells. (B) Effect of FOXA1 and FOXA1 / ESR1 transfection on the target genes ( CMYB , B-cell lymphoma 2, SIAH2 and PS2 ) at 24 h post-transient transfection in T47D cells. The bar diagram represents data derived from triplicate experiments. *P≤0.05, **P≤0.001, ***P≤0.001, ****P≤0.0001. CTRL, control; FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpression; PS2 , trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.
    Figure Legend Snippet: Quantification of target genes regulated by FOXA1 and FOXA / ESR1 extracted from multi-array analysis. RT-qPCR was performed from FOXA1 and FOXA1 / ESR1 -transfected samples in the T47D cell line. (A) Overexpression of FOXA1 and ESR1 was confirmed by RT-qPCR in FOXA1 and FOXA1 / ESR1 -co-transfected cells. (B) Effect of FOXA1 and FOXA1 / ESR1 transfection on the target genes ( CMYB , B-cell lymphoma 2, SIAH2 and PS2 ) at 24 h post-transient transfection in T47D cells. The bar diagram represents data derived from triplicate experiments. *P≤0.05, **P≤0.001, ***P≤0.001, ****P≤0.0001. CTRL, control; FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpression; PS2 , trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.

    Techniques Used: Quantitative RT-PCR, Transfection, Over Expression, Derivative Assay, Real-time Polymerase Chain Reaction

    Effect of FOXA1 and ESR1 on the PS2 promoter. For ChIP assay, MCF7 cells in the absence or presence of estradiol stimulation were sonicated, lysed and pre-cleared. ChIP with specific antibodies against FOXA1 and ESR1 along with corresponding control Ig G was performed. The nucleotide positions −573 to −315 and −506 to −344 represent the ERE and FOXA1 binding sequences, respectively, in the PS2 promoter. The eluted ChIP DNA samples were subjected to PCR analysis using ERE or FOXA1 site-specific primers. The PCR samples were electrophoresed in a 2% agarose gel. E2, estradiol; Ntd, nucleotide; IP, immunoprecipitation; IgG, immunoglobulin G; FOXA1 , forkhead box protein A1; ERE, estrogen response element; ESR1, estrogen receptor 1; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; PS2 , trefoil factor 1.
    Figure Legend Snippet: Effect of FOXA1 and ESR1 on the PS2 promoter. For ChIP assay, MCF7 cells in the absence or presence of estradiol stimulation were sonicated, lysed and pre-cleared. ChIP with specific antibodies against FOXA1 and ESR1 along with corresponding control Ig G was performed. The nucleotide positions −573 to −315 and −506 to −344 represent the ERE and FOXA1 binding sequences, respectively, in the PS2 promoter. The eluted ChIP DNA samples were subjected to PCR analysis using ERE or FOXA1 site-specific primers. The PCR samples were electrophoresed in a 2% agarose gel. E2, estradiol; Ntd, nucleotide; IP, immunoprecipitation; IgG, immunoglobulin G; FOXA1 , forkhead box protein A1; ERE, estrogen response element; ESR1, estrogen receptor 1; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; PS2 , trefoil factor 1.

    Techniques Used: Chromatin Immunoprecipitation, Sonication, Binding Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunoprecipitation

    Analysis of overlapping FOXA1 and ESR1 co-expression genes. Venn diagrams depicting genes overlapping with FOXA1 and ESR1 with a cut-off frequency of (A) 4 (~16%) and (B) 5 (~20%) by meta-analysis with Oncomine™. (C) Pie chart of functional categories for FOXA1:ESR1 overlapping genes with a cut-off frequency of ≥5 studies. The Oncomine™ data analyzed consisted of 4 normal and 20 breast cancer microarrays data sets. FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1.
    Figure Legend Snippet: Analysis of overlapping FOXA1 and ESR1 co-expression genes. Venn diagrams depicting genes overlapping with FOXA1 and ESR1 with a cut-off frequency of (A) 4 (~16%) and (B) 5 (~20%) by meta-analysis with Oncomine™. (C) Pie chart of functional categories for FOXA1:ESR1 overlapping genes with a cut-off frequency of ≥5 studies. The Oncomine™ data analyzed consisted of 4 normal and 20 breast cancer microarrays data sets. FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1.

    Techniques Used: Expressing, Functional Assay

    Analysis of overlapping FOXA1 , ESR1 and GATA 3 co-expression genes. (A) Pie chart of FOXA1 , ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. (B) Pathway pie chart of FOXA1 , ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. The FOXA1 and ESR1 Oncomine™ microarray analysis consisted of 4 normal and 20 breast cancer microarray data. The GATA 3 microarray data was extracted from published data by Wilson and Giguère ( 31 ). ESR1 , estrogen receptor 1; FOXA1 , forkhead box protein A1; GATA3 , GATA binding protein 3.
    Figure Legend Snippet: Analysis of overlapping FOXA1 , ESR1 and GATA 3 co-expression genes. (A) Pie chart of FOXA1 , ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. (B) Pathway pie chart of FOXA1 , ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. The FOXA1 and ESR1 Oncomine™ microarray analysis consisted of 4 normal and 20 breast cancer microarray data. The GATA 3 microarray data was extracted from published data by Wilson and Giguère ( 31 ). ESR1 , estrogen receptor 1; FOXA1 , forkhead box protein A1; GATA3 , GATA binding protein 3.

    Techniques Used: Expressing, Microarray, Binding Assay

    19) Product Images from "A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays"

    Article Title: A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6329

    Gene expression analysis using RT-PCR and RT-qPCR. Several identified genes from the FOXA1:ESR1 overlapping cluster were examined following ectopic FOXA1 expression in ESR -positive MCF7 and T47D cell lines at 24 h post-transfection. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (A) Gene expression of FOXA1:ESR1 overlapping genes using RT-PCR. (B) Gene expression of FOXA1:ESR1 overlapping genes using RT-qPCR. (C) FOXA1 overexpression following FOXA1 ectopic expression, as determined by RT-qPCR. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. the control. ns, not significant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCL2 , B-cell lymphoma 2; PGR , progesterone receptor; GATA3 , GATA binding protein 3; FOXA1 , forkhead box protein A1; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; CTRL, control; OE, overexpression; PS 2, trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.
    Figure Legend Snippet: Gene expression analysis using RT-PCR and RT-qPCR. Several identified genes from the FOXA1:ESR1 overlapping cluster were examined following ectopic FOXA1 expression in ESR -positive MCF7 and T47D cell lines at 24 h post-transfection. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (A) Gene expression of FOXA1:ESR1 overlapping genes using RT-PCR. (B) Gene expression of FOXA1:ESR1 overlapping genes using RT-qPCR. (C) FOXA1 overexpression following FOXA1 ectopic expression, as determined by RT-qPCR. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. the control. ns, not significant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCL2 , B-cell lymphoma 2; PGR , progesterone receptor; GATA3 , GATA binding protein 3; FOXA1 , forkhead box protein A1; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; CTRL, control; OE, overexpression; PS 2, trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Electron Paramagnetic Resonance, Transfection, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay

    Quantification of target genes regulated by FOXA1 and FOXA / ESR1 extracted from multi-array analysis. RT-qPCR was performed from FOXA1 and FOXA1 / ESR1 -transfected samples in the T47D cell line. (A) Overexpression of FOXA1 and ESR1 was confirmed by RT-qPCR in FOXA1 and FOXA1 / ESR1 -co-transfected cells. (B) Effect of FOXA1 and FOXA1 / ESR1 transfection on the target genes ( CMYB , B-cell lymphoma 2, SIAH2 and PS2 ) at 24 h post-transient transfection in T47D cells. The bar diagram represents data derived from triplicate experiments. *P≤0.05, **P≤0.001, ***P≤0.001, ****P≤0.0001. CTRL, control; FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpression; PS2 , trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.
    Figure Legend Snippet: Quantification of target genes regulated by FOXA1 and FOXA / ESR1 extracted from multi-array analysis. RT-qPCR was performed from FOXA1 and FOXA1 / ESR1 -transfected samples in the T47D cell line. (A) Overexpression of FOXA1 and ESR1 was confirmed by RT-qPCR in FOXA1 and FOXA1 / ESR1 -co-transfected cells. (B) Effect of FOXA1 and FOXA1 / ESR1 transfection on the target genes ( CMYB , B-cell lymphoma 2, SIAH2 and PS2 ) at 24 h post-transient transfection in T47D cells. The bar diagram represents data derived from triplicate experiments. *P≤0.05, **P≤0.001, ***P≤0.001, ****P≤0.0001. CTRL, control; FOXA1 , forkhead box protein A1; ESR1 , estrogen receptor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpression; PS2 , trefoil factor 1; SIAH2 , seven in absentia homolog 2; CMYB , cellular myeloblastosis viral oncogene homolog.

    Techniques Used: Quantitative RT-PCR, Transfection, Over Expression, Derivative Assay, Real-time Polymerase Chain Reaction

    20) Product Images from "Bone Marrow CD133+ Stem Cells Ameliorate Visual Dysfunction in Streptozotocin-induced Diabetic Mice with Early Diabetic Retinopathy"

    Article Title: Bone Marrow CD133+ Stem Cells Ameliorate Visual Dysfunction in Streptozotocin-induced Diabetic Mice with Early Diabetic Retinopathy

    Journal: Cell Transplantation

    doi: 10.1177/0963689718759463

    Isolation and characterization of mouse bone marrow-derived CD133 + stem cells. (A, B) Flow cytometry analysis of the ratio of CD133 + cells (red) (A) before isolation in whole BMNCs and (B) the purity after isolation, compared with isotype control (blue) ( n =6). (C) Immunofluorescent staining showed freshly isolated CD133 + cells (defined as passage 0; P0) were positive for CD133 (green). DAPI (blue) was used to visualize cell nuclei. (D) Flow cytometry analysis displayed expression of (D1) CD45 (90.17±4.01%, n =4), (D2) CD117 (75.21±2.61%, n =3), (D3) CD34 (24.26±21.43, n =3), (D4) CD184 (6.04±6.093%, n =3), (D5) CD31 (24.59±5.60%, n =3), (D6) CD309 (11.53±1.42%, n =3), (D7) CD90 (1.91±1.30%, n =4), and (D8) MHC II (8.33±5.65%, n =3) in the freshly isolated CD133 + cells after gated CD133 + cells. The isotype control is represented by blue while a shift was represented by red. (E) Plots summarizing the changes over time of phenotypic markers above including (E1) CD133, (E2) CD45, CD117, CD34, CD184, (E3) CD31, CD309, CD90 and MHC II in freshly isolated (P0) and cultured CD133 + cells passaged three (P3) and six (P6) times. (F) Phase-contrast images showing the typical morphology changes of cultured CD133 + cells from (F1) P0, (F2) P3, and (F3) P6. (G) Representative line graph showing population doubling time plotted as the average days needed for the growth of CD133 + cells to next generation. (H) In vitro, CD133 + cells cultured in differentiation media for 14 days (H1) and stained with neural marker (H2) βIII-tubulin (Tuj1; red) and (H3) glial marker GFAP (red) and counterstained with DAPI (blue). (I) Gene expression profiles of neurotrophic factors in CD133 + cells compared with CD133 – cells and BMNCs by RT-qPCR, including (I1) NGF, (I2) BDNF, (I3) GDNF, and (I4) CNTF ( n =3). Each mRNA expression of CD133 + cells and CD133 – cells was normalized to the mRNA expression level in BMNCs. The expression level was gated by the black line and represented by mean percentage ± SEM (A, B, D). The plot was generated by mean percentage ± SEM (E, G, I). Statistical analysis: one-way analysis of variance followed by Tukey’s multiple comparisons test for (I). * P
    Figure Legend Snippet: Isolation and characterization of mouse bone marrow-derived CD133 + stem cells. (A, B) Flow cytometry analysis of the ratio of CD133 + cells (red) (A) before isolation in whole BMNCs and (B) the purity after isolation, compared with isotype control (blue) ( n =6). (C) Immunofluorescent staining showed freshly isolated CD133 + cells (defined as passage 0; P0) were positive for CD133 (green). DAPI (blue) was used to visualize cell nuclei. (D) Flow cytometry analysis displayed expression of (D1) CD45 (90.17±4.01%, n =4), (D2) CD117 (75.21±2.61%, n =3), (D3) CD34 (24.26±21.43, n =3), (D4) CD184 (6.04±6.093%, n =3), (D5) CD31 (24.59±5.60%, n =3), (D6) CD309 (11.53±1.42%, n =3), (D7) CD90 (1.91±1.30%, n =4), and (D8) MHC II (8.33±5.65%, n =3) in the freshly isolated CD133 + cells after gated CD133 + cells. The isotype control is represented by blue while a shift was represented by red. (E) Plots summarizing the changes over time of phenotypic markers above including (E1) CD133, (E2) CD45, CD117, CD34, CD184, (E3) CD31, CD309, CD90 and MHC II in freshly isolated (P0) and cultured CD133 + cells passaged three (P3) and six (P6) times. (F) Phase-contrast images showing the typical morphology changes of cultured CD133 + cells from (F1) P0, (F2) P3, and (F3) P6. (G) Representative line graph showing population doubling time plotted as the average days needed for the growth of CD133 + cells to next generation. (H) In vitro, CD133 + cells cultured in differentiation media for 14 days (H1) and stained with neural marker (H2) βIII-tubulin (Tuj1; red) and (H3) glial marker GFAP (red) and counterstained with DAPI (blue). (I) Gene expression profiles of neurotrophic factors in CD133 + cells compared with CD133 – cells and BMNCs by RT-qPCR, including (I1) NGF, (I2) BDNF, (I3) GDNF, and (I4) CNTF ( n =3). Each mRNA expression of CD133 + cells and CD133 – cells was normalized to the mRNA expression level in BMNCs. The expression level was gated by the black line and represented by mean percentage ± SEM (A, B, D). The plot was generated by mean percentage ± SEM (E, G, I). Statistical analysis: one-way analysis of variance followed by Tukey’s multiple comparisons test for (I). * P

    Techniques Used: Isolation, Derivative Assay, Flow Cytometry, Cytometry, Staining, Expressing, Cell Culture, In Vitro, Marker, Quantitative RT-PCR, Generated

    Transplanted CD133 + cells suppress inner retinal thinning in STZ-induced diabetic mice with early DR. (A) Representative images of retina from vehicle, STZ-untreated, STZ+CD133 + , and STZ+PBS group and (B) statistical histograms of thickness of TR, IR and individual retina layers, and number of cells in GCL per 100 μm. CD133 + cells prevented thinning of layers in diabetic mice, especially IR, but not ONL. Values expressed as mean ± SEM (B) ( n =4 eyes per group). Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (B). Compared with vehicle group: * P
    Figure Legend Snippet: Transplanted CD133 + cells suppress inner retinal thinning in STZ-induced diabetic mice with early DR. (A) Representative images of retina from vehicle, STZ-untreated, STZ+CD133 + , and STZ+PBS group and (B) statistical histograms of thickness of TR, IR and individual retina layers, and number of cells in GCL per 100 μm. CD133 + cells prevented thinning of layers in diabetic mice, especially IR, but not ONL. Values expressed as mean ± SEM (B) ( n =4 eyes per group). Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (B). Compared with vehicle group: * P

    Techniques Used: Mouse Assay

    Neurotrophic potential of CD133 + cells in STZ-induced diabetic retina. (A, B) BDNF staining among four groups on Post-D28 and Post-D56 (A) and relative quantification of BDNF level (B). (C) A subset of transplanted CD133 + cells (green) was localized in VC and GCL and expressed BDNF (overlay; yellow) in STZ+CD133 + retina. Values expressed as mean ± SEM ( n =5 per group). White arrows indicate EGFP + transplanted cells and white arrowheads indicate the overlay cells in (A, C). Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparisons test for (B). * P
    Figure Legend Snippet: Neurotrophic potential of CD133 + cells in STZ-induced diabetic retina. (A, B) BDNF staining among four groups on Post-D28 and Post-D56 (A) and relative quantification of BDNF level (B). (C) A subset of transplanted CD133 + cells (green) was localized in VC and GCL and expressed BDNF (overlay; yellow) in STZ+CD133 + retina. Values expressed as mean ± SEM ( n =5 per group). White arrows indicate EGFP + transplanted cells and white arrowheads indicate the overlay cells in (A, C). Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparisons test for (B). * P

    Techniques Used: Staining

    Transplanted CD133 + cells survive and migrate in STZ-induced diabetic retina with early DR. (A) Chronological diagram of design for animal grouping: 8-week-old C57BL/6 adult male mice were injected i.p. with either 150 mg/kg STZ (STZ group) or citrate buffer (vehicle group). Eyes of STZ mice were further assigned into STZ+133 + group, STZ+PBS group, and STZ-untreated group. The day injected was regarded as D0. The day of D28 before transplantation was regarded as Pre-D0. The day on the 28th and 56th day after transplantation were regarded as Post-D28 and Post-D56, respectively. Animal experiments were performed on D28 (Pre-D0), D56 (Post-D28) and D84 (Post-D56). (B) Line graphs showing the FBG levels and weight changes within experimental time comparing STZ diabetic mice (blue line) with vehicle mice (red line) within experimental time ( n =10 mice per group for each time point). (C) Cultured P3 CD133 + cells were labeled with EGFP (green) before transplantation. Representative images showing green fluorescence labeled cells under the (C1) phase-contrast, (C2) EGFP, and (C3) merge images after incubation with EGFP-loaded lentiviruses for 5 days. (C4) Flow cytometry analysis showing the purity of EGFP + CD133 + cells (green) compared with non-labeled CD133 + cells (gray). (D, E) Representative images of survival and migration of intravitreally transplanted EGFP-labeled D133 + cells (green) from (D) STZ+CD133 + group retina compared with (E) STZ+PBS retina on the 28th (Post-D28) and 56th (Post-D56) day after transplantation ( n =4 eyes per group). (D1–D3) Zoom-in images in (B) display transplanted CD133 + (EGFP + ; green) cells mainly (D1) located in VC and (D2, D3) some of them migrated into GCL and IR. *, # and † represent the location of images taken and zoom-in in (D1), (D2) and (D3), respectively. (F) Histogram showing the number of CD133 + cells (EGFP + ; green) found on Post–D28 and Post–D56 in the retina. (G) Histogram showing the percentage of EGFP + cells found in VC, GCL, IPL, and INL compared with total EGFP + cells on Post-D28 and Post-D56, respectively. The expression level in (C4) is gated by the black line and represented by mean percentage ± SEM. Values expressed as mean ± SEM. White arrows indicate transplanted cells. Statistical analysis: Student’s t test for (B, F). **: P
    Figure Legend Snippet: Transplanted CD133 + cells survive and migrate in STZ-induced diabetic retina with early DR. (A) Chronological diagram of design for animal grouping: 8-week-old C57BL/6 adult male mice were injected i.p. with either 150 mg/kg STZ (STZ group) or citrate buffer (vehicle group). Eyes of STZ mice were further assigned into STZ+133 + group, STZ+PBS group, and STZ-untreated group. The day injected was regarded as D0. The day of D28 before transplantation was regarded as Pre-D0. The day on the 28th and 56th day after transplantation were regarded as Post-D28 and Post-D56, respectively. Animal experiments were performed on D28 (Pre-D0), D56 (Post-D28) and D84 (Post-D56). (B) Line graphs showing the FBG levels and weight changes within experimental time comparing STZ diabetic mice (blue line) with vehicle mice (red line) within experimental time ( n =10 mice per group for each time point). (C) Cultured P3 CD133 + cells were labeled with EGFP (green) before transplantation. Representative images showing green fluorescence labeled cells under the (C1) phase-contrast, (C2) EGFP, and (C3) merge images after incubation with EGFP-loaded lentiviruses for 5 days. (C4) Flow cytometry analysis showing the purity of EGFP + CD133 + cells (green) compared with non-labeled CD133 + cells (gray). (D, E) Representative images of survival and migration of intravitreally transplanted EGFP-labeled D133 + cells (green) from (D) STZ+CD133 + group retina compared with (E) STZ+PBS retina on the 28th (Post-D28) and 56th (Post-D56) day after transplantation ( n =4 eyes per group). (D1–D3) Zoom-in images in (B) display transplanted CD133 + (EGFP + ; green) cells mainly (D1) located in VC and (D2, D3) some of them migrated into GCL and IR. *, # and † represent the location of images taken and zoom-in in (D1), (D2) and (D3), respectively. (F) Histogram showing the number of CD133 + cells (EGFP + ; green) found on Post–D28 and Post–D56 in the retina. (G) Histogram showing the percentage of EGFP + cells found in VC, GCL, IPL, and INL compared with total EGFP + cells on Post-D28 and Post-D56, respectively. The expression level in (C4) is gated by the black line and represented by mean percentage ± SEM. Values expressed as mean ± SEM. White arrows indicate transplanted cells. Statistical analysis: Student’s t test for (B, F). **: P

    Techniques Used: Mouse Assay, Injection, Transplantation Assay, Cell Culture, Labeling, Fluorescence, Incubation, Flow Cytometry, Cytometry, Migration, Expressing

    Transplanted CD133 + cells delay neuronal degeneration in IR in STZ-induced diabetic mice. (A, B, F, G) Representative images showing the rescue of RBCs stained with protein kinase C alpha (PKCα) (red), and RGCs with Tuj1 (red) on Post-D28 and Post-D56 in STZ+CD133 + group mice, respectively (A3, B3, F3, G3), compared with vehicle (A1, B1, F1, G1), STZ-untreated (A2, B2, F2, G2), and STZ+PBS group (A4, B4, F4, G4). Images were taken at the same location among retinas from four groups to make reliable comparisons. (C, H) Histogram showing the number (per 100 μm), dendrite length, and axon length of PKCα + cells in retina from four groups. Related Western blotting is shown in (D, I) and statistical optical density ratio analysis compared with vehicle group (the average value set as 1) in (E, J). β-actin was used as an internal control. Images were representative for at least five images per eye ( n =4 eyes per group). Values expressed as mean ± SEM ( n =3 eyes per group). DAPI stained the nuclei. Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (E, J, C, H). Compared with vehicle group: * P
    Figure Legend Snippet: Transplanted CD133 + cells delay neuronal degeneration in IR in STZ-induced diabetic mice. (A, B, F, G) Representative images showing the rescue of RBCs stained with protein kinase C alpha (PKCα) (red), and RGCs with Tuj1 (red) on Post-D28 and Post-D56 in STZ+CD133 + group mice, respectively (A3, B3, F3, G3), compared with vehicle (A1, B1, F1, G1), STZ-untreated (A2, B2, F2, G2), and STZ+PBS group (A4, B4, F4, G4). Images were taken at the same location among retinas from four groups to make reliable comparisons. (C, H) Histogram showing the number (per 100 μm), dendrite length, and axon length of PKCα + cells in retina from four groups. Related Western blotting is shown in (D, I) and statistical optical density ratio analysis compared with vehicle group (the average value set as 1) in (E, J). β-actin was used as an internal control. Images were representative for at least five images per eye ( n =4 eyes per group). Values expressed as mean ± SEM ( n =3 eyes per group). DAPI stained the nuclei. Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (E, J, C, H). Compared with vehicle group: * P

    Techniques Used: Mouse Assay, Staining, Western Blot

    Intravitreal transplantation of CD133 + cells prevents early visual dysfunction in STZ-induced diabetic mice. (A, B) Transplantation of CD133 + cells improved scotopic ERG responses in diabetic mice especially the b-wave and the sum of oscillatory potentials (∑OPs) up to Post-D56 in diabetic retina. (A) Representative images of scotopic ERG on Pre-D0, Post-D28, and Post-D56 at flash intensity of 0.5 log (cd·s·m −2 ; 0dB). Upper: ERGs. Bottom: OPs. Red dashed line: vehicle; blue dashed line: STZ-untreated; magenta line: STZ+CD133 + ; black dashed line: STZ+PBS. (B) Corresponding statistic graphs displaying the amplitude of (B1) a-wave, (B2) b-wave, and (B3) ∑OPs. (C, D) Scotopic optomotor responses assessed by visual acuity via optomotor illustrated in (C) was improved in STZ+CD133 + group up to Post-D56, compared with the other three groups. (D) Measurement of visual acuity from four groups plotted as a histogram. Number of vehicle (red) versus STZ-untreated (blue) versus STZ+CD133 + (magenta)/PBS (black) eyes: Pre-P0: n =10 versus 10, Post-D28: n =2 versus 12 versus 12, Post-D56: n =10 versus 10 versus 10. Values expressed as mean ± SEM. Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (B) and (D). Compared with vehicle group: * P
    Figure Legend Snippet: Intravitreal transplantation of CD133 + cells prevents early visual dysfunction in STZ-induced diabetic mice. (A, B) Transplantation of CD133 + cells improved scotopic ERG responses in diabetic mice especially the b-wave and the sum of oscillatory potentials (∑OPs) up to Post-D56 in diabetic retina. (A) Representative images of scotopic ERG on Pre-D0, Post-D28, and Post-D56 at flash intensity of 0.5 log (cd·s·m −2 ; 0dB). Upper: ERGs. Bottom: OPs. Red dashed line: vehicle; blue dashed line: STZ-untreated; magenta line: STZ+CD133 + ; black dashed line: STZ+PBS. (B) Corresponding statistic graphs displaying the amplitude of (B1) a-wave, (B2) b-wave, and (B3) ∑OPs. (C, D) Scotopic optomotor responses assessed by visual acuity via optomotor illustrated in (C) was improved in STZ+CD133 + group up to Post-D56, compared with the other three groups. (D) Measurement of visual acuity from four groups plotted as a histogram. Number of vehicle (red) versus STZ-untreated (blue) versus STZ+CD133 + (magenta)/PBS (black) eyes: Pre-P0: n =10 versus 10, Post-D28: n =2 versus 12 versus 12, Post-D56: n =10 versus 10 versus 10. Values expressed as mean ± SEM. Statistical analysis: One-way analysis of variance followed by Tukey’s multiple comparisons test for (B) and (D). Compared with vehicle group: * P

    Techniques Used: Transplantation Assay, Mouse Assay

    Neural differentiation ability of CD133 + cells in STZ-induced diabetic mice. (A) Representative images showing intravitreal transplantation of CD133 + (EGFP + ; green) cells in diabetic retina were found on Post-D28 and Post-D56, and expressed RGC marker Tuj1 (magenta) but not retina specific RBC marker PKCα (red). (B, C) Corresponding histograms showing the percentage of number of EGFP + Tuj1 + cells versus EGFP + cells (E) in total and (F) in GCL layer on Post-D28 and Post-D56, respectively. (D) Only scatter CD133 + cells expressed GFAP (magenta). DAPI (blue) in (A, D) show the nuclei. Isolectin B4 (IB4) (red) in (D) show the vessel walls. White arrows indicate EGFP + transplanted cells and white arrowheads indicate the overlay cells (A, D). Images were representative of six random fields ( n =3). Values expressed as mean ± SEM ( n =3; B, C). OPN: optic nerve. Scale bars represent 50 μm (D) and 20 μm (A). DAPI: 4’,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; RBC: rod-on bipolar cell; RGC: retinal ganglion cell; SEM: standard error of the mean.
    Figure Legend Snippet: Neural differentiation ability of CD133 + cells in STZ-induced diabetic mice. (A) Representative images showing intravitreal transplantation of CD133 + (EGFP + ; green) cells in diabetic retina were found on Post-D28 and Post-D56, and expressed RGC marker Tuj1 (magenta) but not retina specific RBC marker PKCα (red). (B, C) Corresponding histograms showing the percentage of number of EGFP + Tuj1 + cells versus EGFP + cells (E) in total and (F) in GCL layer on Post-D28 and Post-D56, respectively. (D) Only scatter CD133 + cells expressed GFAP (magenta). DAPI (blue) in (A, D) show the nuclei. Isolectin B4 (IB4) (red) in (D) show the vessel walls. White arrows indicate EGFP + transplanted cells and white arrowheads indicate the overlay cells (A, D). Images were representative of six random fields ( n =3). Values expressed as mean ± SEM ( n =3; B, C). OPN: optic nerve. Scale bars represent 50 μm (D) and 20 μm (A). DAPI: 4’,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; RBC: rod-on bipolar cell; RGC: retinal ganglion cell; SEM: standard error of the mean.

    Techniques Used: Mouse Assay, Transplantation Assay, Marker

    21) Product Images from "Posttranscriptional Suppression of Proto-Oncogene c-fms Expression by Vigilin in Breast Cancer ▿ Expression by Vigilin in Breast Cancer ▿ §"

    Article Title: Posttranscriptional Suppression of Proto-Oncogene c-fms Expression by Vigilin in Breast Cancer ▿ Expression by Vigilin in Breast Cancer ▿ §

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01031-10

    Vigilin abundance affects the distribution of c- fms mRNA on polysomes. Dex-treated BT20 cells were transfected with either control or vigilin shRNA or QC-CMV-FLAG-Vigilin or pCMV-FLAG. Polysome profiles were prepared by sucrose density gradient ultracentrifugation.
    Figure Legend Snippet: Vigilin abundance affects the distribution of c- fms mRNA on polysomes. Dex-treated BT20 cells were transfected with either control or vigilin shRNA or QC-CMV-FLAG-Vigilin or pCMV-FLAG. Polysome profiles were prepared by sucrose density gradient ultracentrifugation.

    Techniques Used: Transfection, shRNA

    22) Product Images from "Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors"

    Article Title: Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors

    Journal: BioMed Research International

    doi: 10.1155/2013/186068

    Expression of mGluR1 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and LY 367385 (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR1 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized against actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mGluR1 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and LY 367385 (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR1 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized against actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Expression of mGluR5 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control rats and rats with EAE at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and MPEP (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR5 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mGluR5 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control rats and rats with EAE at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and MPEP (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR5 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Expression of mRNA of NMDARs (a, c, and e) and protein (b, d, and f) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of NMDA receptors: amantadine (c and d) and memantine (e and f). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. Levels of NMDA mRNAs were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), and (e) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mRNA of NMDARs (a, c, and e) and protein (b, d, and f) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of NMDA receptors: amantadine (c and d) and memantine (e and f). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. Levels of NMDA mRNAs were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), and (e) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    23) Product Images from "PCBP1 depletion promotes tumorigenesis through attenuation of p27Kip1 mRNA stability and translation"

    Article Title: PCBP1 depletion promotes tumorigenesis through attenuation of p27Kip1 mRNA stability and translation

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0840-1

    PCBP1 up-regulates p27 expression by enhancing the mRNA stability and translation. ( a ) Immunoblot of endogenous p27 protein levels upon transcription inhibition or/and proteasome degradation repression. A2780 cells overexpressing GFP-PCBP1 or GFP alone were treated with DMSO, Act D at 0.5 μg/ml for 8 h, MG132 at 20 μM for 4 h, or with their combination for 4 h, and analyzed. Results are representative of at least three independent experiments. ( b ) Immunoblot of endogenous p27 protein levels in A2780 cells with endogenous PCBP1 knockdown. Cells were treated with MG132 as in ( a ) ( c ) Immunoblot of p27 ubiquitin ligase Skp2 and p27 expression in the indicated cells. No evident Skp2 expression alteration is shown. ( d ) Immunoblot of the indicated proteins possibly related to p27 protein expression and cell survival. ( e ) Semi-quantitative RT-PCR analysis of p27 or c-myc mRNA stability in A2780 cells overexpressing PCBP1 or GFP control, on condition of Act D treatment to terminate the novel mRNA transcription. c-myc and GAPDH were used as positive and negative controls. The PCR of p27 were performed with 20 cycles, c-Myc with 25 and GAPDH for 20 cycles, respectively. Relative p27 and c-myc level against GAPDH were further normalized against DMSO treated GFP and shown at the bottom. ( f ) Semi-quantitative RT-PCR analyses of p27 or c-myc mRNA stability in A2780 cells with endogenous PCBP1 knockdown by specific shRNAs as the method in E. * p
    Figure Legend Snippet: PCBP1 up-regulates p27 expression by enhancing the mRNA stability and translation. ( a ) Immunoblot of endogenous p27 protein levels upon transcription inhibition or/and proteasome degradation repression. A2780 cells overexpressing GFP-PCBP1 or GFP alone were treated with DMSO, Act D at 0.5 μg/ml for 8 h, MG132 at 20 μM for 4 h, or with their combination for 4 h, and analyzed. Results are representative of at least three independent experiments. ( b ) Immunoblot of endogenous p27 protein levels in A2780 cells with endogenous PCBP1 knockdown. Cells were treated with MG132 as in ( a ) ( c ) Immunoblot of p27 ubiquitin ligase Skp2 and p27 expression in the indicated cells. No evident Skp2 expression alteration is shown. ( d ) Immunoblot of the indicated proteins possibly related to p27 protein expression and cell survival. ( e ) Semi-quantitative RT-PCR analysis of p27 or c-myc mRNA stability in A2780 cells overexpressing PCBP1 or GFP control, on condition of Act D treatment to terminate the novel mRNA transcription. c-myc and GAPDH were used as positive and negative controls. The PCR of p27 were performed with 20 cycles, c-Myc with 25 and GAPDH for 20 cycles, respectively. Relative p27 and c-myc level against GAPDH were further normalized against DMSO treated GFP and shown at the bottom. ( f ) Semi-quantitative RT-PCR analyses of p27 or c-myc mRNA stability in A2780 cells with endogenous PCBP1 knockdown by specific shRNAs as the method in E. * p

    Techniques Used: Expressing, Inhibition, Activated Clotting Time Assay, Quantitative RT-PCR, Polymerase Chain Reaction

    PCBP1 binds to p27 transcript to increase its expression. ( a ) Identification of the purely PCBP1-bound mRNAs by RNA sequencing. mRNA numbers are indicated as copy number. ( b ) RT-PCR validation of PCBP1-associated p27 mRNA from the precipitated mRNA pools in A. p21 and GAPDH transcripts are respectively used as the positive and negative controls of PCBP1-bound mRNA. ( c ) RT-PCR detection of endogenous p27 mRNA immunoprecipitated by PCBP1-specific antibody. Normal IgG is used as negative control for RIP. c-Myc and GAPDH are used as positive or negative control of PCBP1-bound mRNA, respectively. ( d ) Quantitative RT-PCR detection of p27 mRNA in A2780 cells with overexpressing or silencing endogenous PCBP1. Three independent experiments were carried out and analyzed. Data are shown as means±SD. * p
    Figure Legend Snippet: PCBP1 binds to p27 transcript to increase its expression. ( a ) Identification of the purely PCBP1-bound mRNAs by RNA sequencing. mRNA numbers are indicated as copy number. ( b ) RT-PCR validation of PCBP1-associated p27 mRNA from the precipitated mRNA pools in A. p21 and GAPDH transcripts are respectively used as the positive and negative controls of PCBP1-bound mRNA. ( c ) RT-PCR detection of endogenous p27 mRNA immunoprecipitated by PCBP1-specific antibody. Normal IgG is used as negative control for RIP. c-Myc and GAPDH are used as positive or negative control of PCBP1-bound mRNA, respectively. ( d ) Quantitative RT-PCR detection of p27 mRNA in A2780 cells with overexpressing or silencing endogenous PCBP1. Three independent experiments were carried out and analyzed. Data are shown as means±SD. * p

    Techniques Used: Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Quantitative RT-PCR

    PCBP1 interacts with p27 mRNA 3'-UTR mainly through its KH1 domain. ( a ) Luciferase activities induced by the full-length p27 3'-UTR (construct a) and its serial deletions (construct b-m) in PCBP1 over-expressing cells or the control cells. Top panel shows the schematic diagram of human p27 mRNA. Full-length p27 mRNA 3'-UTR fragment and its deleted mutants were fused downstream of luciferase coding region in pGL3 plasmid. A2780 cells were transiently co-transfected with the indicated constructs with pRL-TK plasmid for 24-48 h and lyzed for enzyme activity measurement. Relative luciferase activity (Firefly/Renilla) was normalized from three independent experiments (mean±SD). ( b ) Immunoblot of p27 protein expression affected by the indicated PCBP1 mutations. A2780 cells transiently transfected with the pEGFP vector (Lane 1) or plasmids coding the wild-type PCBP1 (Lane 2) or its mutations (Lane 3-9) as indicated. The protein band intensity was scanned and measured by Image J software. Relative p27 level against GAPDH is further normalized against GFP and shown at the bottom. Results are representative of at least three independent experiments. ( c ) Effect of PCBP1 KH1 domain to Luciferase activity. A2780 cells were triply co-transfected with luciferase reporter containing full-length p27 3'-UTR (construct a) and plasmids encoding GFP-PCBP1 KH1 mutant, GFP-PCBP1, or GFP with pRL-TK, respectively. 48h after transfection, the enzyme activates were measured as in ( a ) Meanwhile, the reporter plasmid without PCBP1-binding p27 3'-UTR region (construct k), as a negative control, was similarly used for the above triple transfection. Relative luciferase activity (Firefly/Renilla) is presented from three independent experiments (mean±SD). * and ** represent p
    Figure Legend Snippet: PCBP1 interacts with p27 mRNA 3'-UTR mainly through its KH1 domain. ( a ) Luciferase activities induced by the full-length p27 3'-UTR (construct a) and its serial deletions (construct b-m) in PCBP1 over-expressing cells or the control cells. Top panel shows the schematic diagram of human p27 mRNA. Full-length p27 mRNA 3'-UTR fragment and its deleted mutants were fused downstream of luciferase coding region in pGL3 plasmid. A2780 cells were transiently co-transfected with the indicated constructs with pRL-TK plasmid for 24-48 h and lyzed for enzyme activity measurement. Relative luciferase activity (Firefly/Renilla) was normalized from three independent experiments (mean±SD). ( b ) Immunoblot of p27 protein expression affected by the indicated PCBP1 mutations. A2780 cells transiently transfected with the pEGFP vector (Lane 1) or plasmids coding the wild-type PCBP1 (Lane 2) or its mutations (Lane 3-9) as indicated. The protein band intensity was scanned and measured by Image J software. Relative p27 level against GAPDH is further normalized against GFP and shown at the bottom. Results are representative of at least three independent experiments. ( c ) Effect of PCBP1 KH1 domain to Luciferase activity. A2780 cells were triply co-transfected with luciferase reporter containing full-length p27 3'-UTR (construct a) and plasmids encoding GFP-PCBP1 KH1 mutant, GFP-PCBP1, or GFP with pRL-TK, respectively. 48h after transfection, the enzyme activates were measured as in ( a ) Meanwhile, the reporter plasmid without PCBP1-binding p27 3'-UTR region (construct k), as a negative control, was similarly used for the above triple transfection. Relative luciferase activity (Firefly/Renilla) is presented from three independent experiments (mean±SD). * and ** represent p

    Techniques Used: Luciferase, Construct, Expressing, Plasmid Preparation, Transfection, Activity Assay, Software, Mutagenesis, Binding Assay, Negative Control

    24) Product Images from "Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein"

    Article Title: Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2017.01.002

    In Vitro and In Vivo Furin Cleavage of RD114-TRco (A) Western blot analysis of whole-cell proteins (30 μg) extracted from PK-7 cells, transfected with either RD114-TRWT or RD114-TRco plasmid and treated overnight at 16°C with 4 U/sample of recombinant furin. The filter was probed with the anti-TM Ab and, after stripping, with anti-HIV serum as an internal control. (B) Western blot analysis of whole-cell proteins (30 μg) extracted from HEK293T cells transfected with the indicated amounts of plasmid DNA encoding either RD114-TRWT or RD114-TRco. The filter was stained with anti-TM Ab and, after stripping, with anti-β actin Ab as an internal control.
    Figure Legend Snippet: In Vitro and In Vivo Furin Cleavage of RD114-TRco (A) Western blot analysis of whole-cell proteins (30 μg) extracted from PK-7 cells, transfected with either RD114-TRWT or RD114-TRco plasmid and treated overnight at 16°C with 4 U/sample of recombinant furin. The filter was probed with the anti-TM Ab and, after stripping, with anti-HIV serum as an internal control. (B) Western blot analysis of whole-cell proteins (30 μg) extracted from HEK293T cells transfected with the indicated amounts of plasmid DNA encoding either RD114-TRWT or RD114-TRco. The filter was stained with anti-TM Ab and, after stripping, with anti-β actin Ab as an internal control.

    Techniques Used: In Vitro, In Vivo, Western Blot, Transfection, Plasmid Preparation, Recombinant, Stripping Membranes, Staining

    Subcellular Localization of RD114-TRWT and RD114-TRco (A–D) COS-7 cells transfected with pIRES-RD114-TR plasmids were fixed, permeabilized, and stained at room temperature with the Abs as indicated. Endobrevin/VAMP8 is an early and late endosomal marker, and calnexin is an ER marker. Nuclei were stained with DAPI. Scale bar, 40 μm.
    Figure Legend Snippet: Subcellular Localization of RD114-TRWT and RD114-TRco (A–D) COS-7 cells transfected with pIRES-RD114-TR plasmids were fixed, permeabilized, and stained at room temperature with the Abs as indicated. Endobrevin/VAMP8 is an early and late endosomal marker, and calnexin is an ER marker. Nuclei were stained with DAPI. Scale bar, 40 μm.

    Techniques Used: Transfection, Staining, Marker

    RD114-TRWT Expression on RD3-MolPack-GFP Producer Cells (A) PD assay of cell surface-biotinylated proteins followed by western blot analysis. Membrane biotinylation was performed on intact cells. 1 mg of total cell extracts was incubated with 60 μL of streptavidin-conjugated Dynabeads for PD. The PD deglycosylated TM subunit migrated slightly higher than expected because of erroneous migration of the proteins in SDS-PAGE. (B) PNGaseF and EndoH deglycosylations were performed on cellular (30 μg whole-cell extracts) and viral (50 ng p24Gag equivalent) proteins obtained from RD3-MolPack-GFP cells, and their derived viral vectors were collected after 48 hr from cell seeding. Anti-calnexin and anti-ERK staining are internal controls for nonspecific biotinylation and PD; anti-p24Gag staining is for internal loading control. A long exposure of the film is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lane 3) in the vectors. * indicates non-specific staining of the anti-TM Abs recognizing BSA present in the supernatants. ‡ refers to EndoH-resistant SU and TM proteins.
    Figure Legend Snippet: RD114-TRWT Expression on RD3-MolPack-GFP Producer Cells (A) PD assay of cell surface-biotinylated proteins followed by western blot analysis. Membrane biotinylation was performed on intact cells. 1 mg of total cell extracts was incubated with 60 μL of streptavidin-conjugated Dynabeads for PD. The PD deglycosylated TM subunit migrated slightly higher than expected because of erroneous migration of the proteins in SDS-PAGE. (B) PNGaseF and EndoH deglycosylations were performed on cellular (30 μg whole-cell extracts) and viral (50 ng p24Gag equivalent) proteins obtained from RD3-MolPack-GFP cells, and their derived viral vectors were collected after 48 hr from cell seeding. Anti-calnexin and anti-ERK staining are internal controls for nonspecific biotinylation and PD; anti-p24Gag staining is for internal loading control. A long exposure of the film is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lane 3) in the vectors. * indicates non-specific staining of the anti-TM Abs recognizing BSA present in the supernatants. ‡ refers to EndoH-resistant SU and TM proteins.

    Techniques Used: Expressing, Western Blot, Incubation, Migration, SDS Page, Derivative Assay, Staining

    Prediction of RD114-TRWT and RD114-TRco mRNA Secondary Structures (A and B) Schematics of the most thermodynamically stable (lowest free energy value) secondary structures of RD114-TRWT (A) and RD114-TRco (B) mRNA, predicted by the MFOLD software. Data are annotated using color-based characters, indicating the probability of base-pairing from 0 (light blue) to 1 (red), where 1 is the maximum pairing level. (C and D) The 5′ end substructures of 320 bp (C) and 330 bp (D) of RD114-TRWT and RD114-TRco mRNA, respectively, are shown in the insets. (E) Boxplot of RD114-TRWT and RD114-TRco mRNA secondary structure ΔG, calculated by MFOLD software. Statistical analysis was performed by Wilcoxon rank-sum test. Statistical significance was set as ***p
    Figure Legend Snippet: Prediction of RD114-TRWT and RD114-TRco mRNA Secondary Structures (A and B) Schematics of the most thermodynamically stable (lowest free energy value) secondary structures of RD114-TRWT (A) and RD114-TRco (B) mRNA, predicted by the MFOLD software. Data are annotated using color-based characters, indicating the probability of base-pairing from 0 (light blue) to 1 (red), where 1 is the maximum pairing level. (C and D) The 5′ end substructures of 320 bp (C) and 330 bp (D) of RD114-TRWT and RD114-TRco mRNA, respectively, are shown in the insets. (E) Boxplot of RD114-TRWT and RD114-TRco mRNA secondary structure ΔG, calculated by MFOLD software. Statistical analysis was performed by Wilcoxon rank-sum test. Statistical significance was set as ***p

    Techniques Used: Software

    RD114-TRWT and RD114-TRco Expression in Transiently Transfected PK-7 Cells and Generated LVs (A) Western blot analysis of whole-cell proteins (30 μg) obtained from PK-7 cells after transfection of SIN-EGFP TV and envelope plasmids encoding either RD114-TRWT or RD114-TRco, treated or not treated with either PNGaseF or EndoH enzymes and then probed with anti-SU and anti-TM Abs. (B) Vector proteins (160 ng of p24Gag equivalent) obtained from the virions produced by the transfected cells shown in (A), treated or not treated with either PNGaseF or EndoH enzymes, and then probed with anti-SU and anti-TM Abs. Anti-HIV protein staining (p55Gag and p24Gag) was used as an internal control. A long exposure of the films is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lanes 3 and 6). ‡ refers to EndoH-resistant SU and TM proteins.
    Figure Legend Snippet: RD114-TRWT and RD114-TRco Expression in Transiently Transfected PK-7 Cells and Generated LVs (A) Western blot analysis of whole-cell proteins (30 μg) obtained from PK-7 cells after transfection of SIN-EGFP TV and envelope plasmids encoding either RD114-TRWT or RD114-TRco, treated or not treated with either PNGaseF or EndoH enzymes and then probed with anti-SU and anti-TM Abs. (B) Vector proteins (160 ng of p24Gag equivalent) obtained from the virions produced by the transfected cells shown in (A), treated or not treated with either PNGaseF or EndoH enzymes, and then probed with anti-SU and anti-TM Abs. Anti-HIV protein staining (p55Gag and p24Gag) was used as an internal control. A long exposure of the films is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lanes 3 and 6). ‡ refers to EndoH-resistant SU and TM proteins.

    Techniques Used: Expressing, Transfection, Generated, Western Blot, Plasmid Preparation, Produced, Staining

    Northern Blot Analysis of RD114-TRWT and RD114-TRco mRNA (A) Total RNA (5 μg) extracted from PK-7 cells transiently transfected with SIN-eGFP TV and either SIN-RD114-TRWT or SIN-RD114-TRco plasmids was tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively, and the common ψ probe as an internal control. Three bands were detected for both mRNAs, corresponding to the full-length (ψ and specific RD114-TR probes), the spliced, and the internal cassette transcripts, respectively (specific RD114-TR probes). Bottom: ethidium bromide (EtBr) staining of the agarose gel showing 28S and 18S RNAs. (B) Total, nuclear, and cytosolic mRNA (5 μg) extracted from PK-7 cells transiently mock-transfected (mo) and transfected with the pIRES-puro3-based plasmids encoding either RD114-TRWT or RD114-TRco were tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively. A single band derived from the expression cassette was detected for both samples. Bottom: EtBr staining of the agarose gel showing 28S and 18S RNAs. (C) qRT-PCR analysis of the nuclear and cytoplasmic distribution of RD114-TRWT and RD114-TRco mRNA. Nuclear and cytoplasmic mRNA was retro-transcribed, and then qPCR was carried out on the corresponding cDNA using specific primers for RD114-TR WT and co genes and, as internal normalizers, specific primers for the U6 and GAPDH genes. The data were derived from a single qRT-PCR experiment in which each sample was run in sestuplicate.
    Figure Legend Snippet: Northern Blot Analysis of RD114-TRWT and RD114-TRco mRNA (A) Total RNA (5 μg) extracted from PK-7 cells transiently transfected with SIN-eGFP TV and either SIN-RD114-TRWT or SIN-RD114-TRco plasmids was tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively, and the common ψ probe as an internal control. Three bands were detected for both mRNAs, corresponding to the full-length (ψ and specific RD114-TR probes), the spliced, and the internal cassette transcripts, respectively (specific RD114-TR probes). Bottom: ethidium bromide (EtBr) staining of the agarose gel showing 28S and 18S RNAs. (B) Total, nuclear, and cytosolic mRNA (5 μg) extracted from PK-7 cells transiently mock-transfected (mo) and transfected with the pIRES-puro3-based plasmids encoding either RD114-TRWT or RD114-TRco were tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively. A single band derived from the expression cassette was detected for both samples. Bottom: EtBr staining of the agarose gel showing 28S and 18S RNAs. (C) qRT-PCR analysis of the nuclear and cytoplasmic distribution of RD114-TRWT and RD114-TRco mRNA. Nuclear and cytoplasmic mRNA was retro-transcribed, and then qPCR was carried out on the corresponding cDNA using specific primers for RD114-TR WT and co genes and, as internal normalizers, specific primers for the U6 and GAPDH genes. The data were derived from a single qRT-PCR experiment in which each sample was run in sestuplicate.

    Techniques Used: Northern Blot, Transfection, Sequencing, Staining, Agarose Gel Electrophoresis, Derivative Assay, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    25) Product Images from "Vitamin D alleviates lipopolysaccharide-induced acute lung injury via regulation of the renin-angiotensin system"

    Article Title: Vitamin D alleviates lipopolysaccharide-induced acute lung injury via regulation of the renin-angiotensin system

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7546

    Cal inhibits ACE and AT1R expression, and induces ACE2 expression in LPS-treated rat PMVECs. (A) Morphology of rat PMVECs and immunostaining for PMVEC marker FITC-PHA. Phase: Normal PMVEC morphometrics under phase-contrast microscopy (magnification, ×200). The cells grew initially as capillary-like structures and assumed typical cobblestone morphology of endothelial cells at confluence. PHA: PMVECs bound to FITC-PHA under fluorescence microscopy to reveal yellow green fluorescence (magnification, ×400). Reverse transcription-quantitative polymerase chain reaction analysis of (B) ACE and ACE2, and (C) AT1R and AT2R mRNA expression levels in rat PMVECs in various treatment groups. ACE2 is a counter-regulator of ACE and AT1R is a downstream effector of ACE. AT2R was employed as a control. (D) Western blot analysis of the protein levels of ACE, ACE2, AT1R and AT2R in rat PMVECs in various treatment groups. (E) Densitometric analysis of the relative protein expression levels of ACE, ACE2, AT1R and AT2R. Data are presented as the mean + standard deviation of three biological replicates. *P
    Figure Legend Snippet: Cal inhibits ACE and AT1R expression, and induces ACE2 expression in LPS-treated rat PMVECs. (A) Morphology of rat PMVECs and immunostaining for PMVEC marker FITC-PHA. Phase: Normal PMVEC morphometrics under phase-contrast microscopy (magnification, ×200). The cells grew initially as capillary-like structures and assumed typical cobblestone morphology of endothelial cells at confluence. PHA: PMVECs bound to FITC-PHA under fluorescence microscopy to reveal yellow green fluorescence (magnification, ×400). Reverse transcription-quantitative polymerase chain reaction analysis of (B) ACE and ACE2, and (C) AT1R and AT2R mRNA expression levels in rat PMVECs in various treatment groups. ACE2 is a counter-regulator of ACE and AT1R is a downstream effector of ACE. AT2R was employed as a control. (D) Western blot analysis of the protein levels of ACE, ACE2, AT1R and AT2R in rat PMVECs in various treatment groups. (E) Densitometric analysis of the relative protein expression levels of ACE, ACE2, AT1R and AT2R. Data are presented as the mean + standard deviation of three biological replicates. *P

    Techniques Used: Expressing, Immunostaining, Marker, Microscopy, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

    Cal suppresses REN and Ang II levels in LPS-treated rat PMVECs. (A) RT-qPCR analysis of REN and Ang II mRNA expression in rat PMVECs in various treatment groups. (B) Levels of REN and Ang II in the culture medium of rat PMVECs were quantified by ELISA. Data are presented as the mean + standard deviation of three biological replicates. *P
    Figure Legend Snippet: Cal suppresses REN and Ang II levels in LPS-treated rat PMVECs. (A) RT-qPCR analysis of REN and Ang II mRNA expression in rat PMVECs in various treatment groups. (B) Levels of REN and Ang II in the culture medium of rat PMVECs were quantified by ELISA. Data are presented as the mean + standard deviation of three biological replicates. *P

    Techniques Used: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    26) Product Images from "Epigenetic silencing of IRF1 dysregulates type III interferon responses to respiratory virus infection in epithelial to mesenchymal transition"

    Article Title: Epigenetic silencing of IRF1 dysregulates type III interferon responses to respiratory virus infection in epithelial to mesenchymal transition

    Journal: Nature microbiology

    doi: 10.1038/nmicrobiol.2017.86

    IRF1 silencing dysregulates the IFN response in TGFβ-induced EMT a – b , Q-RT-PCR analysis of IRF1 ( a ) and IRF7 ( b ) in hSAECs (Con) and EMT-hSAECs (EMT), stimulated with 50 µg/mL poly(I:C) for 0h, 4h and 6h. c , Western blot analysis of IRF1 and IRF7 in cellular nuclear fractions of hSAECs (Con) and EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 0h, 1h and 3h. LaminB2 was used as a loading control. Shown are representative blots from three experiments. d , Microaffinity capture of poly(I:C)-inducible ISG/DNA-binding proteins. Nuclear extracts from hSAECs (Con) and EMT-hSAECs (EMT) in the absence or presence of poly(I:C) stimulation (50 µg/mL for 3h) were affinity-purified by biotinylated ISG duplex DNA or non-biotinylated competitor, captured by streptavidin beads and probed with the indicated Abs on Western blots. Shown are representative blots from two experiments. e , Confocal immunofluorescence imaging for IRF1 in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3h. The secondary Ab was Alex Fluo 488 (green). Shown is the representative staining from five images. f , XChIP assay for IRF3 and RelA binding to the IRF1 promoter in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3 h or infected with RSV at MOI 0.5 for 15 h. Data were quantified relative to the input signal and shown as fold-change normalized to unstimulated samples (Con). g , XChIP assay of IRF1 binding to the IFNB1 and IFNL1 promoters in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3h. h , XChIP assay of IRF7 binding to IFNB1 and IFNL1 promoters in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL (polyI:C) for 3h. i-j , Q-RT-PCR analysis of MX1 ( i ) and RIG-I ( j ) in control siRNA (EMT-siCon)- or IRF7 siRNA (EMT-siIRF7)- transfected EMT-hSAECs, stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. k – l , Q-RT-PCR analysis of IFNB1 ( k ) and IFNL1 ( l ) in lentiviral IRF1 stably transduced EMT-hSAECs (EMT-IRF1) stimulated with 50 µg/mL poly(I:C) for 0 h, 2 h, 4 h and 6 h. The empty lentiviral-transduced EMT-hSAECs were used as controls (EMT-Con). m , Q-RT-PCR analysis of RV16 viral RNA 5’ UTR in EMT-Con and EMT-IRF1 cells infected with RV16 (MOI = 1) for 0 h, 8 h and 24 h. Data are the mean ± S.D. from n=3 biological replicates.
    Figure Legend Snippet: IRF1 silencing dysregulates the IFN response in TGFβ-induced EMT a – b , Q-RT-PCR analysis of IRF1 ( a ) and IRF7 ( b ) in hSAECs (Con) and EMT-hSAECs (EMT), stimulated with 50 µg/mL poly(I:C) for 0h, 4h and 6h. c , Western blot analysis of IRF1 and IRF7 in cellular nuclear fractions of hSAECs (Con) and EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 0h, 1h and 3h. LaminB2 was used as a loading control. Shown are representative blots from three experiments. d , Microaffinity capture of poly(I:C)-inducible ISG/DNA-binding proteins. Nuclear extracts from hSAECs (Con) and EMT-hSAECs (EMT) in the absence or presence of poly(I:C) stimulation (50 µg/mL for 3h) were affinity-purified by biotinylated ISG duplex DNA or non-biotinylated competitor, captured by streptavidin beads and probed with the indicated Abs on Western blots. Shown are representative blots from two experiments. e , Confocal immunofluorescence imaging for IRF1 in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3h. The secondary Ab was Alex Fluo 488 (green). Shown is the representative staining from five images. f , XChIP assay for IRF3 and RelA binding to the IRF1 promoter in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3 h or infected with RSV at MOI 0.5 for 15 h. Data were quantified relative to the input signal and shown as fold-change normalized to unstimulated samples (Con). g , XChIP assay of IRF1 binding to the IFNB1 and IFNL1 promoters in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL poly(I:C) for 3h. h , XChIP assay of IRF7 binding to IFNB1 and IFNL1 promoters in hSAECs (Con) or EMT-hSAECs (EMT) stimulated with 50 µg/mL (polyI:C) for 3h. i-j , Q-RT-PCR analysis of MX1 ( i ) and RIG-I ( j ) in control siRNA (EMT-siCon)- or IRF7 siRNA (EMT-siIRF7)- transfected EMT-hSAECs, stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. k – l , Q-RT-PCR analysis of IFNB1 ( k ) and IFNL1 ( l ) in lentiviral IRF1 stably transduced EMT-hSAECs (EMT-IRF1) stimulated with 50 µg/mL poly(I:C) for 0 h, 2 h, 4 h and 6 h. The empty lentiviral-transduced EMT-hSAECs were used as controls (EMT-Con). m , Q-RT-PCR analysis of RV16 viral RNA 5’ UTR in EMT-Con and EMT-IRF1 cells infected with RV16 (MOI = 1) for 0 h, 8 h and 24 h. Data are the mean ± S.D. from n=3 biological replicates.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, DNA Binding Assay, Affinity Purification, Immunofluorescence, Imaging, Staining, Binding Assay, Infection, Transfection, Stable Transfection

    CRISPR/Cas9-mediated IRF1 gene knockout a , Western blot analysis of CRISPR/Cas9-mediated IRF1 knockout. hSAECs stably transduced by empty lentiCRISPR (Con) or lentiCRISPRv2 with IRF1 sgRNA (CRISPR) were stimulated with 50 µg/mL poly(I:C) for 0h (−) and 3h (+). Total cell lysates were detected using IRF1 Ab. α-Tubulin blot is shown as the loading control. Shown are representative blots from two experiments. b – c , Q-RT-PCR analysis of IFNB1 ( b ) and IFNL1 ( c ) in cells used in a and stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. d – f , Q-RT-PCR analysis of IFNB1 ( d ), IFNL1 ( e ) and RV16 viral RNA 5’ UTR ( f ) in cells used in a and infected with RV16 for 0h, 8h and 24h. Data are the mean ± S.D. from n=3 biological replicates.
    Figure Legend Snippet: CRISPR/Cas9-mediated IRF1 gene knockout a , Western blot analysis of CRISPR/Cas9-mediated IRF1 knockout. hSAECs stably transduced by empty lentiCRISPR (Con) or lentiCRISPRv2 with IRF1 sgRNA (CRISPR) were stimulated with 50 µg/mL poly(I:C) for 0h (−) and 3h (+). Total cell lysates were detected using IRF1 Ab. α-Tubulin blot is shown as the loading control. Shown are representative blots from two experiments. b – c , Q-RT-PCR analysis of IFNB1 ( b ) and IFNL1 ( c ) in cells used in a and stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. d – f , Q-RT-PCR analysis of IFNB1 ( d ), IFNL1 ( e ) and RV16 viral RNA 5’ UTR ( f ) in cells used in a and infected with RV16 for 0h, 8h and 24h. Data are the mean ± S.D. from n=3 biological replicates.

    Techniques Used: CRISPR, Gene Knockout, Western Blot, Knock-Out, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Infection

    ZEB1 downregulates the IFN response in hSAECs and TGFβ-induced EMT-hSAECs a , Q-RT-PCR analysis of EMT core transcription factors ( SNAIL1 TWIST1 ZEB1 and ZEB2 ) in hSAECs (Con) and EMT-hSAECs (EMT). b , Western blot analysis of ZEB1 and ZEB2, using cellular nuclear exacts from hSAECs (Con) and EMT-hSAECS (EMT) stimulated with 50 µg/mL poly(I:C) for 0h and 3h. LaminB2 was used as the loading control. s.e., short exposure; l.e., long exposure. c , Confocal immunofluorescence imaging for ZEB1 in hSAECs (Con) or EMT-hSAECs (EMT). The secondary Ab was Alex Fluo 488 (green). d , Confocal immunofluorescence staining of ZEB1 in mouse lung from chronic TGFβ-induced fibrosis/EMT. Con, PBS-treated mouse lung; TGFβ, TGFβ—treated mouse lung. Shown is the representative staining from five images. e , Western blot analysis of ZEB1, and α-tubulin as loading control, using total protein exacts from hSAECs stably transduced with Lentiviral ZEB1. The empty lentiviral-transduced hSAECs were used as controls. Shown are representative blots from two experiments. n.s, nonspecific bands. f – g , Q-RT-PCR analysis of IFNB1 ( f ) and IFNL1 ( g ) in Con and ZEB1 cells used in c , stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. h-j , Q-RT-PCR analysis of IFNL1 ( h ), IFNL2 / 3 ( i ) and RV16 viral RNA 5’ UTR ( j ) in Con and ZEB1 cells used in c , infected with RV16 (MOI = 1) for 24h. Data are shown as fold change normalized to unstimulated cells (Con). k , Western blot analysis of ZEB1, and α-tubulin as loading control, using total protein exacts from hSAECs stably transduced with 3 inducible lentiviral ZEB1 shRNAs (shR1, shR2 and shR3). Cells were first induced to EMT by 10 ng/mL of TGFβ for 15 days and then treated with 2 µg/mL doxycycline for 72 h. Cellular samples were treated and collected in biological duplicates. Non-silencing shRNA-transduced cells under the same conditions were used as controls (EMT-Con). Shown are representative blots from two experiments. l – m , Q-RT-PCR analysis of IFNB1 ( l ) and IFNL1 ( m ) in ZEB1 shRNA (shR1)-depleted EMT-hSAECs (same treatment as in k ) stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. Data are the mean ± S.D. from n=3 biological replicates.
    Figure Legend Snippet: ZEB1 downregulates the IFN response in hSAECs and TGFβ-induced EMT-hSAECs a , Q-RT-PCR analysis of EMT core transcription factors ( SNAIL1 TWIST1 ZEB1 and ZEB2 ) in hSAECs (Con) and EMT-hSAECs (EMT). b , Western blot analysis of ZEB1 and ZEB2, using cellular nuclear exacts from hSAECs (Con) and EMT-hSAECS (EMT) stimulated with 50 µg/mL poly(I:C) for 0h and 3h. LaminB2 was used as the loading control. s.e., short exposure; l.e., long exposure. c , Confocal immunofluorescence imaging for ZEB1 in hSAECs (Con) or EMT-hSAECs (EMT). The secondary Ab was Alex Fluo 488 (green). d , Confocal immunofluorescence staining of ZEB1 in mouse lung from chronic TGFβ-induced fibrosis/EMT. Con, PBS-treated mouse lung; TGFβ, TGFβ—treated mouse lung. Shown is the representative staining from five images. e , Western blot analysis of ZEB1, and α-tubulin as loading control, using total protein exacts from hSAECs stably transduced with Lentiviral ZEB1. The empty lentiviral-transduced hSAECs were used as controls. Shown are representative blots from two experiments. n.s, nonspecific bands. f – g , Q-RT-PCR analysis of IFNB1 ( f ) and IFNL1 ( g ) in Con and ZEB1 cells used in c , stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. h-j , Q-RT-PCR analysis of IFNL1 ( h ), IFNL2 / 3 ( i ) and RV16 viral RNA 5’ UTR ( j ) in Con and ZEB1 cells used in c , infected with RV16 (MOI = 1) for 24h. Data are shown as fold change normalized to unstimulated cells (Con). k , Western blot analysis of ZEB1, and α-tubulin as loading control, using total protein exacts from hSAECs stably transduced with 3 inducible lentiviral ZEB1 shRNAs (shR1, shR2 and shR3). Cells were first induced to EMT by 10 ng/mL of TGFβ for 15 days and then treated with 2 µg/mL doxycycline for 72 h. Cellular samples were treated and collected in biological duplicates. Non-silencing shRNA-transduced cells under the same conditions were used as controls (EMT-Con). Shown are representative blots from two experiments. l – m , Q-RT-PCR analysis of IFNB1 ( l ) and IFNL1 ( m ) in ZEB1 shRNA (shR1)-depleted EMT-hSAECs (same treatment as in k ) stimulated with 50 µg/mL poly(I:C) for 0h, 2h, 4h and 6h. Data are the mean ± S.D. from n=3 biological replicates.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Imaging, Staining, Stable Transfection, Transduction, Infection, shRNA

    Deficient type I/III IFN responses in TGFβ-induced EMT a , Confocal immunofluorescence imaging of hSAECs (Con) and EMT-hSAECs (EMT, induced by 10 ng/mL TGFβ treatment for 15 days). Distribution of F-actin, E-Cadherin (E-Cad) and vimentin (VIM) was shown by staining with Alexa 568-conjugated phalloidin or secondary antibody (red); nuclei were counterstained with DAPI (blue). Shown is the representative staining from five images. b – g , Q-RT-PCR analysis of: type I interferons IFNA1 ( b ) and IFNB1 ( c ), type III interferons IFNL1 ( d ) and IFNL2/3 ( e ), and interferon-stimulated genes (ISGs) MX1 ( f ) and RIG-I ( g ) in hSAECs (Con) and EMT-hSAECs (EMT), stimulated by poly IC (50 µg/mL) for 0h, 4h and 6h. The results were quantified relative to the signal of DNA polymerase beta ( POLB ) and shown as fold-change of mRNA abundance normalized to unstimulated samples (Con). h – i , Q-RT-PCR analysis of the interferon-stimulated genes (ISGs) MX1 ( h ) and RIG-I ( i ) in hSAECs (Con) and EMT-hSAECs (EMT) infected by RV16 for 0h, 4h, 6h and 24h. j – k , Q-RT-PCR analysis of relative virus load in hSAECs (Con) and EMT-hSAECs (EMT), infected by RSV ( j ) or RV16 ( k ) for 0h, 12h and 24h. The results are shown as fold changes of RV16 viral RNA 5’ UTR and RSV N viral RNA, respectively. Data are the means ± S.D. from n=3 biological replicates. l , Virus plaque assays of hSAECs (Con) and EMT-hSAECs (EMT) infected by RSV or RV16 for 24h (MOI = 1). Virus plaques were detected in triplicate in HEp-2 (RSV) or H1HeLa cells (RV16), using serial 2-fold dilutions of cell culture supernatants or freeze-thawed cellular lysates, respectively. Plaques were stained at 5 days (RSV) or 9 days (RV16) post-infection. Shown are the representative triplicates at the same dilution. The complete view of these virus plaque data can be seen in Supplementary Figure 1 .
    Figure Legend Snippet: Deficient type I/III IFN responses in TGFβ-induced EMT a , Confocal immunofluorescence imaging of hSAECs (Con) and EMT-hSAECs (EMT, induced by 10 ng/mL TGFβ treatment for 15 days). Distribution of F-actin, E-Cadherin (E-Cad) and vimentin (VIM) was shown by staining with Alexa 568-conjugated phalloidin or secondary antibody (red); nuclei were counterstained with DAPI (blue). Shown is the representative staining from five images. b – g , Q-RT-PCR analysis of: type I interferons IFNA1 ( b ) and IFNB1 ( c ), type III interferons IFNL1 ( d ) and IFNL2/3 ( e ), and interferon-stimulated genes (ISGs) MX1 ( f ) and RIG-I ( g ) in hSAECs (Con) and EMT-hSAECs (EMT), stimulated by poly IC (50 µg/mL) for 0h, 4h and 6h. The results were quantified relative to the signal of DNA polymerase beta ( POLB ) and shown as fold-change of mRNA abundance normalized to unstimulated samples (Con). h – i , Q-RT-PCR analysis of the interferon-stimulated genes (ISGs) MX1 ( h ) and RIG-I ( i ) in hSAECs (Con) and EMT-hSAECs (EMT) infected by RV16 for 0h, 4h, 6h and 24h. j – k , Q-RT-PCR analysis of relative virus load in hSAECs (Con) and EMT-hSAECs (EMT), infected by RSV ( j ) or RV16 ( k ) for 0h, 12h and 24h. The results are shown as fold changes of RV16 viral RNA 5’ UTR and RSV N viral RNA, respectively. Data are the means ± S.D. from n=3 biological replicates. l , Virus plaque assays of hSAECs (Con) and EMT-hSAECs (EMT) infected by RSV or RV16 for 24h (MOI = 1). Virus plaques were detected in triplicate in HEp-2 (RSV) or H1HeLa cells (RV16), using serial 2-fold dilutions of cell culture supernatants or freeze-thawed cellular lysates, respectively. Plaques were stained at 5 days (RSV) or 9 days (RV16) post-infection. Shown are the representative triplicates at the same dilution. The complete view of these virus plaque data can be seen in Supplementary Figure 1 .

    Techniques Used: Immunofluorescence, Imaging, Staining, Reverse Transcription Polymerase Chain Reaction, Infection, Cell Culture

    27) Product Images from "Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension"

    Article Title: Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090544

    The mRNA expression level of Cu transporters (CTR1 and ATP7A) and lysyl oxidase (LOX) is increased in whole-lung and pulmonary artery (PA) tissues of mice with chronically hypoxia-induced pulmonary hypertension (HPH). Whole lung tissues and isolated PA tissue from normoxic (Nor, 21% O 2 ) and hypoxic (Hyp, 10% O 2 for 5 weeks) mice were homogenized and their mRNA transcripts evaluated by RT-PCR utilizing primers specific for ATOX1, ATP7A, CCS, CTR1, LOX, GAPDH or 18s rRNA (internal controls). A: RT-PCR products from whole-lung tissues were separated on 2% agarose gels (upper panel) and the band intensities quantitated by ImageJ, normalized to intensity of GAPDH, and graphed relative to Nor (n = 4 Nor mouse lungs; n = 8 Hyp mouse lungs). B: PA dissected from Nor and Hyp mice were used for RNA extraction (n = 5) and analyzed by quantitative PCR. Real-time PCR reaction was set with primers specific for the indicated genes. The cycle threshold C(t) values were normalized to 18s rRNA to obtain ΔC(t) , quantified relative to normoxic control for each of the indicated genes (ΔΔC(t)), and graphed as % of normoxic control. C: Representative records of right ventricular pressure (RVP, left panel) and summarized data (mean±SE) showing RV systolic pressure (RVSP) in Nor (n = 6) and Hyp (n = 13) mice. D: Representative records (left panel) and summarized data (right panel, mean±SE) of right ventricular contractility (RV-±dp/dt max ) in Nor and Hyp mice. E: Summarized data (mean±SE) showing the ratio of right ventricle (RV) weight to left ventricle (LV) and septum (S) weight [RV/(LV+S)] in Nor (n = 7) and Hyp (n = 7) mice. * P
    Figure Legend Snippet: The mRNA expression level of Cu transporters (CTR1 and ATP7A) and lysyl oxidase (LOX) is increased in whole-lung and pulmonary artery (PA) tissues of mice with chronically hypoxia-induced pulmonary hypertension (HPH). Whole lung tissues and isolated PA tissue from normoxic (Nor, 21% O 2 ) and hypoxic (Hyp, 10% O 2 for 5 weeks) mice were homogenized and their mRNA transcripts evaluated by RT-PCR utilizing primers specific for ATOX1, ATP7A, CCS, CTR1, LOX, GAPDH or 18s rRNA (internal controls). A: RT-PCR products from whole-lung tissues were separated on 2% agarose gels (upper panel) and the band intensities quantitated by ImageJ, normalized to intensity of GAPDH, and graphed relative to Nor (n = 4 Nor mouse lungs; n = 8 Hyp mouse lungs). B: PA dissected from Nor and Hyp mice were used for RNA extraction (n = 5) and analyzed by quantitative PCR. Real-time PCR reaction was set with primers specific for the indicated genes. The cycle threshold C(t) values were normalized to 18s rRNA to obtain ΔC(t) , quantified relative to normoxic control for each of the indicated genes (ΔΔC(t)), and graphed as % of normoxic control. C: Representative records of right ventricular pressure (RVP, left panel) and summarized data (mean±SE) showing RV systolic pressure (RVSP) in Nor (n = 6) and Hyp (n = 13) mice. D: Representative records (left panel) and summarized data (right panel, mean±SE) of right ventricular contractility (RV-±dp/dt max ) in Nor and Hyp mice. E: Summarized data (mean±SE) showing the ratio of right ventricle (RV) weight to left ventricle (LV) and septum (S) weight [RV/(LV+S)] in Nor (n = 7) and Hyp (n = 7) mice. * P

    Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Real-time Polymerase Chain Reaction

    PASMC migration is dependent on Cu and inhibited by knockdown of CTR1. Cell migration was determined by the modified Boyden chamber assay. Cells were plated on top of the porous (8-µm pore) membrane. After 48 hrs, the membrane was fixed and stained using Diff-Quick and the migrated cells in randomly chosen fields were counted at 200× magnification. A: Representative images ( a ) showing human PASMC cultured under normoxic (Nor) or hypoxic (Hyp) conditions in the absence (Control) or presence (BCS) of 200 µM BCS (a Cu chelator). Summarized data (mean±SE) showing migrated cell counts ( b ) in normoxic (Nor) and hypoxic (Hyp) PASMC treated with (BCS) or without (Control) BCS. ** P
    Figure Legend Snippet: PASMC migration is dependent on Cu and inhibited by knockdown of CTR1. Cell migration was determined by the modified Boyden chamber assay. Cells were plated on top of the porous (8-µm pore) membrane. After 48 hrs, the membrane was fixed and stained using Diff-Quick and the migrated cells in randomly chosen fields were counted at 200× magnification. A: Representative images ( a ) showing human PASMC cultured under normoxic (Nor) or hypoxic (Hyp) conditions in the absence (Control) or presence (BCS) of 200 µM BCS (a Cu chelator). Summarized data (mean±SE) showing migrated cell counts ( b ) in normoxic (Nor) and hypoxic (Hyp) PASMC treated with (BCS) or without (Control) BCS. ** P

    Techniques Used: Migration, Modification, Boyden Chamber Assay, Staining, Diff-Quik, Cell Culture

    Hypoxia-mediated upregulation of mRNA expression of Cu transporters (CTR1, ATP7A) and lysyl oxidase (LOX) is associated with an increase in Cu transportation in human pulmonary arterial smooth muscle cells (PASMC). A: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and 64 Cu uptake (mean±SE) in human PASMC exposed to normoxia (Nor) and hypoxia (Hyp, 3% O 2 for 48 hrs, n = 3; right panel). B: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and 64 Cu uptake (mean±SE, right panel) in human PASMC treated with vehicle (Cont) and CoCl 2 (100 µM for 48 hrs, n = 3; right pane). Lactate dehydrogenase (LDH) and erythropoietin (EPO) were used as positive controls. * P
    Figure Legend Snippet: Hypoxia-mediated upregulation of mRNA expression of Cu transporters (CTR1, ATP7A) and lysyl oxidase (LOX) is associated with an increase in Cu transportation in human pulmonary arterial smooth muscle cells (PASMC). A: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and 64 Cu uptake (mean±SE) in human PASMC exposed to normoxia (Nor) and hypoxia (Hyp, 3% O 2 for 48 hrs, n = 3; right panel). B: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and 64 Cu uptake (mean±SE, right panel) in human PASMC treated with vehicle (Cont) and CoCl 2 (100 µM for 48 hrs, n = 3; right pane). Lactate dehydrogenase (LDH) and erythropoietin (EPO) were used as positive controls. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Downregulation of HIF-1α by siRNA significantly attenuates mRNA expression of CTR1 in hypoxic PASMC. A: Real-time RT-PCR analysis on HIF-1α ( a ), HIF-2α ( b ) and CTR1 ( c ) in hypoxic PASMC treated with (50–200 pmol) or without (0 pmol) siRNA specifically targeting HIF-1α, HIF-2α and CTR1, respectively. Data are shown in mean±SE. *** P
    Figure Legend Snippet: Downregulation of HIF-1α by siRNA significantly attenuates mRNA expression of CTR1 in hypoxic PASMC. A: Real-time RT-PCR analysis on HIF-1α ( a ), HIF-2α ( b ) and CTR1 ( c ) in hypoxic PASMC treated with (50–200 pmol) or without (0 pmol) siRNA specifically targeting HIF-1α, HIF-2α and CTR1, respectively. Data are shown in mean±SE. *** P

    Techniques Used: Expressing, Quantitative RT-PCR

    Chelation of Cu and knockdown of CTR1 both decrease Bcl-2 expression in human PASMC during hypoxia. A: Western blot analysis on PCNA (a marker for cell proliferation) and Bcl-2 (an anti-apoptotic protein) in PASMC transfected with scrambled siRNA (Cont) or human CTR1-siRNA (siRNA) and PASMC treated with the Cu chelator BCS. B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl-2 (right panel) protein levels in PASMC transfected with Control-siRNA or hCTR1-siRNA and PASMC treated with BCS. * P
    Figure Legend Snippet: Chelation of Cu and knockdown of CTR1 both decrease Bcl-2 expression in human PASMC during hypoxia. A: Western blot analysis on PCNA (a marker for cell proliferation) and Bcl-2 (an anti-apoptotic protein) in PASMC transfected with scrambled siRNA (Cont) or human CTR1-siRNA (siRNA) and PASMC treated with the Cu chelator BCS. B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl-2 (right panel) protein levels in PASMC transfected with Control-siRNA or hCTR1-siRNA and PASMC treated with BCS. * P

    Techniques Used: Expressing, Western Blot, Marker, Transfection

    The protein expression level of CTR1, pro-LOX and HIF-1α is increased in whole-lung tissues of mice with HPH. A–C: Western blot analysis of mouse CTR1 (A), pro-LOX (B) and HIF-1α (C) in total and membrane proteins extracted from whole-lung lung tissues of normoxic control mice (Nor, n = 5) and chronically hypoxic (Nor, n = 5) mice. Proteins from Nor and Hyp mouse lungs were solubilized in 3% DDM/1× RIPA buffer and utilized for Western blot analysis using antibodies specific for mouse CTR1, pro-LOX, and HIF-1α. β-actin or β-tubulin was used as a loading control. D: Summarized data (mean±SE) showing protein expression levels of CTR1, pro-LOX and HIF-1α in lungs tissues isolated from Nor and Hyp mice. The band intensity was quantitated with ImageJ software, normalized with respect to the loading control, and then shown relative to control (% of Nor). ** P
    Figure Legend Snippet: The protein expression level of CTR1, pro-LOX and HIF-1α is increased in whole-lung tissues of mice with HPH. A–C: Western blot analysis of mouse CTR1 (A), pro-LOX (B) and HIF-1α (C) in total and membrane proteins extracted from whole-lung lung tissues of normoxic control mice (Nor, n = 5) and chronically hypoxic (Nor, n = 5) mice. Proteins from Nor and Hyp mouse lungs were solubilized in 3% DDM/1× RIPA buffer and utilized for Western blot analysis using antibodies specific for mouse CTR1, pro-LOX, and HIF-1α. β-actin or β-tubulin was used as a loading control. D: Summarized data (mean±SE) showing protein expression levels of CTR1, pro-LOX and HIF-1α in lungs tissues isolated from Nor and Hyp mice. The band intensity was quantitated with ImageJ software, normalized with respect to the loading control, and then shown relative to control (% of Nor). ** P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Isolation, Software

    28) Product Images from "An M1-like macrophage polarization in decidual tissue during spontaneous preterm labor that is attenuated by rosiglitazone treatment"

    Article Title: An M1-like macrophage polarization in decidual tissue during spontaneous preterm labor that is attenuated by rosiglitazone treatment

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1502055

    Rosiglitazone treatment reduces the mRNA expression of Nfκb1, Tnf , and Il10 in decidual and myometrial macrophages
    Figure Legend Snippet: Rosiglitazone treatment reduces the mRNA expression of Nfκb1, Tnf , and Il10 in decidual and myometrial macrophages

    Techniques Used: Expressing

    29) Product Images from "Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein"

    Article Title: Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2017.01.002

    In Vitro and In Vivo Furin Cleavage of RD114-TRco (A) Western blot analysis of whole-cell proteins (30 μg) extracted from PK-7 cells, transfected with either RD114-TRWT or RD114-TRco plasmid and treated overnight at 16°C with 4 U/sample of recombinant furin. The filter was probed with the anti-TM Ab and, after stripping, with anti-HIV serum as an internal control. (B) Western blot analysis of whole-cell proteins (30 μg) extracted from HEK293T cells transfected with the indicated amounts of plasmid DNA encoding either RD114-TRWT or RD114-TRco. The filter was stained with anti-TM Ab and, after stripping, with anti-β actin Ab as an internal control.
    Figure Legend Snippet: In Vitro and In Vivo Furin Cleavage of RD114-TRco (A) Western blot analysis of whole-cell proteins (30 μg) extracted from PK-7 cells, transfected with either RD114-TRWT or RD114-TRco plasmid and treated overnight at 16°C with 4 U/sample of recombinant furin. The filter was probed with the anti-TM Ab and, after stripping, with anti-HIV serum as an internal control. (B) Western blot analysis of whole-cell proteins (30 μg) extracted from HEK293T cells transfected with the indicated amounts of plasmid DNA encoding either RD114-TRWT or RD114-TRco. The filter was stained with anti-TM Ab and, after stripping, with anti-β actin Ab as an internal control.

    Techniques Used: In Vitro, In Vivo, Western Blot, Transfection, Plasmid Preparation, Recombinant, Stripping Membranes, Staining

    Subcellular Localization of RD114-TRWT and RD114-TRco (A–D) COS-7 cells transfected with pIRES-RD114-TR plasmids were fixed, permeabilized, and stained at room temperature with the Abs as indicated. Endobrevin/VAMP8 is an early and late endosomal marker, and calnexin is an ER marker. Nuclei were stained with DAPI. Scale bar, 40 μm.
    Figure Legend Snippet: Subcellular Localization of RD114-TRWT and RD114-TRco (A–D) COS-7 cells transfected with pIRES-RD114-TR plasmids were fixed, permeabilized, and stained at room temperature with the Abs as indicated. Endobrevin/VAMP8 is an early and late endosomal marker, and calnexin is an ER marker. Nuclei were stained with DAPI. Scale bar, 40 μm.

    Techniques Used: Transfection, Staining, Marker

    Prediction of RD114-TRWT and RD114-TRco mRNA Secondary Structures (A and B) Schematics of the most thermodynamically stable (lowest free energy value) secondary structures of RD114-TRWT (A) and RD114-TRco (B) mRNA, predicted by the MFOLD software. Data are annotated using color-based characters, indicating the probability of base-pairing from 0 (light blue) to 1 (red), where 1 is the maximum pairing level. (C and D) The 5′ end substructures of 320 bp (C) and 330 bp (D) of RD114-TRWT and RD114-TRco mRNA, respectively, are shown in the insets. (E) Boxplot of RD114-TRWT and RD114-TRco mRNA secondary structure ΔG, calculated by MFOLD software. Statistical analysis was performed by Wilcoxon rank-sum test. Statistical significance was set as ***p
    Figure Legend Snippet: Prediction of RD114-TRWT and RD114-TRco mRNA Secondary Structures (A and B) Schematics of the most thermodynamically stable (lowest free energy value) secondary structures of RD114-TRWT (A) and RD114-TRco (B) mRNA, predicted by the MFOLD software. Data are annotated using color-based characters, indicating the probability of base-pairing from 0 (light blue) to 1 (red), where 1 is the maximum pairing level. (C and D) The 5′ end substructures of 320 bp (C) and 330 bp (D) of RD114-TRWT and RD114-TRco mRNA, respectively, are shown in the insets. (E) Boxplot of RD114-TRWT and RD114-TRco mRNA secondary structure ΔG, calculated by MFOLD software. Statistical analysis was performed by Wilcoxon rank-sum test. Statistical significance was set as ***p

    Techniques Used: Software

    RD114-TRWT and RD114-TRco Expression in Transiently Transfected PK-7 Cells and Generated LVs (A) Western blot analysis of whole-cell proteins (30 μg) obtained from PK-7 cells after transfection of SIN-EGFP TV and envelope plasmids encoding either RD114-TRWT or RD114-TRco, treated or not treated with either PNGaseF or EndoH enzymes and then probed with anti-SU and anti-TM Abs. (B) Vector proteins (160 ng of p24Gag equivalent) obtained from the virions produced by the transfected cells shown in (A), treated or not treated with either PNGaseF or EndoH enzymes, and then probed with anti-SU and anti-TM Abs. Anti-HIV protein staining (p55Gag and p24Gag) was used as an internal control. A long exposure of the films is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lanes 3 and 6). ‡ refers to EndoH-resistant SU and TM proteins.
    Figure Legend Snippet: RD114-TRWT and RD114-TRco Expression in Transiently Transfected PK-7 Cells and Generated LVs (A) Western blot analysis of whole-cell proteins (30 μg) obtained from PK-7 cells after transfection of SIN-EGFP TV and envelope plasmids encoding either RD114-TRWT or RD114-TRco, treated or not treated with either PNGaseF or EndoH enzymes and then probed with anti-SU and anti-TM Abs. (B) Vector proteins (160 ng of p24Gag equivalent) obtained from the virions produced by the transfected cells shown in (A), treated or not treated with either PNGaseF or EndoH enzymes, and then probed with anti-SU and anti-TM Abs. Anti-HIV protein staining (p55Gag and p24Gag) was used as an internal control. A long exposure of the films is included to better visualize the absence of the EndoH-resistant band of the TM subunit (lanes 3 and 6). ‡ refers to EndoH-resistant SU and TM proteins.

    Techniques Used: Expressing, Transfection, Generated, Western Blot, Plasmid Preparation, Produced, Staining

    Northern Blot Analysis of RD114-TRWT and RD114-TRco mRNA (A) Total RNA (5 μg) extracted from PK-7 cells transiently transfected with SIN-eGFP TV and either SIN-RD114-TRWT or SIN-RD114-TRco plasmids was tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively, and the common ψ probe as an internal control. Three bands were detected for both mRNAs, corresponding to the full-length (ψ and specific RD114-TR probes), the spliced, and the internal cassette transcripts, respectively (specific RD114-TR probes). Bottom: ethidium bromide (EtBr) staining of the agarose gel showing 28S and 18S RNAs. (B) Total, nuclear, and cytosolic mRNA (5 μg) extracted from PK-7 cells transiently mock-transfected (mo) and transfected with the pIRES-puro3-based plasmids encoding either RD114-TRWT or RD114-TRco were tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively. A single band derived from the expression cassette was detected for both samples. Bottom: EtBr staining of the agarose gel showing 28S and 18S RNAs. (C) qRT-PCR analysis of the nuclear and cytoplasmic distribution of RD114-TRWT and RD114-TRco mRNA. Nuclear and cytoplasmic mRNA was retro-transcribed, and then qPCR was carried out on the corresponding cDNA using specific primers for RD114-TR WT and co genes and, as internal normalizers, specific primers for the U6 and GAPDH genes. The data were derived from a single qRT-PCR experiment in which each sample was run in sestuplicate.
    Figure Legend Snippet: Northern Blot Analysis of RD114-TRWT and RD114-TRco mRNA (A) Total RNA (5 μg) extracted from PK-7 cells transiently transfected with SIN-eGFP TV and either SIN-RD114-TRWT or SIN-RD114-TRco plasmids was tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively, and the common ψ probe as an internal control. Three bands were detected for both mRNAs, corresponding to the full-length (ψ and specific RD114-TR probes), the spliced, and the internal cassette transcripts, respectively (specific RD114-TR probes). Bottom: ethidium bromide (EtBr) staining of the agarose gel showing 28S and 18S RNAs. (B) Total, nuclear, and cytosolic mRNA (5 μg) extracted from PK-7 cells transiently mock-transfected (mo) and transfected with the pIRES-puro3-based plasmids encoding either RD114-TRWT or RD114-TRco were tested with two sequence-specific RD114-TRWT and RD114-TRco probes, respectively. A single band derived from the expression cassette was detected for both samples. Bottom: EtBr staining of the agarose gel showing 28S and 18S RNAs. (C) qRT-PCR analysis of the nuclear and cytoplasmic distribution of RD114-TRWT and RD114-TRco mRNA. Nuclear and cytoplasmic mRNA was retro-transcribed, and then qPCR was carried out on the corresponding cDNA using specific primers for RD114-TR WT and co genes and, as internal normalizers, specific primers for the U6 and GAPDH genes. The data were derived from a single qRT-PCR experiment in which each sample was run in sestuplicate.

    Techniques Used: Northern Blot, Transfection, Sequencing, Staining, Agarose Gel Electrophoresis, Derivative Assay, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    30) Product Images from "Epsc Involved in the Encoding of Exopolysaccharides Produced by Bacillus amyloliquefaciens FZB42 Act to Boost the Drought Tolerance of Arabidopsis thaliana"

    Article Title: Epsc Involved in the Encoding of Exopolysaccharides Produced by Bacillus amyloliquefaciens FZB42 Act to Boost the Drought Tolerance of Arabidopsis thaliana

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123795

    The relative expression levels of drought-responsive marker genes in the leaves of A. thaliana . A qRT-PCR assay was used to estimate the expression levels of the stress response-associated marker genes, RD29A , RD17 , ERD1 , and LEA14 , in drought plants inoculated with either wild type FZB42 or the epsC mutant. Values are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant ( p
    Figure Legend Snippet: The relative expression levels of drought-responsive marker genes in the leaves of A. thaliana . A qRT-PCR assay was used to estimate the expression levels of the stress response-associated marker genes, RD29A , RD17 , ERD1 , and LEA14 , in drought plants inoculated with either wild type FZB42 or the epsC mutant. Values are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant ( p

    Techniques Used: Expressing, Marker, Quantitative RT-PCR, Mutagenesis

    31) Product Images from "Use of Graph Theory to Characterize Human and Arthropod Vector Cell Protein Response to Infection With Anaplasma phagocytophilum"

    Article Title: Use of Graph Theory to Characterize Human and Arthropod Vector Cell Protein Response to Infection With Anaplasma phagocytophilum

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00265

    Functional analysis by RNAi supports a role for tick and human Rab14 in A. phagocytophilum infection of host cells. (A) A 75–83% knockdown by RNAi of tick rab14 (B7QHS7) in tick cells resulted in a 40% decrease in A. phagocytophilum infection levels, suggesting that A. phagocytophilum increases the levels of Rab14 to facilitate infection. Tick ISE6 cells were treated with rab14 dsRNA and control cells were treated with the unrelated Rs86 dsRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 ( msp4 ) gene-specific primers. Normalized Ct values were compared between groups by Student's t -test with unequal variance ( p = 0.02; n = 6 biological replicates). (B) Tick rab14 knockdown did not affect cell viability. The percent of apoptotic tick ISE6 cells was determined after RNAi with rab14 test and Rs86 control dsRNAs by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit. The percentage of apoptotic cells was compared between both test and control dsRNA treated UtC and ItC by Student's t -test with unequal variance ( p > 0.05; n = 6 biological replicates). (C) Representative images of immunofluorescence analysis of UhC and IhC incubated with either ON-TARGETplus SMARTpool Human rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. Cells were stained with rabbit anti- A. phagocytophilum msp4 antibodies, labeled with FITC (green, arrows) and DAPI (blue). To confirm the uptake of siRNA, cells were treated with Accell Red Non-targeting Control siRNA (red, arrows) and labeled with DAPI (blue). (D) Human rab14 was up-regulated at the mRNA level in response to infection. The RNA levels of human rab14 (P61106) were determined by real-time RT-PCR in UhC and IhC. Normalized Ct values were compared between groups by Student's t -test with unequal variance ( p = 0.03; n = 4 biological replicates). (E) A 31–52% knockdown by RNAi of rab14 in human HL60 cells did not affect A. phagocytophilum infection levels, suggesting that Rab14 protein levels decrease post-transcriptionally in human neutrophils to control A. phagocytophilum infection. Human HL60 cells were treated with rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 ( msp4 ) gene-specific primers. Normalized Ct values were compared between groups by Student's t -test with unequal variance (non-significant, p > 0.05; n = 4 biological replicates). (F) Proposed model of ras-related protein function in A. phagocytophilum -infected tick and human cells. In tick cells, A. phagocytophilum ( Ap ) increases the levels of active ras-related proteins Rab14 in phagosomal membranes to prevent the transfer of bacteria from phagosomes to lysosomes and hijacks Rab10 and other endoplasmic reticulum membrane proteins to its vacuole to complete the infection cycle and favor pathogen survival and facilitate infection. In human neutrophils, the decrease in Rab10 levels appears as a post-transcriptional mechanism to control A. phagocytophilum infection.
    Figure Legend Snippet: Functional analysis by RNAi supports a role for tick and human Rab14 in A. phagocytophilum infection of host cells. (A) A 75–83% knockdown by RNAi of tick rab14 (B7QHS7) in tick cells resulted in a 40% decrease in A. phagocytophilum infection levels, suggesting that A. phagocytophilum increases the levels of Rab14 to facilitate infection. Tick ISE6 cells were treated with rab14 dsRNA and control cells were treated with the unrelated Rs86 dsRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 ( msp4 ) gene-specific primers. Normalized Ct values were compared between groups by Student's t -test with unequal variance ( p = 0.02; n = 6 biological replicates). (B) Tick rab14 knockdown did not affect cell viability. The percent of apoptotic tick ISE6 cells was determined after RNAi with rab14 test and Rs86 control dsRNAs by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit. The percentage of apoptotic cells was compared between both test and control dsRNA treated UtC and ItC by Student's t -test with unequal variance ( p > 0.05; n = 6 biological replicates). (C) Representative images of immunofluorescence analysis of UhC and IhC incubated with either ON-TARGETplus SMARTpool Human rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. Cells were stained with rabbit anti- A. phagocytophilum msp4 antibodies, labeled with FITC (green, arrows) and DAPI (blue). To confirm the uptake of siRNA, cells were treated with Accell Red Non-targeting Control siRNA (red, arrows) and labeled with DAPI (blue). (D) Human rab14 was up-regulated at the mRNA level in response to infection. The RNA levels of human rab14 (P61106) were determined by real-time RT-PCR in UhC and IhC. Normalized Ct values were compared between groups by Student's t -test with unequal variance ( p = 0.03; n = 4 biological replicates). (E) A 31–52% knockdown by RNAi of rab14 in human HL60 cells did not affect A. phagocytophilum infection levels, suggesting that Rab14 protein levels decrease post-transcriptionally in human neutrophils to control A. phagocytophilum infection. Human HL60 cells were treated with rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 ( msp4 ) gene-specific primers. Normalized Ct values were compared between groups by Student's t -test with unequal variance (non-significant, p > 0.05; n = 4 biological replicates). (F) Proposed model of ras-related protein function in A. phagocytophilum -infected tick and human cells. In tick cells, A. phagocytophilum ( Ap ) increases the levels of active ras-related proteins Rab14 in phagosomal membranes to prevent the transfer of bacteria from phagosomes to lysosomes and hijacks Rab10 and other endoplasmic reticulum membrane proteins to its vacuole to complete the infection cycle and favor pathogen survival and facilitate infection. In human neutrophils, the decrease in Rab10 levels appears as a post-transcriptional mechanism to control A. phagocytophilum infection.

    Techniques Used: Functional Assay, Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Immunofluorescence, Immunohistochemistry, Incubation, Staining, Labeling, Quantitative RT-PCR

    32) Product Images from "Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer"

    Article Title: Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4528-x

    ERα interacts to ERRβ promoter in-vitro . a Schematic representation of two functional half ERE sites present in ERRβ promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of ERRβ promoter. b Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in ERRβ promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32 P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed
    Figure Legend Snippet: ERα interacts to ERRβ promoter in-vitro . a Schematic representation of two functional half ERE sites present in ERRβ promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of ERRβ promoter. b Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in ERRβ promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32 P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed

    Techniques Used: In Vitro, Functional Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Incubation

    ERRβ is a regulator of cell cycle and inhibition of ERRβ leads to cell proliferation. a Western blots and densitometry analysis showing changes in the expression of cell cycle markers, such as p21 cip , p18 and cyclin D1 upon the over expression of ERRβ in MCF7 cells. b , c ERRβ was ectopically expressed in ER + ve breast cancer cells, after 48 h the mRNA levels of p21 cip were examined by RT-PCR and RT-qPCR, p21 cip was significantly up-regulated. All the results were obtained from three independent experiments and each done in triplicates, 2-group unpaired t-test was used to obtain p -values and p
    Figure Legend Snippet: ERRβ is a regulator of cell cycle and inhibition of ERRβ leads to cell proliferation. a Western blots and densitometry analysis showing changes in the expression of cell cycle markers, such as p21 cip , p18 and cyclin D1 upon the over expression of ERRβ in MCF7 cells. b , c ERRβ was ectopically expressed in ER + ve breast cancer cells, after 48 h the mRNA levels of p21 cip were examined by RT-PCR and RT-qPCR, p21 cip was significantly up-regulated. All the results were obtained from three independent experiments and each done in triplicates, 2-group unpaired t-test was used to obtain p -values and p

    Techniques Used: Inhibition, Western Blot, Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Estrogen facilitates binding of ERRβ on half EREs through ERα. a ChIP assay showing ERα binding to half EREs on ERRβ promoter in-vivo in the presence of estrogen in (i) MCF7 and (ii) T47D cells. b ERRβ binding in the upstream region of ERRβ promoter upon estrogen treatment in (i) MCF7 and (ii) T47D cells. c ER-ve breast cancer cells (MDA-MB 231) showing no binding of ERα on ERRβ promoter and used as a negative control. d Re-ChIP shows binding of ERα and ERRβ heterodimer complex on half ERE sites present on ERRβ promoter
    Figure Legend Snippet: Estrogen facilitates binding of ERRβ on half EREs through ERα. a ChIP assay showing ERα binding to half EREs on ERRβ promoter in-vivo in the presence of estrogen in (i) MCF7 and (ii) T47D cells. b ERRβ binding in the upstream region of ERRβ promoter upon estrogen treatment in (i) MCF7 and (ii) T47D cells. c ER-ve breast cancer cells (MDA-MB 231) showing no binding of ERα on ERRβ promoter and used as a negative control. d Re-ChIP shows binding of ERα and ERRβ heterodimer complex on half ERE sites present on ERRβ promoter

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, In Vivo, Multiple Displacement Amplification, Negative Control

    Effect of ERα and ERRβ on ERRβ promoter. a Schematic representation of ERRβ promoter showing two half ERE sites. b ERα regulates ERRβ classically in the presence of estrogen. c MCF7 cells were transfected with ERα , ERRβ along with ERRβ promoter and luciferase readings were obtained in the presence and absence of estrogen stimulation. Renilla readings were taken as a control and all the experiments were conducted in triplicates; statistical significance was analyzed using One-way ANOVA test and p
    Figure Legend Snippet: Effect of ERα and ERRβ on ERRβ promoter. a Schematic representation of ERRβ promoter showing two half ERE sites. b ERα regulates ERRβ classically in the presence of estrogen. c MCF7 cells were transfected with ERα , ERRβ along with ERRβ promoter and luciferase readings were obtained in the presence and absence of estrogen stimulation. Renilla readings were taken as a control and all the experiments were conducted in triplicates; statistical significance was analyzed using One-way ANOVA test and p

    Techniques Used: Transfection, Luciferase

    Estrogen regulates the expression of ERRβ. a Western blots and densitometry analyses showing up-regulation of ERRβ upon estrogen treatment at different concentrations [10 nM (i) 100 nM (ii)] for different time points (0, 6, 12, 24, 48 h) in MCF7 cells. MCF7 cells showed > 2 fold high expression of ERRβ upon the treatment of 100 nM E2 treatment. b Combinatorial treatment of MCF7 cells with estrogen and tamoxifen decrease ERRβ expression. The association between normalized percentage expression in different groups were analyzed using One-way ANOVA test (ns- no significance, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)
    Figure Legend Snippet: Estrogen regulates the expression of ERRβ. a Western blots and densitometry analyses showing up-regulation of ERRβ upon estrogen treatment at different concentrations [10 nM (i) 100 nM (ii)] for different time points (0, 6, 12, 24, 48 h) in MCF7 cells. MCF7 cells showed > 2 fold high expression of ERRβ upon the treatment of 100 nM E2 treatment. b Combinatorial treatment of MCF7 cells with estrogen and tamoxifen decrease ERRβ expression. The association between normalized percentage expression in different groups were analyzed using One-way ANOVA test (ns- no significance, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)

    Techniques Used: Expressing, Western Blot

    Expression of ERRβ is ERα dependent. Efficient knockdown of ERα showing significant decrease in the expression of ERRβ in MCF7 cells. a , b Quantitative Real-time PCR (qRT-PCR) and Reverse transcription polymerase chain reaction (RT-PCR) results showing decreased expression of ERRβ in ERα depleted MCF7 cells. Housekeeping gene GAPDH treated as control and ΔCt, ΔΔCt, 2 -ΔΔCt values were calculated and graph was plotted using 2 -ΔΔCt values. Fold change ≥ 2 was considered as significant. p -values were calculated using 2-group t-test (* p
    Figure Legend Snippet: Expression of ERRβ is ERα dependent. Efficient knockdown of ERα showing significant decrease in the expression of ERRβ in MCF7 cells. a , b Quantitative Real-time PCR (qRT-PCR) and Reverse transcription polymerase chain reaction (RT-PCR) results showing decreased expression of ERRβ in ERα depleted MCF7 cells. Housekeeping gene GAPDH treated as control and ΔCt, ΔΔCt, 2 -ΔΔCt values were calculated and graph was plotted using 2 -ΔΔCt values. Fold change ≥ 2 was considered as significant. p -values were calculated using 2-group t-test (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    33) Product Images from "Modulation of Alpha-synuclein Expression and Associated Effects by MicroRNA Let-7 in Transgenic C. elegans"

    Article Title: Modulation of Alpha-synuclein Expression and Associated Effects by MicroRNA Let-7 in Transgenic C. elegans

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00328

    Graph depicting relative expression of let-7 miRNA and its targets, studied through real-time PCR (qPCR). (A) Expression level of let-7 miRNA in PD model vs. wild type. (B) Expression level of let-7 miRNA and their targets in let-7 knockdown worms. ∗ p
    Figure Legend Snippet: Graph depicting relative expression of let-7 miRNA and its targets, studied through real-time PCR (qPCR). (A) Expression level of let-7 miRNA in PD model vs. wild type. (B) Expression level of let-7 miRNA and their targets in let-7 knockdown worms. ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    34) Product Images from "Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes"

    Article Title: Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes

    Journal: PPAR Research

    doi: 10.1155/2013/476049

    The effect of forskolin and rosiglitazone on mRNA expression of (a) UCP1, (b) PGC1 α , (c) Cidea, (d) PRDM16, (e) C/EBP β , and (f) PPAR γ is mediated by PKA and PPAR γ dependent pathways. HIB-1B cells were grown to confluence and then treated with H89 (10 μ M) for 1 hour or GW9662 (30 μ M) for 3 hours prior to and during addition of rosiglitazone (Rosi) (10 μ M) for 24 hours, or forskolin (Fosk) (10 μ M) for the final 3 hours of rosiglitazone treatment, before RNA extraction, as indicated. All drugs were added in serum-free medium. Controls were treated with DMSO. Gene expression levels were analysed by quantitative real-time PCR and normalized against 36B4 expression. Error bar means the mean ± SEM of triplicate observations within a single experiment performed in triplicate. *** Significant difference P
    Figure Legend Snippet: The effect of forskolin and rosiglitazone on mRNA expression of (a) UCP1, (b) PGC1 α , (c) Cidea, (d) PRDM16, (e) C/EBP β , and (f) PPAR γ is mediated by PKA and PPAR γ dependent pathways. HIB-1B cells were grown to confluence and then treated with H89 (10 μ M) for 1 hour or GW9662 (30 μ M) for 3 hours prior to and during addition of rosiglitazone (Rosi) (10 μ M) for 24 hours, or forskolin (Fosk) (10 μ M) for the final 3 hours of rosiglitazone treatment, before RNA extraction, as indicated. All drugs were added in serum-free medium. Controls were treated with DMSO. Gene expression levels were analysed by quantitative real-time PCR and normalized against 36B4 expression. Error bar means the mean ± SEM of triplicate observations within a single experiment performed in triplicate. *** Significant difference P

    Techniques Used: Expressing, RNA Extraction, Real-time Polymerase Chain Reaction

    35) Product Images from "Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation"

    Article Title: Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation

    Journal: Scientific Reports

    doi: 10.1038/srep39211

    The effect of 11,12-EET or 19,20-EDP plus sEH inhibition on TNFα-induced VCAM-1 and ICAM-1 protein levels. ( a ) Representative blots from HRMEC treated with TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM); and ( b ) quantification of 3 individual blots. Each bar represents the mean ± SEM (n = 3).
    Figure Legend Snippet: The effect of 11,12-EET or 19,20-EDP plus sEH inhibition on TNFα-induced VCAM-1 and ICAM-1 protein levels. ( a ) Representative blots from HRMEC treated with TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM); and ( b ) quantification of 3 individual blots. Each bar represents the mean ± SEM (n = 3).

    Techniques Used: Inhibition

    The effect of 11,12-EET, 19,20-EDP, AUDA, or the corresponding diols on TNFα-induced adhesion molecule expression. HRMEC were treated with TNFα in the presence or absence of ( a ) 11,12-EET (0.5 μM), 19,20-EDP (0.5 μM), AUDA (10 μM), 11,12-DHET (0.5 μM), or 19,20-DHDP (0.5 μM); or ( b ) combinations of 11,12-EET or 19,20-EDP with AUDA. Expression of VCAM-1 and ICAM-1 was assessed by qRT-PCR analysis. Each bar represents the mean ± SEM (a: n = 6; b: n = 12).
    Figure Legend Snippet: The effect of 11,12-EET, 19,20-EDP, AUDA, or the corresponding diols on TNFα-induced adhesion molecule expression. HRMEC were treated with TNFα in the presence or absence of ( a ) 11,12-EET (0.5 μM), 19,20-EDP (0.5 μM), AUDA (10 μM), 11,12-DHET (0.5 μM), or 19,20-DHDP (0.5 μM); or ( b ) combinations of 11,12-EET or 19,20-EDP with AUDA. Expression of VCAM-1 and ICAM-1 was assessed by qRT-PCR analysis. Each bar represents the mean ± SEM (a: n = 6; b: n = 12).

    Techniques Used: Expressing, Quantitative RT-PCR

    The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced NFκB Activation. HRMEC were transfected with luciferase constructs and treated with TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM). NFκB activity was determined by measuring the ratio of firefly-to-Renilla luciferase. Each bar represents the mean ± SEM (n = 20).
    Figure Legend Snippet: The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced NFκB Activation. HRMEC were transfected with luciferase constructs and treated with TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM). NFκB activity was determined by measuring the ratio of firefly-to-Renilla luciferase. Each bar represents the mean ± SEM (n = 20).

    Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Activity Assay

    The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced leukocyte adhesion to HRMEC monolayers. HRMEC monolayers were treated TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM), and PBMC were then flowed over the treated monolayers in a parallel plate flow chamber. Each bar represents the mean ± SEM (vehicle: n = 8; TNFα: n = 9; 11,12-EET + AUDA: n = 6; 19,20-EDP: n = 4).
    Figure Legend Snippet: The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced leukocyte adhesion to HRMEC monolayers. HRMEC monolayers were treated TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM), and PBMC were then flowed over the treated monolayers in a parallel plate flow chamber. Each bar represents the mean ± SEM (vehicle: n = 8; TNFα: n = 9; 11,12-EET + AUDA: n = 6; 19,20-EDP: n = 4).

    Techniques Used: Flow Cytometry

    The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced retinal leukostasis. Mice were injected intravitreally with 50 ng/ml TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM). ( a ) Representative images of retinal flatmounts with Concanavalin-A perfusion; yellow arrows indicate adhered leukocytes; ( b ) quantification of adherent leukocytes normalized to retinal area. Bars represent mean ± SD (vehicle: n = 8; TNFα: n = 16; 11,12-EET + AUDA: n = 4; 19,20-EDP: n = 12).
    Figure Legend Snippet: The effect of 11,12-EET or 19,20-EDP plus AUDA on TNFα-induced retinal leukostasis. Mice were injected intravitreally with 50 ng/ml TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM). ( a ) Representative images of retinal flatmounts with Concanavalin-A perfusion; yellow arrows indicate adhered leukocytes; ( b ) quantification of adherent leukocytes normalized to retinal area. Bars represent mean ± SD (vehicle: n = 8; TNFα: n = 16; 11,12-EET + AUDA: n = 4; 19,20-EDP: n = 12).

    Techniques Used: Mouse Assay, Injection

    The effect of TNFα on epoxygenated fatty acid levels. HRMEC were treated with vehicle or TNFα (1 ng/ml), arachidonic acid and docosahexaenoic acid substrates were provided, and the level of their epoxygenated products was measured in the conditioned media by LC-MS/MS. These data are normalized to the total protein of the cell lysates. Each bar represents the mean ± SEM (n = 8 for EET measurements; n = 9 for EDP measurements).
    Figure Legend Snippet: The effect of TNFα on epoxygenated fatty acid levels. HRMEC were treated with vehicle or TNFα (1 ng/ml), arachidonic acid and docosahexaenoic acid substrates were provided, and the level of their epoxygenated products was measured in the conditioned media by LC-MS/MS. These data are normalized to the total protein of the cell lysates. Each bar represents the mean ± SEM (n = 8 for EET measurements; n = 9 for EDP measurements).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    36) Product Images from "CHEK2 represses breast stromal fibroblasts and their paracrine tumor-promoting effects through suppressing SDF-1 and IL-6"

    Article Title: CHEK2 represses breast stromal fibroblasts and their paracrine tumor-promoting effects through suppressing SDF-1 and IL-6

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2614-5

    CHEK2 expression is down-regulated in cancer-associated fibroblasts. a Whole cell lysates were prepared from the indicated cells and 50 μg of proteins were used for immunoblotting analysis using antibodies against the indicated proteins. b Total RNA was extracted from the indicated cells and the amount of the CHEK2 mRNA was assessed by qRT-PCR. Error bars represent means ± S.D
    Figure Legend Snippet: CHEK2 expression is down-regulated in cancer-associated fibroblasts. a Whole cell lysates were prepared from the indicated cells and 50 μg of proteins were used for immunoblotting analysis using antibodies against the indicated proteins. b Total RNA was extracted from the indicated cells and the amount of the CHEK2 mRNA was assessed by qRT-PCR. Error bars represent means ± S.D

    Techniques Used: Expressing, Quantitative RT-PCR

    CHEK2 represses the expression/secretion of SDF-1 and IL-6 . a Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis. The numbers below the bands indicate the corresponding expression levels after loading correction against GAPDH. b Total RNA was extracted from the indicated cells and used to assess the mRNA levels of SDF-1 and IL-1 using qRT-PCR and specific primers. c Conditioned media from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA, and were presented in the respective histograms. Error bars represent means ± S.D, *, p value
    Figure Legend Snippet: CHEK2 represses the expression/secretion of SDF-1 and IL-6 . a Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis. The numbers below the bands indicate the corresponding expression levels after loading correction against GAPDH. b Total RNA was extracted from the indicated cells and used to assess the mRNA levels of SDF-1 and IL-1 using qRT-PCR and specific primers. c Conditioned media from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA, and were presented in the respective histograms. Error bars represent means ± S.D, *, p value

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

    37) Product Images from "Arachidonic acid-dependent gene regulation during preadipocyte differentiation controls adipocyte potential [S]"

    Article Title: Arachidonic acid-dependent gene regulation during preadipocyte differentiation controls adipocyte potential [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M049551

    The calcium and PKC signaling pathway mediates the effect of AA on aP2 expression. A: 3T3-L1 cells (day 0) were loaded with calcium-sensitive Fluo-4 before stimulation with 10 μM AA (red line) or BSA-vehicle (black line), and calcium mobilization was determined by live cell imaging for 10–15 min (top panel). Cells were pretreated with indomethacin (10 μM) (middle panel) or AL 8810 (10 μM) (lower panel) for 30 min before stimulation with AA (10 μM). A representative experiment from three independent experiments is shown. B, C: 3T3-L1 cells (day 0) were pretreated with BAPTA-AM (30 μM) or GF109203X (10 μM), as indicated, for 30 min prior to 100 μM AA treatment for 24 h. Total RNA was extracted and subjected to RT-PCR for aP2 mRNA transcript. Data are presented as mean ± SEM of n = 3. *** P
    Figure Legend Snippet: The calcium and PKC signaling pathway mediates the effect of AA on aP2 expression. A: 3T3-L1 cells (day 0) were loaded with calcium-sensitive Fluo-4 before stimulation with 10 μM AA (red line) or BSA-vehicle (black line), and calcium mobilization was determined by live cell imaging for 10–15 min (top panel). Cells were pretreated with indomethacin (10 μM) (middle panel) or AL 8810 (10 μM) (lower panel) for 30 min before stimulation with AA (10 μM). A representative experiment from three independent experiments is shown. B, C: 3T3-L1 cells (day 0) were pretreated with BAPTA-AM (30 μM) or GF109203X (10 μM), as indicated, for 30 min prior to 100 μM AA treatment for 24 h. Total RNA was extracted and subjected to RT-PCR for aP2 mRNA transcript. Data are presented as mean ± SEM of n = 3. *** P

    Techniques Used: Expressing, Live Cell Imaging, Reverse Transcription Polymerase Chain Reaction

    AA induces the expression of Fra-1 after 24 h of treatment in 3T3-L1 cells. A: 3T3-L1 cells (day 0) were treated with increasing doses of AA (10 μM, 100 μM, or 1 mM) for 24 h in the presence of MDI. Total RNA (left panel) or total cell lysates (right panel, 100 μM) were isolated and subjected to RT-PCR or Western blotting, respectively. B: Western blot of Fra-1 in cells pretreated with indomethacin (10 μM) for 30 min prior to AA (100 μM) treatment for 24 h. C: Cells were treated for 24 h with PGF 2α (10 nM) and total RNA (left panel) or whole cell lysates (right panel, 24 h) were harvested at the indicated time points and subjected to RT-PCR or Western blotting, respectively. D: Cells were pretreated with AL 8810 (10 μM) for 30 min prior to AA (100 μM) for 24 h. Total RNA was extracted and subjected to RT-PCR for Fra-1 mRNA transcript. E: 80% confluent 3T3-L1 cells were transfected with siControl or siFP. On the third day after transfection, cells were treated with 100 μM AA for 24 h in the presence of MDI. Western blot was performed with antibodies against Fra-1 and GAPDH. F, G: Cells were pretreated with GF109203X (10 μM) or U0126 (10 μM) for 30 min prior to AA (100 μM) stimulation for 24 h. Western blot was performed using antibodies against Fra-1 and GAPDH. Fra-1 mRNA levels are presented as fold change of the vehicle control from n = 3. Error bars represent ±SEM. ** P
    Figure Legend Snippet: AA induces the expression of Fra-1 after 24 h of treatment in 3T3-L1 cells. A: 3T3-L1 cells (day 0) were treated with increasing doses of AA (10 μM, 100 μM, or 1 mM) for 24 h in the presence of MDI. Total RNA (left panel) or total cell lysates (right panel, 100 μM) were isolated and subjected to RT-PCR or Western blotting, respectively. B: Western blot of Fra-1 in cells pretreated with indomethacin (10 μM) for 30 min prior to AA (100 μM) treatment for 24 h. C: Cells were treated for 24 h with PGF 2α (10 nM) and total RNA (left panel) or whole cell lysates (right panel, 24 h) were harvested at the indicated time points and subjected to RT-PCR or Western blotting, respectively. D: Cells were pretreated with AL 8810 (10 μM) for 30 min prior to AA (100 μM) for 24 h. Total RNA was extracted and subjected to RT-PCR for Fra-1 mRNA transcript. E: 80% confluent 3T3-L1 cells were transfected with siControl or siFP. On the third day after transfection, cells were treated with 100 μM AA for 24 h in the presence of MDI. Western blot was performed with antibodies against Fra-1 and GAPDH. F, G: Cells were pretreated with GF109203X (10 μM) or U0126 (10 μM) for 30 min prior to AA (100 μM) stimulation for 24 h. Western blot was performed using antibodies against Fra-1 and GAPDH. Fra-1 mRNA levels are presented as fold change of the vehicle control from n = 3. Error bars represent ±SEM. ** P

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    Fra-1 mediates the inhibition of adipocyte differentiation by AA. A: 3T3-L1 cells were treated with increasing doses of AA for 24 h in the presence of MDI. After the 24 h, AA was withdrawn and total RNA was isolated on day 10 of the differentiation and subjected to RT-PCR for aP2, PPARγ2, and C/EBPα. Data are presented as mean ± SEM based on triplicate determinations. * P
    Figure Legend Snippet: Fra-1 mediates the inhibition of adipocyte differentiation by AA. A: 3T3-L1 cells were treated with increasing doses of AA for 24 h in the presence of MDI. After the 24 h, AA was withdrawn and total RNA was isolated on day 10 of the differentiation and subjected to RT-PCR for aP2, PPARγ2, and C/EBPα. Data are presented as mean ± SEM based on triplicate determinations. * P

    Techniques Used: Inhibition, Isolation, Reverse Transcription Polymerase Chain Reaction

    PGF 2α mediates the effect of AA on aP2 expression in 3T3-L1 cells. 3T3-L1 cells (day 0) were pretreated with indomethacin (10 μM), SC-236 (10 μM), and SC-560 (10 μM) (A) and baicalein (10 μM) or 17-ODYA (10 μM) (B) for 30 min prior to AA (100 μM) treatment for 24 h in the presence of MDI. Total RNA was isolated and RT-PCR was performed. C: 3T3-L1 cells (day 0) were treated with 1 nM, 10 nM, 100 nM, or 1 μM of PGF 2α , PGE 2 , cPGI 2 , and 15-deoxy-Δ 12,14 PGJ 2 for 24 h in the presence of MDI. The red line is the PGF 2α treatment. D: 3T3-L1 cells (day 0) were treated with PGF 2α (10 nM) in the presence of MDI for 24 h. E: 3T3-L1 cells (day 0) were pretreated with AL 8810 (10 μM), L-161,982 (10 μM), or CAY10441 (10 μM) for 30 min prior to 100 μM AA treatment for 24 h. F: Eighty percent confluent 3T3-L1 cells were transfected with a nontargeting control siRNA (siControl) or siRNA targeting the FP receptor (siFP). On the third day after transfection, cells were treated with 100 μM AA for 24 h in the presence of MDI. RT-PCR was conducted for aP2 mRNA and FP receptor transcripts. Data are presented as mean ± SEM of n = 3. * P
    Figure Legend Snippet: PGF 2α mediates the effect of AA on aP2 expression in 3T3-L1 cells. 3T3-L1 cells (day 0) were pretreated with indomethacin (10 μM), SC-236 (10 μM), and SC-560 (10 μM) (A) and baicalein (10 μM) or 17-ODYA (10 μM) (B) for 30 min prior to AA (100 μM) treatment for 24 h in the presence of MDI. Total RNA was isolated and RT-PCR was performed. C: 3T3-L1 cells (day 0) were treated with 1 nM, 10 nM, 100 nM, or 1 μM of PGF 2α , PGE 2 , cPGI 2 , and 15-deoxy-Δ 12,14 PGJ 2 for 24 h in the presence of MDI. The red line is the PGF 2α treatment. D: 3T3-L1 cells (day 0) were treated with PGF 2α (10 nM) in the presence of MDI for 24 h. E: 3T3-L1 cells (day 0) were pretreated with AL 8810 (10 μM), L-161,982 (10 μM), or CAY10441 (10 μM) for 30 min prior to 100 μM AA treatment for 24 h. F: Eighty percent confluent 3T3-L1 cells were transfected with a nontargeting control siRNA (siControl) or siRNA targeting the FP receptor (siFP). On the third day after transfection, cells were treated with 100 μM AA for 24 h in the presence of MDI. RT-PCR was conducted for aP2 mRNA and FP receptor transcripts. Data are presented as mean ± SEM of n = 3. * P

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection

    AA induces the expression of aP2 after 24 h of treatment in 3T3-L1 cells. A: Oil Red O stain­ing of 2 day postconfluent 3T3-L1 cells (day 0) upon AA treatment (10 μM, 100 μM, and 1 mM) or fatty acid-free BSA (vehicle for AA) for 24 h. Cells were captured under a light microscope using 20× magnification. B: 3T3-L1 cells (day 0) were incubated for 24 h with BSA vehicle (set as 1, dashed line) or AA (10 μM, 100 μM, and 1 mM) in the presence of MDI. Total RNA was harvested and RT-PCR was performed. Mean values are shown of n = 3 and error bars represent ±SEM. Statistical significance was determined by a Student’s t -test (two-tailed). ** P
    Figure Legend Snippet: AA induces the expression of aP2 after 24 h of treatment in 3T3-L1 cells. A: Oil Red O stain­ing of 2 day postconfluent 3T3-L1 cells (day 0) upon AA treatment (10 μM, 100 μM, and 1 mM) or fatty acid-free BSA (vehicle for AA) for 24 h. Cells were captured under a light microscope using 20× magnification. B: 3T3-L1 cells (day 0) were incubated for 24 h with BSA vehicle (set as 1, dashed line) or AA (10 μM, 100 μM, and 1 mM) in the presence of MDI. Total RNA was harvested and RT-PCR was performed. Mean values are shown of n = 3 and error bars represent ±SEM. Statistical significance was determined by a Student’s t -test (two-tailed). ** P

    Techniques Used: Expressing, Light Microscopy, Incubation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    38) Product Images from "OmpA-like proteins of Porphyromonas gingivalis contribute to serum resistance and prevent Toll-like receptor 4-mediated host cell activation"

    Article Title: OmpA-like proteins of Porphyromonas gingivalis contribute to serum resistance and prevent Toll-like receptor 4-mediated host cell activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202791

    Involvement of TLR2 and TLR4 in the cell-stimulatory activities of the OmpALP-deficient strain of P . gingivalis . (A-C) HGFs, HUVECs, and RAW264.7 cells (10 5 ) were grown in 12-well plates in an antibiotic-free culture medium containing 10% of inactivated FBS. Bacterial cells of WT, Δ 695 , Δ 694 , or Δ 695–694 resuspended in PBS were added to the cell cultures (at MOI: 100 for HGFs and HUVECs; at MOI: 10 for RAW264.7 cells). The cells were incubated for 12 h, followed by RNA extraction. Relative expression levels of IL6 and IL8 in HGFs (A) and HUVECs (B) and, Il6 and Cxcl2 in RAW264.7 cells (C) were determined by qRT-PCR. Each value, expressed as a fold increase, is mean ± SD (n = 3); *p
    Figure Legend Snippet: Involvement of TLR2 and TLR4 in the cell-stimulatory activities of the OmpALP-deficient strain of P . gingivalis . (A-C) HGFs, HUVECs, and RAW264.7 cells (10 5 ) were grown in 12-well plates in an antibiotic-free culture medium containing 10% of inactivated FBS. Bacterial cells of WT, Δ 695 , Δ 694 , or Δ 695–694 resuspended in PBS were added to the cell cultures (at MOI: 100 for HGFs and HUVECs; at MOI: 10 for RAW264.7 cells). The cells were incubated for 12 h, followed by RNA extraction. Relative expression levels of IL6 and IL8 in HGFs (A) and HUVECs (B) and, Il6 and Cxcl2 in RAW264.7 cells (C) were determined by qRT-PCR. Each value, expressed as a fold increase, is mean ± SD (n = 3); *p

    Techniques Used: Incubation, RNA Extraction, Expressing, Quantitative RT-PCR

    39) Product Images from "Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells"

    Article Title: Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00194-13

    Gene expression in I. scapularis ISE6 cells and tick developmental stages. The CG2, CG8, CG10, T1, T2, and T3 mRNA levels in ISE6 cells and ticks were determined by real-time RT-PCR. Amplification efficiencies were normalized against the amplification efficiency for tick 16S rRNA, and normalized mRNA levels (average ± SD) are expressed in arbitrary units.
    Figure Legend Snippet: Gene expression in I. scapularis ISE6 cells and tick developmental stages. The CG2, CG8, CG10, T1, T2, and T3 mRNA levels in ISE6 cells and ticks were determined by real-time RT-PCR. Amplification efficiencies were normalized against the amplification efficiency for tick 16S rRNA, and normalized mRNA levels (average ± SD) are expressed in arbitrary units.

    Techniques Used: Expressing, Quantitative RT-PCR, Amplification

    T2 and CG8 protein levels decrease in A. phagocytophilum -infected ISE6 tick cells. (A) Flow cytometry profile histogram showing in cells from early and late infection (infected cells [IC]) and uninfected cells (UC) the isotype control (IgG IC, green; IgG UC, yellow), T2 (T2 IC, purple; T2 UC, orange), and CG8 (CG8 IC, red; CG8 UC, blue) MFI peaks visualized by use of an FITC-conjugated secondary antibody and CellQuest Pro software. (B) Ratio of MFI for infected cells to MFI for uninfected cells for MSP4, subolesin (SUB), T2, and CG8 in cells from early and late infection and uninfected cells. Positive and negative values denote higher and lower protein levels in infected cells, respectively, with respect to the levels in the uninfected controls. MFI was calculated as the MFI of the test labeled sample minus the MFI of the isotype control. (C) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum -infected ISE6 tick cells. Tick cells were stained with rabbit anti-tick proteins antibodies (green, FITC). (a and b) Bright-field image of infected cells (a) and preimmune control serum-treated infected cells (b), which gave results to similar those for uninfected cells; (c and d) uninfected (c) and infected (d) cells stained with antisubolesin antibodies; (e and f) uninfected (e) and infected (f) cells stained with anti-MSP4 antibodies; (g and h) uninfected (g) and infected (h) cells stained with anti-T2 antibodies; (i and j) uninfected (i) and infected (j) cells stained with anti-CG8 antibodies. Bars, 10 μm (a, b) and 20 μm (c to j).
    Figure Legend Snippet: T2 and CG8 protein levels decrease in A. phagocytophilum -infected ISE6 tick cells. (A) Flow cytometry profile histogram showing in cells from early and late infection (infected cells [IC]) and uninfected cells (UC) the isotype control (IgG IC, green; IgG UC, yellow), T2 (T2 IC, purple; T2 UC, orange), and CG8 (CG8 IC, red; CG8 UC, blue) MFI peaks visualized by use of an FITC-conjugated secondary antibody and CellQuest Pro software. (B) Ratio of MFI for infected cells to MFI for uninfected cells for MSP4, subolesin (SUB), T2, and CG8 in cells from early and late infection and uninfected cells. Positive and negative values denote higher and lower protein levels in infected cells, respectively, with respect to the levels in the uninfected controls. MFI was calculated as the MFI of the test labeled sample minus the MFI of the isotype control. (C) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum -infected ISE6 tick cells. Tick cells were stained with rabbit anti-tick proteins antibodies (green, FITC). (a and b) Bright-field image of infected cells (a) and preimmune control serum-treated infected cells (b), which gave results to similar those for uninfected cells; (c and d) uninfected (c) and infected (d) cells stained with antisubolesin antibodies; (e and f) uninfected (e) and infected (f) cells stained with anti-MSP4 antibodies; (g and h) uninfected (g) and infected (h) cells stained with anti-T2 antibodies; (i and j) uninfected (i) and infected (j) cells stained with anti-CG8 antibodies. Bars, 10 μm (a, b) and 20 μm (c to j).

    Techniques Used: Infection, Flow Cytometry, Cytometry, Software, Labeling, Immunofluorescence, Staining

    40) Product Images from "Molecular and cytological features of the mouse B-cell lymphoma line iMycEμ-1"

    Article Title: Molecular and cytological features of the mouse B-cell lymphoma line iMycEμ-1

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-4-40

    Myc expression and Myc -driven gene expression changes in iMyc Eμ -1 cells . A , expression of Myc in iMyc Eμ -1 cells, LBL, and normal controls. The average gene expression was calculated and plotted according to the microarray measurements shown in Figure 3. B , RT-PCR of Myc , Myc His and Aktb mRNA levels (top) and Western blotting of Myc protein (bottom) in normal B cells (lane 1), LBL from an iMyc Eμ mouse (lane 2), and the iMyc Eμ -1 cell line (lane 3; SM, size marker). C , real-time qPCR analysis of Myc mRNA levels in iMyc Eμ -1 and LBL cells. Mean values and standard deviations based on three independent iMyc Eμ -1 and five LBL samples are shown. D , expression of proliferation signature genes in iMyc Eμ samples and normal controls. The expression of genes that fall in the proliferation signature defined in Figure 3 was averaged for each cell population and plotted. E , differentially expressed Myc targets in iMyc Eμ -1 cells (left column) and LBL (right column) compared to normal resting B cells. Relative gene expression levels are depicted according to the color scale at the bottom. Gene designations and names are listed to the right.
    Figure Legend Snippet: Myc expression and Myc -driven gene expression changes in iMyc Eμ -1 cells . A , expression of Myc in iMyc Eμ -1 cells, LBL, and normal controls. The average gene expression was calculated and plotted according to the microarray measurements shown in Figure 3. B , RT-PCR of Myc , Myc His and Aktb mRNA levels (top) and Western blotting of Myc protein (bottom) in normal B cells (lane 1), LBL from an iMyc Eμ mouse (lane 2), and the iMyc Eμ -1 cell line (lane 3; SM, size marker). C , real-time qPCR analysis of Myc mRNA levels in iMyc Eμ -1 and LBL cells. Mean values and standard deviations based on three independent iMyc Eμ -1 and five LBL samples are shown. D , expression of proliferation signature genes in iMyc Eμ samples and normal controls. The expression of genes that fall in the proliferation signature defined in Figure 3 was averaged for each cell population and plotted. E , differentially expressed Myc targets in iMyc Eμ -1 cells (left column) and LBL (right column) compared to normal resting B cells. Relative gene expression levels are depicted according to the color scale at the bottom. Gene designations and names are listed to the right.

    Techniques Used: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker, Real-time Polymerase Chain Reaction

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    Transfection:

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    Recombinant:

    Article Title: Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression
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    Isolation:

    Article Title: Anthocyanins from Cornus kousa ethanolic extract attenuate obesity in association with anti-angiogenic activities in 3T3-L1 cells by down-regulating adipogeneses and lipogenesis
    Article Snippet: .. Total RNA was isolated from HUVECs using TRI reagent (Sigma Aldrich). .. Then 2 μg of total RNA was reverse transcribed to cDNA for RT-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).

    Article Title: Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes
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    Mouse Assay:

    Article Title: Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes
    Article Snippet: .. To determine the reactivity of sera from immunized and non-immunized mice reacting to different endothelium, HLCVECs, HUVECs and activated HUVECs were seeded onto glass coverslips in 6-cm plates, then fixed in cold acetone and incubated with serially diluted sera isolated from immunized or non-immunized mice at 37 °C for 1 h, fluorescein-conjugated goat-anti-mouse IgG (H + L) (Sigma) was subsequently applied to them and incubated for another 1h, then to be restained by 0.1 g/L Evens Blue and washed 3 times by PBS. ..

    Blocking Assay:

    Article Title: Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression
    Article Snippet: .. After blocking non-specific binding with 10% (w/v) FBS in PBS for 2 h at 37°C, clear cell supernatants (50 µl) and a concentration series of recombinant mouse IL-12p70 or TNFα were added to the plate in triplicate before incubating at 37°C for 2 h. Between incubation steps, wells were washed thoroughly with ELISA wash buffer (PBS containing 0.05% (v/v) Tween-20 [Sigma]). .. Bound cytokines were detected using a biotin-conjugated antibody followed by StreptAvidin-Horse radish peroxidase (HRP), with 1 h incubation at 37°C.

    Purification:

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    Article Title: A Novel Lipopolysaccharide Recognition Mechanism Mediated by Internalization in Teleost Macrophages
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    Enzyme-linked Immunosorbent Assay:

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    Concentration Assay:

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    Incubation:

    Article Title: Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression
    Article Snippet: .. After blocking non-specific binding with 10% (w/v) FBS in PBS for 2 h at 37°C, clear cell supernatants (50 µl) and a concentration series of recombinant mouse IL-12p70 or TNFα were added to the plate in triplicate before incubating at 37°C for 2 h. Between incubation steps, wells were washed thoroughly with ELISA wash buffer (PBS containing 0.05% (v/v) Tween-20 [Sigma]). .. Bound cytokines were detected using a biotin-conjugated antibody followed by StreptAvidin-Horse radish peroxidase (HRP), with 1 h incubation at 37°C.

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    Binding Assay:

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  • 94
    Millipore monoclonal anti p53
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    Monoclonal Anti P53, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rt pcr analysis total rna
    Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, <t>RNA</t> was isolated and cDNA made for real-time <t>PCR</t> analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p
    Rt Pcr Analysis Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore first strand cdna synthesis kit
    Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, <t>RNA</t> was isolated and cDNA made for real-time <t>PCR</t> analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p
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    Integration of chromatin immunoprecipitation on DNA chip and gene expression profiling . (a) Small interfering RNA (siRNA)-mediated knockdown of p53 and/or p73 in HCT16-3(6) cells. Immunoblot of lysates from untreated or hydroxyurea-treated HCT116-3(6) cells (1 mM, 16 hours) following transfection with chemically synthesized siRNA oligos against lacZ, p53, and/or p73. (b) Flow chart for the integrated analysis of chromatin immunoprecipitation on DNA chip with expression profiling. (c) Summary of the relationship between p53/p73 binding and gene expression. Column 4: expression in p53 and p73 siRNA-transfected cells is lower than that of lacZ-transfected cells. Column 5: expression in p53 and p73 siRNA-transfected cells is higher than that of lacZ-transfected cells.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Integration of chromatin immunoprecipitation on DNA chip and gene expression profiling . (a) Small interfering RNA (siRNA)-mediated knockdown of p53 and/or p73 in HCT16-3(6) cells. Immunoblot of lysates from untreated or hydroxyurea-treated HCT116-3(6) cells (1 mM, 16 hours) following transfection with chemically synthesized siRNA oligos against lacZ, p53, and/or p73. (b) Flow chart for the integrated analysis of chromatin immunoprecipitation on DNA chip with expression profiling. (c) Summary of the relationship between p53/p73 binding and gene expression. Column 4: expression in p53 and p73 siRNA-transfected cells is lower than that of lacZ-transfected cells. Column 5: expression in p53 and p73 siRNA-transfected cells is higher than that of lacZ-transfected cells.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Expressing, Small Interfering RNA, Transfection, Synthesized, Flow Cytometry, Binding Assay

    Model-based analysis for promoter array . (a) Scheme for chromatin immunoprecipitation (ChIP) on DNA chip analysis. (b) Confirmation of ChIP and ligation-mediated -polymerase chain reaction amplified amplicons. ChIPs were performed as described in Figure 1(e). Enrichment of the p21cip1 distal promoter region was used to check the specificity of the samples prior to hybridization, M = mouse immunoglobulin (IgG); R = rabbit IgG. (c) Outline of the bioinformatics analysis for the identification of p53 and p73 ChIP-enriched promoters. (d) Definition of window and promoter-scores. a-g: window score; *: promoter-score. (e) Pair-wise scatter plots of the promoter-scores for the input channels (top). R 2 values are summarized in the table. (f) Pair-wise scatter plots of the promoter-scores between the ChIP and input channels (top). R 2 values are summarized in the table. (g) Pair-wise scatter plots of the promoter-scores between the ChIP and IgG channels (top). R 2 values are summarized in the table. (h) Correlation between the ChIP channels and OCT4 ChIP control. R 2 values are summarized in the table.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Model-based analysis for promoter array . (a) Scheme for chromatin immunoprecipitation (ChIP) on DNA chip analysis. (b) Confirmation of ChIP and ligation-mediated -polymerase chain reaction amplified amplicons. ChIPs were performed as described in Figure 1(e). Enrichment of the p21cip1 distal promoter region was used to check the specificity of the samples prior to hybridization, M = mouse immunoglobulin (IgG); R = rabbit IgG. (c) Outline of the bioinformatics analysis for the identification of p53 and p73 ChIP-enriched promoters. (d) Definition of window and promoter-scores. a-g: window score; *: promoter-score. (e) Pair-wise scatter plots of the promoter-scores for the input channels (top). R 2 values are summarized in the table. (f) Pair-wise scatter plots of the promoter-scores between the ChIP and input channels (top). R 2 values are summarized in the table. (g) Pair-wise scatter plots of the promoter-scores between the ChIP and IgG channels (top). R 2 values are summarized in the table. (h) Correlation between the ChIP channels and OCT4 ChIP control. R 2 values are summarized in the table.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Ligation, Polymerase Chain Reaction, Amplification, Hybridization

    Comparison of p53 and p73 promoter occupancy profiles . (a) Summary of the correlation analysis on selected promoters using model-based algorithm for promoter arrays promoter-scores (column 3), or NimbleGen log2 ratios (column 4). R 2 values represent the degree of correlation and P values represent the significance of correlation. (b) Venn diagrams depicting the number of common and distinct promoters occupied by p53 and/or p73 using FDR MAP

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Comparison of p53 and p73 promoter occupancy profiles . (a) Summary of the correlation analysis on selected promoters using model-based algorithm for promoter arrays promoter-scores (column 3), or NimbleGen log2 ratios (column 4). R 2 values represent the degree of correlation and P values represent the significance of correlation. (b) Venn diagrams depicting the number of common and distinct promoters occupied by p53 and/or p73 using FDR MAP

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques:

    Summary of quantitative chromatin immunoprecipitation analysis of selected promoters for p53 and p73 binding before and after hydroxyurea treatment . Cross-linked chromatin from untreated hydroxyurea (-HU) or hydroxyurea-treated (+HU) (1 mM, 16 hours) HCT116-3(6) cells were immunoprecipitated with the indicated antibodies and followed by polymerase chain reaction analysis using the primers flanking the sequences corresponding to the model-based algorithm for promoter array promoter-score window in each of the promoters. Data shown are log2 occupancy units from two independent chromatin immunoprecipitation experiments +/- standard deviation. log2 occupancy units > 1: significant enrichment of the selected promoter sequences in the anti-p53 or anti-p73 chromatin immunoprecipitation.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Summary of quantitative chromatin immunoprecipitation analysis of selected promoters for p53 and p73 binding before and after hydroxyurea treatment . Cross-linked chromatin from untreated hydroxyurea (-HU) or hydroxyurea-treated (+HU) (1 mM, 16 hours) HCT116-3(6) cells were immunoprecipitated with the indicated antibodies and followed by polymerase chain reaction analysis using the primers flanking the sequences corresponding to the model-based algorithm for promoter array promoter-score window in each of the promoters. Data shown are log2 occupancy units from two independent chromatin immunoprecipitation experiments +/- standard deviation. log2 occupancy units > 1: significant enrichment of the selected promoter sequences in the anti-p53 or anti-p73 chromatin immunoprecipitation.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation

    MLH3 is a p73-specific target induced by hydroxyurea . (a) Western blotting analysis of lysates from untreated or hydroxyurea (HU)-treated HCT116-3(6) cells (1 mM, 16 hours) stably expressing lacZ, p53, or p73 small hairpin RNA (shRNA). (b) Effect of p53 or p73 knockdown on p21cip1 gene expression. Real-time RT-PCR analysis of the steady-state p21cip1 mRNA level in HCT116-3(6) cells stably expressing p53 or p73 shRNA *P

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: MLH3 is a p73-specific target induced by hydroxyurea . (a) Western blotting analysis of lysates from untreated or hydroxyurea (HU)-treated HCT116-3(6) cells (1 mM, 16 hours) stably expressing lacZ, p53, or p73 small hairpin RNA (shRNA). (b) Effect of p53 or p73 knockdown on p21cip1 gene expression. Real-time RT-PCR analysis of the steady-state p21cip1 mRNA level in HCT116-3(6) cells stably expressing p53 or p73 shRNA *P

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR

    Hydroxyurea-induced accumulation of p53 and p73 and increased occupancy of the p21cip1 promoter in HCT116-3(6) cells . (a) The indicated cells were treated with or without HU (1 mM) for 16 hours, and the indicated proteins were detected by immunoblotting as described in Methods. (b) Higher levels of p53 and p73 induced by HU correlates with increased association with chromatin. HCT116-3(6) cells with or without HU treatment were subjected to chromatin immunoprecipitation (ChIP) using the indicated antibodies followed by immunoblotting with the indicated antibodies. (c) Polymerase chain reaction (PCR) primer designed for the distal p53 binding site in the p21cip1 promoter. (d) ChIP specificity. p73 -/- 3T3 cells reconstituted with either the empty vector or human p73α were treated with or without HU as in (a) and were subjected to ChIP using the indicated antibodies. Enrichment of the p21cip1 promoters was assessed by PCR using primers encompassing the p53-binding site in the distal region of the p21cip1 promoter as shown in (c). (e) Increased occupancy of the p21cip1 promoter by p53 and p73 following HU treatment. Chromatin from HCT116-3(6) cells with or without HU treatment were immunoprecipitated with the indicated antibodies (lanes 2, 4, 6, and 8) and followed by PCR analysis using the primers as shown in (c). M = mouse immunoglobulin G (IgG); R = rabbit IgG. Fold enrichment relative to the IgG sample was determined by quantitative PCR (f) Quantitative real time-PCR analysis of (e) shown as percent total input DNA.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Hydroxyurea-induced accumulation of p53 and p73 and increased occupancy of the p21cip1 promoter in HCT116-3(6) cells . (a) The indicated cells were treated with or without HU (1 mM) for 16 hours, and the indicated proteins were detected by immunoblotting as described in Methods. (b) Higher levels of p53 and p73 induced by HU correlates with increased association with chromatin. HCT116-3(6) cells with or without HU treatment were subjected to chromatin immunoprecipitation (ChIP) using the indicated antibodies followed by immunoblotting with the indicated antibodies. (c) Polymerase chain reaction (PCR) primer designed for the distal p53 binding site in the p21cip1 promoter. (d) ChIP specificity. p73 -/- 3T3 cells reconstituted with either the empty vector or human p73α were treated with or without HU as in (a) and were subjected to ChIP using the indicated antibodies. Enrichment of the p21cip1 promoters was assessed by PCR using primers encompassing the p53-binding site in the distal region of the p21cip1 promoter as shown in (c). (e) Increased occupancy of the p21cip1 promoter by p53 and p73 following HU treatment. Chromatin from HCT116-3(6) cells with or without HU treatment were immunoprecipitated with the indicated antibodies (lanes 2, 4, 6, and 8) and followed by PCR analysis using the primers as shown in (c). M = mouse immunoglobulin G (IgG); R = rabbit IgG. Fold enrichment relative to the IgG sample was determined by quantitative PCR (f) Quantitative real time-PCR analysis of (e) shown as percent total input DNA.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p

    Journal: Molecular Cancer

    Article Title: Identification of novel Notch target genes in T cell leukaemia

    doi: 10.1186/1476-4598-8-35

    Figure Lengend Snippet: Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p

    Article Snippet: RT-PCR analysis Total RNA was isolated from GFP+ transduced cell lines or cells treated with gamma secretase inhibitor (GSI IX; Calbiochem) and reverse transcribed to cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, Warrington, UK).

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing