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    Name:
    Branched PEI g PEG
    Description:

    Catalog Number:
    900743
    Price:
    None
    Applications:
    Branched PEI-g-PEG, PEG Mn 5000 was derived from Sigma-Aldrich Product 408727. Based upon an approximate Mw of 25,000 for the branched PEI, Product 900926 contains approximately 15 grafted PEG chains per PEI unit (by NMR).
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    Branched PEI g PEG

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    Average 97 stars, based on 15699 article reviews
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    pei - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Enhancement of 5-FU sensitivity by the proapoptotic rpL3 gene in p53 null colon cancer cells through combined polymer nanoparticles"

    Article Title: Enhancement of 5-FU sensitivity by the proapoptotic rpL3 gene in p53 null colon cancer cells through combined polymer nanoparticles

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13216

    Structure and characterization of NPs delivering 5-FU+pL3 ( A ) Sketched representation of NPs delivering 5-FU+pL3. ( B ) Emission spectra (excitation = 530 nm) of ethidium bromide in the presence of pL3-loaded PLGA@PEI (0–100 μg/mL). ( C ) Gel retardation assay before and after sample treatment with DNAse: free pL3 (a), pL3-loaded PLGA@PEI (b), pL3-loaded HA-coated PLGA@PEI (c), (pL3 was 2 μg/mL). ( D ) Size distribution of the sample during the layering procedure. ( E ) Release profile of 5-FU and pL3 from combined NPs in DMEM FBS + .
    Figure Legend Snippet: Structure and characterization of NPs delivering 5-FU+pL3 ( A ) Sketched representation of NPs delivering 5-FU+pL3. ( B ) Emission spectra (excitation = 530 nm) of ethidium bromide in the presence of pL3-loaded PLGA@PEI (0–100 μg/mL). ( C ) Gel retardation assay before and after sample treatment with DNAse: free pL3 (a), pL3-loaded PLGA@PEI (b), pL3-loaded HA-coated PLGA@PEI (c), (pL3 was 2 μg/mL). ( D ) Size distribution of the sample during the layering procedure. ( E ) Release profile of 5-FU and pL3 from combined NPs in DMEM FBS + .

    Techniques Used: Electrophoretic Mobility Shift Assay

    2) Product Images from "Requirements for gene silencing mediated by U1 snRNA binding to a target sequence"

    Article Title: Requirements for gene silencing mediated by U1 snRNA binding to a target sequence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn068

    Inhibitory activity of U1 snRNAs having extended 5′ends. ( A ). Exogenous 5′-end mutated U1 snRNA inhibits the expression of the targeted Renilla reporter in a dose-dependent manner. HeLa cells were cotransfected with a Renilla pRL/87mtU1 reporter that contains a mtU1 binding site at position –87 and variable amounts (1, 2 or 3 µg) of mtU1/+0 plasmid that expresses a 5′-end mutated U1 snRNA designed to base pair to the mtU1-binding site as indicated. ‘+0’ serves to indicate a normal length 5′ end as opposed to an extended 5′ end. As a control, a plasmid expressing wtU1 was used in place of the mtU1/+0 plasmid. In all cases, a firefly plasmid was also cotransfected for normalization purposes. The inhibitory activity of the mtU1/+0 plasmid was calculated by dividing the normalized Renilla activity of the transfection with the pRL/87mtU1 + wtU1 plasmids by the normalized Renilla activity of the transfection with the pRL/87mtU1 + mtU1/+0 plasmids. The wtU1 plasmid did not affect expression of pRL/87mtU1 and therefore had an inhibitory value set to 1.0. The standard deviations were calculated from three independent experiments. ( B ) 5′ end extended U1 snRNAs do not increase inhibitory activity. The plasmid that expresses mtU1/+0 was used as a parental vector to construct plasmids that express U1 snRNA extended at the 5′-end +1, +2, +3, +4, +5, +6 and +15 nt (mutU1/+1, mutU1/+2, etc.). These extensions increase the length of the duplex formed between pRL/87mtU1 pre-mRNA and the mtU1-derived snRNA. The sequence of the 5′ end of the U1 snRNA and the duplex length is indicated for each case. As described in (A), all plasmids were transfected along with pRL/87mtU1 and firefly luciferase and their relative inhibitory activities were calculated. The inhibitory activities are derived from five different experiments. Standard deviations are not shown but were
    Figure Legend Snippet: Inhibitory activity of U1 snRNAs having extended 5′ends. ( A ). Exogenous 5′-end mutated U1 snRNA inhibits the expression of the targeted Renilla reporter in a dose-dependent manner. HeLa cells were cotransfected with a Renilla pRL/87mtU1 reporter that contains a mtU1 binding site at position –87 and variable amounts (1, 2 or 3 µg) of mtU1/+0 plasmid that expresses a 5′-end mutated U1 snRNA designed to base pair to the mtU1-binding site as indicated. ‘+0’ serves to indicate a normal length 5′ end as opposed to an extended 5′ end. As a control, a plasmid expressing wtU1 was used in place of the mtU1/+0 plasmid. In all cases, a firefly plasmid was also cotransfected for normalization purposes. The inhibitory activity of the mtU1/+0 plasmid was calculated by dividing the normalized Renilla activity of the transfection with the pRL/87mtU1 + wtU1 plasmids by the normalized Renilla activity of the transfection with the pRL/87mtU1 + mtU1/+0 plasmids. The wtU1 plasmid did not affect expression of pRL/87mtU1 and therefore had an inhibitory value set to 1.0. The standard deviations were calculated from three independent experiments. ( B ) 5′ end extended U1 snRNAs do not increase inhibitory activity. The plasmid that expresses mtU1/+0 was used as a parental vector to construct plasmids that express U1 snRNA extended at the 5′-end +1, +2, +3, +4, +5, +6 and +15 nt (mutU1/+1, mutU1/+2, etc.). These extensions increase the length of the duplex formed between pRL/87mtU1 pre-mRNA and the mtU1-derived snRNA. The sequence of the 5′ end of the U1 snRNA and the duplex length is indicated for each case. As described in (A), all plasmids were transfected along with pRL/87mtU1 and firefly luciferase and their relative inhibitory activities were calculated. The inhibitory activities are derived from five different experiments. Standard deviations are not shown but were

    Techniques Used: Activity Assay, Expressing, Binding Assay, Plasmid Preparation, Transfection, Construct, Derivative Assay, Sequencing, Luciferase

    Effect of U1-binding site:U1 snRNA duplex length on inhibition. ( A ) Shown is a series of Renilla luciferase plasmids with different U1-binding sites cloned at 145 nt from the poly(A) signal (pRL/145/x). Shown in red are nucleotides from the U1-binding site able to bind endogenous U1 snRNA. The U1-binding site:U1 snRNA duplex length is indicated for each case. As was done in the Supplementary Data Figure S1 , HeLa cells were transfected with these plasmids along with a firefly luciferase plasmid for normalization purposes. Inhibitory activities were calculated as in the Supplementary Data Figure S1 and the bar graph summarizes five independent experiments. The pRL/145mtU1 is the reference control plasmid that matches pRL/145/+16 except for three point mutations in the U1-binding site. ( B ) An electrophoretic mobility shift assay (EMSA) was used to detect binding of purified U1 snRNP to 0.03 pmol 32P-radiolabeled RNAs with various types of U1-binding sites. EMSA conditions were as previously described ( 18 ). The RNAs are all matching except for differences in the U1-binding site sequence. Lanes 1–9 contain 32P labeled U1-10 RNA that has a 10-nt wt U1-binding site. Lanes 10 and 11 contain U1-7 RNA that matches U1-10 RNA except the U1-binding site is 7-nt long. Lanes 12–15 contain U1-8 RNA that matches U1-10 RNA except the U1-binding site is 8-nt long. The amounts of purified U1 snRNP added are indicated (note 100 ng U1 snRNP = 0.3 pmol). The purification of U1 snRNP is described in the Supplementary Data Figure S2 . The experiment was repeated ×5, quantitated by phosphorimagery and a kDa of 4 +/− 1.5 nM was calculated for the U1 snRNP:U1-10 RNA complex. The U1-8 RNA bound about 3× weaker than the U1-10 RNA and no detectable binding to the U1-7 RNA was observed under these conditions.
    Figure Legend Snippet: Effect of U1-binding site:U1 snRNA duplex length on inhibition. ( A ) Shown is a series of Renilla luciferase plasmids with different U1-binding sites cloned at 145 nt from the poly(A) signal (pRL/145/x). Shown in red are nucleotides from the U1-binding site able to bind endogenous U1 snRNA. The U1-binding site:U1 snRNA duplex length is indicated for each case. As was done in the Supplementary Data Figure S1 , HeLa cells were transfected with these plasmids along with a firefly luciferase plasmid for normalization purposes. Inhibitory activities were calculated as in the Supplementary Data Figure S1 and the bar graph summarizes five independent experiments. The pRL/145mtU1 is the reference control plasmid that matches pRL/145/+16 except for three point mutations in the U1-binding site. ( B ) An electrophoretic mobility shift assay (EMSA) was used to detect binding of purified U1 snRNP to 0.03 pmol 32P-radiolabeled RNAs with various types of U1-binding sites. EMSA conditions were as previously described ( 18 ). The RNAs are all matching except for differences in the U1-binding site sequence. Lanes 1–9 contain 32P labeled U1-10 RNA that has a 10-nt wt U1-binding site. Lanes 10 and 11 contain U1-7 RNA that matches U1-10 RNA except the U1-binding site is 7-nt long. Lanes 12–15 contain U1-8 RNA that matches U1-10 RNA except the U1-binding site is 8-nt long. The amounts of purified U1 snRNP added are indicated (note 100 ng U1 snRNP = 0.3 pmol). The purification of U1 snRNP is described in the Supplementary Data Figure S2 . The experiment was repeated ×5, quantitated by phosphorimagery and a kDa of 4 +/− 1.5 nM was calculated for the U1 snRNP:U1-10 RNA complex. The U1-8 RNA bound about 3× weaker than the U1-10 RNA and no detectable binding to the U1-7 RNA was observed under these conditions.

    Techniques Used: Binding Assay, Inhibition, Luciferase, Clone Assay, Transfection, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Labeling

    Analysis of the role of RS domains in U1i. ( A ) Schematic of mutU1/+0/MS2, the modified U1 snRNA used to test the inhibitory activity of various MS2 fusion proteins. mutU1/+0/MS2 is identical to U1 snRNP shown in the Supplementary Data Figure S1A but loop 1 has been replaced by an MS2-binding sequence and the 5′-end binds a mutated sequence in the 3′ terminal exon of the Renilla reporter plasmid as shown. ( B ) Analysis of the expression of MS2 fusion proteins. HeLa cells were transfected with the plasmids that express a control protein, MS2 alone or MS2 fused to the RS region of U1-70K (MS2/70K) or the RS region of U2AF65 (MS2/U2AF65) and extracts were collected at 48 h posttransfection. MS2 expression was evaluated in the extracts by western blot analysis. ( C ) Reconstitution of functional U1i complexes by tethering RS domains to the loop 1 of U1 snRNA. HeLa cells were cotransfected with three plasmids: (i) a Renilla construct that binds mutU1/+0/MS2, (ii) a plasmid that expresses either a control protein, MS2 alone or MS2 fused to the RS region of U1-70K (MS2/70K) or the RS region of U2AF65 (MS2/U2AF65) and (iii) a plasmid that expresses either mutU1/+0/MS2 or a control U1 snRNA. In all cases, a plasmid expressing firefly luciferase was also cotransfected as a control. Extracts were collected at 48 h posttransfection and luciferase activity was evaluated. All data were normalized to firefly luciferase expression. Renilla expression in the presence of a control U1 snRNA was similar in all cases and was used to calculate the fold inhibition. ( D ) Design of a Renilla reporter that tethers RS domains upstream of an active wtU1-binding site. The two MS2 stem–loops are 42-nt apart and collectively are 17-nt upstream of the wtU1-binding site. ( E ) Disruption of the inhibitory activity of the wtU1-binding site by a cotransfected MS2 fusion protein. Shown are the results where the reporter in (D) is cotransfected with either an empty vector ‘no MS2’ or an MS2 fusion expression plasmid that expresses MS2 protein or MS2 fused to RS domains from various SR proteins as indicated. The MS2/mtU2AF65 and MS2/mtASF/SF2 are controls that express a mutated RS domain from U2AF65 or ASF/SF2, respectively. The mutations and the RS domain are as previously described ( 46 ). Western blotting was used to confirm that the MS2 fusion proteins were expressed to a similar level (data not shown).
    Figure Legend Snippet: Analysis of the role of RS domains in U1i. ( A ) Schematic of mutU1/+0/MS2, the modified U1 snRNA used to test the inhibitory activity of various MS2 fusion proteins. mutU1/+0/MS2 is identical to U1 snRNP shown in the Supplementary Data Figure S1A but loop 1 has been replaced by an MS2-binding sequence and the 5′-end binds a mutated sequence in the 3′ terminal exon of the Renilla reporter plasmid as shown. ( B ) Analysis of the expression of MS2 fusion proteins. HeLa cells were transfected with the plasmids that express a control protein, MS2 alone or MS2 fused to the RS region of U1-70K (MS2/70K) or the RS region of U2AF65 (MS2/U2AF65) and extracts were collected at 48 h posttransfection. MS2 expression was evaluated in the extracts by western blot analysis. ( C ) Reconstitution of functional U1i complexes by tethering RS domains to the loop 1 of U1 snRNA. HeLa cells were cotransfected with three plasmids: (i) a Renilla construct that binds mutU1/+0/MS2, (ii) a plasmid that expresses either a control protein, MS2 alone or MS2 fused to the RS region of U1-70K (MS2/70K) or the RS region of U2AF65 (MS2/U2AF65) and (iii) a plasmid that expresses either mutU1/+0/MS2 or a control U1 snRNA. In all cases, a plasmid expressing firefly luciferase was also cotransfected as a control. Extracts were collected at 48 h posttransfection and luciferase activity was evaluated. All data were normalized to firefly luciferase expression. Renilla expression in the presence of a control U1 snRNA was similar in all cases and was used to calculate the fold inhibition. ( D ) Design of a Renilla reporter that tethers RS domains upstream of an active wtU1-binding site. The two MS2 stem–loops are 42-nt apart and collectively are 17-nt upstream of the wtU1-binding site. ( E ) Disruption of the inhibitory activity of the wtU1-binding site by a cotransfected MS2 fusion protein. Shown are the results where the reporter in (D) is cotransfected with either an empty vector ‘no MS2’ or an MS2 fusion expression plasmid that expresses MS2 protein or MS2 fused to RS domains from various SR proteins as indicated. The MS2/mtU2AF65 and MS2/mtASF/SF2 are controls that express a mutated RS domain from U2AF65 or ASF/SF2, respectively. The mutations and the RS domain are as previously described ( 46 ). Western blotting was used to confirm that the MS2 fusion proteins were expressed to a similar level (data not shown).

    Techniques Used: Modification, Activity Assay, Binding Assay, Sequencing, Plasmid Preparation, Expressing, Transfection, Western Blot, Functional Assay, Construct, Luciferase, Inhibition

    Splicing regulatory sequences interfere with U1 snRNP's poly(A) site inhibitory activity. ( A ) Plasmid pRL/145mtU1, whose binding to endogenous U1 snRNP is schematized at the top of the figure, served as a parental plasmid to insert specific sequences upstream or downstream of the U1 target site, as indicated. Sequences chosen are control sequences (UpX, UpXX or Down X), or sequences A, J, SF2, SR and TIA-1, which bind unknown factors (A and J), SF2/ASF, SRp40 and TIA-1, respectively, in the nucleotides shown in bold. The rest of the sequence has been included to keep the context that is known to affect splicing activity. ( B ) Each Renilla plasmid was transfected into HeLa cells along with a firefly luciferase expressing plasmid as a transfection control and a U1 snRNA expression plasmid: either wtU1, or mtU1/0 (same as in Figure 1) that should form a 10-bp duplex or 8bpmtU1 that should form an 8-bp duplex. Indicated at the top of the panel is the predicted duplex formed between the exogenous U1 snRNA and the U1 target site sequence. Luciferase activity was measured and normalized to calculate the inhibitory activity in each case. The results show the average of three independent experiments.
    Figure Legend Snippet: Splicing regulatory sequences interfere with U1 snRNP's poly(A) site inhibitory activity. ( A ) Plasmid pRL/145mtU1, whose binding to endogenous U1 snRNP is schematized at the top of the figure, served as a parental plasmid to insert specific sequences upstream or downstream of the U1 target site, as indicated. Sequences chosen are control sequences (UpX, UpXX or Down X), or sequences A, J, SF2, SR and TIA-1, which bind unknown factors (A and J), SF2/ASF, SRp40 and TIA-1, respectively, in the nucleotides shown in bold. The rest of the sequence has been included to keep the context that is known to affect splicing activity. ( B ) Each Renilla plasmid was transfected into HeLa cells along with a firefly luciferase expressing plasmid as a transfection control and a U1 snRNA expression plasmid: either wtU1, or mtU1/0 (same as in Figure 1) that should form a 10-bp duplex or 8bpmtU1 that should form an 8-bp duplex. Indicated at the top of the panel is the predicted duplex formed between the exogenous U1 snRNA and the U1 target site sequence. Luciferase activity was measured and normalized to calculate the inhibitory activity in each case. The results show the average of three independent experiments.

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

    3) Product Images from "Requirements for gene silencing mediated by U1 snRNA binding to a target sequence"

    Article Title: Requirements for gene silencing mediated by U1 snRNA binding to a target sequence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn068

    Analysis of U1-binding sites with double-point mutations at positions 1 and 2, 1 and 10 or 9 and 10. A saturation mutagenesis analysis was performed where all possible double mutations were introduced at positions 1 and 2 ( A ) 1 and 10 ( B ) and 9 and 10 ( C ) of the 10-nt wtU1-binding site of the pRL/180wtU1 plasmid shown in the Supplementary Data Figure S1 . The mutations were analyzed and graphed as in Figure 3 . The sequence of the wtU1-binding site and its base pairing with the 11 nt of the 5′-end of U1 snRNA (blue font) is schematized above the graph. Each pair of letters indicates a particular mutant where the lowercase red letters correspond to a mutation, while the uppercase black letters match the wtU1-binding site. Each pair of letters is positioned above its corresponding bar graph representing the inhibitory activity of that mutated U1 site. Also indicated are the N H values that are defined as the number of continuous hydrogen bonds (see Discussion section). To facilitate comparison we included the single-point mutations from Figure 2 as red histograms. All double mutants (totals) or double mutants from (A) (1 and 2), (B) (1 and 10) and (C) (1 and 10) were classified according to their good, low or no inhibitory activity ( > 5-, 2–5- and ≤ 2-fold inhibition, respectively). Shown is the number of double mutants in each group ( D ).
    Figure Legend Snippet: Analysis of U1-binding sites with double-point mutations at positions 1 and 2, 1 and 10 or 9 and 10. A saturation mutagenesis analysis was performed where all possible double mutations were introduced at positions 1 and 2 ( A ) 1 and 10 ( B ) and 9 and 10 ( C ) of the 10-nt wtU1-binding site of the pRL/180wtU1 plasmid shown in the Supplementary Data Figure S1 . The mutations were analyzed and graphed as in Figure 3 . The sequence of the wtU1-binding site and its base pairing with the 11 nt of the 5′-end of U1 snRNA (blue font) is schematized above the graph. Each pair of letters indicates a particular mutant where the lowercase red letters correspond to a mutation, while the uppercase black letters match the wtU1-binding site. Each pair of letters is positioned above its corresponding bar graph representing the inhibitory activity of that mutated U1 site. Also indicated are the N H values that are defined as the number of continuous hydrogen bonds (see Discussion section). To facilitate comparison we included the single-point mutations from Figure 2 as red histograms. All double mutants (totals) or double mutants from (A) (1 and 2), (B) (1 and 10) and (C) (1 and 10) were classified according to their good, low or no inhibitory activity ( > 5-, 2–5- and ≤ 2-fold inhibition, respectively). Shown is the number of double mutants in each group ( D ).

    Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, Sequencing, Activity Assay, Inhibition

    Effect of secondary structure on U1-binding site inhibition. ( A ) The pRL/145/stem0 plasmid has a 13-nt wtU1-binding site (the canonical 10 nt are highlighted in yellow) completely occluded in a stem–loop sequence. The ‘0’ indicates 0 nt of the U1-binding site should be found outside of the stem. As diagrammed, a collection of plasmids were made and tested that match pRL/145/stem0 except the U1-binding site increasingly moves out of the stem. The number below each stem–loop structure indicates the number of U1-binding site nucleotides that should be found outside of the stem (3, 6, 7, 8, 9 or 13). ( B ) The plasmids were analyzed and graphed as indicated in Figure 3 . Error bars indicate standard deviations of four different experiments.
    Figure Legend Snippet: Effect of secondary structure on U1-binding site inhibition. ( A ) The pRL/145/stem0 plasmid has a 13-nt wtU1-binding site (the canonical 10 nt are highlighted in yellow) completely occluded in a stem–loop sequence. The ‘0’ indicates 0 nt of the U1-binding site should be found outside of the stem. As diagrammed, a collection of plasmids were made and tested that match pRL/145/stem0 except the U1-binding site increasingly moves out of the stem. The number below each stem–loop structure indicates the number of U1-binding site nucleotides that should be found outside of the stem (3, 6, 7, 8, 9 or 13). ( B ) The plasmids were analyzed and graphed as indicated in Figure 3 . Error bars indicate standard deviations of four different experiments.

    Techniques Used: Binding Assay, Inhibition, Plasmid Preparation, Sequencing

    Analysis of single-point mutations of the U1-binding site. A saturation mutagenesis analysis was performed where all 30 possible single-point mutations were introduced in the 10-nt wtU1-binding site of the pRL/180wtU1 plasmid shown in the Supplementary Data Figure S1 . Each plasmid was cotransfected with the firefly luciferase control into HeLa cells and inhibitory activities were calculated as described in the Supplementary Data Figure. S1 . The results are plotted in a bars graph where error bars indicate standard deviations of five independent experiments. The sequence of the wtU1-binding site and its base pairing with the 11 nt of the 5′-end of U1 snRNA (blue font) is schematized above the graph. Each dotted line indicates the mutation (in red font) which is positioned above its corresponding bar graph representing the inhibitory activity of that mutation. The inhibitory activity of the reporter with a wtU1-binding site is shown in green to the left of the graph. The 6G and 7G single mutants were combined to give the 6G/7G double mutant whose activity is shown on the far right.
    Figure Legend Snippet: Analysis of single-point mutations of the U1-binding site. A saturation mutagenesis analysis was performed where all 30 possible single-point mutations were introduced in the 10-nt wtU1-binding site of the pRL/180wtU1 plasmid shown in the Supplementary Data Figure S1 . Each plasmid was cotransfected with the firefly luciferase control into HeLa cells and inhibitory activities were calculated as described in the Supplementary Data Figure. S1 . The results are plotted in a bars graph where error bars indicate standard deviations of five independent experiments. The sequence of the wtU1-binding site and its base pairing with the 11 nt of the 5′-end of U1 snRNA (blue font) is schematized above the graph. Each dotted line indicates the mutation (in red font) which is positioned above its corresponding bar graph representing the inhibitory activity of that mutation. The inhibitory activity of the reporter with a wtU1-binding site is shown in green to the left of the graph. The 6G and 7G single mutants were combined to give the 6G/7G double mutant whose activity is shown on the far right.

    Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, Luciferase, Sequencing, Activity Assay

    Effect of U1-binding site:U1 snRNA duplex length on inhibition. ( A ) Shown is a series of Renilla luciferase plasmids with different U1-binding sites cloned at 145 nt from the poly(A) signal (pRL/145/x). Shown in red are nucleotides from the U1-binding site able to bind endogenous U1 snRNA. The U1-binding site:U1 snRNA duplex length is indicated for each case. As was done in the Supplementary Data Figure S1 , HeLa cells were transfected with these plasmids along with a firefly luciferase plasmid for normalization purposes. Inhibitory activities were calculated as in the Supplementary Data Figure S1 and the bar graph summarizes five independent experiments. The pRL/145mtU1 is the reference control plasmid that matches pRL/145/+16 except for three point mutations in the U1-binding site. ( B ) An electrophoretic mobility shift assay (EMSA) was used to detect binding of purified U1 snRNP to 0.03 pmol 32P-radiolabeled RNAs with various types of U1-binding sites. EMSA conditions were as previously described ( 18 ). The RNAs are all matching except for differences in the U1-binding site sequence. Lanes 1–9 contain 32P labeled U1-10 RNA that has a 10-nt wt U1-binding site. Lanes 10 and 11 contain U1-7 RNA that matches U1-10 RNA except the U1-binding site is 7-nt long. Lanes 12–15 contain U1-8 RNA that matches U1-10 RNA except the U1-binding site is 8-nt long. The amounts of purified U1 snRNP added are indicated (note 100 ng U1 snRNP = 0.3 pmol). The purification of U1 snRNP is described in the Supplementary Data Figure S2 . The experiment was repeated ×5, quantitated by phosphorimagery and a kDa of 4 +/− 1.5 nM was calculated for the U1 snRNP:U1-10 RNA complex. The U1-8 RNA bound about 3× weaker than the U1-10 RNA and no detectable binding to the U1-7 RNA was observed under these conditions.
    Figure Legend Snippet: Effect of U1-binding site:U1 snRNA duplex length on inhibition. ( A ) Shown is a series of Renilla luciferase plasmids with different U1-binding sites cloned at 145 nt from the poly(A) signal (pRL/145/x). Shown in red are nucleotides from the U1-binding site able to bind endogenous U1 snRNA. The U1-binding site:U1 snRNA duplex length is indicated for each case. As was done in the Supplementary Data Figure S1 , HeLa cells were transfected with these plasmids along with a firefly luciferase plasmid for normalization purposes. Inhibitory activities were calculated as in the Supplementary Data Figure S1 and the bar graph summarizes five independent experiments. The pRL/145mtU1 is the reference control plasmid that matches pRL/145/+16 except for three point mutations in the U1-binding site. ( B ) An electrophoretic mobility shift assay (EMSA) was used to detect binding of purified U1 snRNP to 0.03 pmol 32P-radiolabeled RNAs with various types of U1-binding sites. EMSA conditions were as previously described ( 18 ). The RNAs are all matching except for differences in the U1-binding site sequence. Lanes 1–9 contain 32P labeled U1-10 RNA that has a 10-nt wt U1-binding site. Lanes 10 and 11 contain U1-7 RNA that matches U1-10 RNA except the U1-binding site is 7-nt long. Lanes 12–15 contain U1-8 RNA that matches U1-10 RNA except the U1-binding site is 8-nt long. The amounts of purified U1 snRNP added are indicated (note 100 ng U1 snRNP = 0.3 pmol). The purification of U1 snRNP is described in the Supplementary Data Figure S2 . The experiment was repeated ×5, quantitated by phosphorimagery and a kDa of 4 +/− 1.5 nM was calculated for the U1 snRNP:U1-10 RNA complex. The U1-8 RNA bound about 3× weaker than the U1-10 RNA and no detectable binding to the U1-7 RNA was observed under these conditions.

    Techniques Used: Binding Assay, Inhibition, Luciferase, Clone Assay, Transfection, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Labeling

    4) Product Images from "Acquisition of temozolomide resistance by the rat C6 glioma cell line increases cell migration and side population phenotype"

    Article Title: Acquisition of temozolomide resistance by the rat C6 glioma cell line increases cell migration and side population phenotype

    Journal: Oncology Reports

    doi: 10.3892/or.2019.7350

    TMZ-resistant rat glioma (C6-PL3) cells do not possess typical properties of cancer stem cells. (A) The ability of C6-PL3 cells and parental rat glioma (C6) cells to form tumor spheres was observed under phase-contrast microscopy. (B) Expression of Nestin, SOX2, BMI1, and β-actin in C6-PL3 and C6 cells was analyzed using western blotting. (C) Expression of nestin in C6-PL3 and C6 cells was analyzed using immunofluorescence. TMZ, temozolomide.
    Figure Legend Snippet: TMZ-resistant rat glioma (C6-PL3) cells do not possess typical properties of cancer stem cells. (A) The ability of C6-PL3 cells and parental rat glioma (C6) cells to form tumor spheres was observed under phase-contrast microscopy. (B) Expression of Nestin, SOX2, BMI1, and β-actin in C6-PL3 and C6 cells was analyzed using western blotting. (C) Expression of nestin in C6-PL3 and C6 cells was analyzed using immunofluorescence. TMZ, temozolomide.

    Techniques Used: Microscopy, Expressing, Western Blot, Immunofluorescence

    Drug resistance in parental rat glioma cells (C6 cells) induced promotion of cell invasion. (A) Transwell assays were carried out for TMZ-resistant rat glioma cells (C6-PL3) and C6 cells. Images of cells stained with Giemsa invading through Matrigel in a representative experiment are presented, and results of 15 different regions in three independent experiments (five regions per experiment) are summarized in (B) with an ×200 magnification. (C-H) Rats were allografted with C6-PL3 and C6 cells for 20 days. Brain slices were collected and stained. Representative images of brain slices collected from different rats are presented, with arrows indicating C6 glioma cells. Dotted lines indicate the boundary between tumor and normal brain tissue. (I) Quantitative results from both groups (three rats/group) are summarized. **P
    Figure Legend Snippet: Drug resistance in parental rat glioma cells (C6 cells) induced promotion of cell invasion. (A) Transwell assays were carried out for TMZ-resistant rat glioma cells (C6-PL3) and C6 cells. Images of cells stained with Giemsa invading through Matrigel in a representative experiment are presented, and results of 15 different regions in three independent experiments (five regions per experiment) are summarized in (B) with an ×200 magnification. (C-H) Rats were allografted with C6-PL3 and C6 cells for 20 days. Brain slices were collected and stained. Representative images of brain slices collected from different rats are presented, with arrows indicating C6 glioma cells. Dotted lines indicate the boundary between tumor and normal brain tissue. (I) Quantitative results from both groups (three rats/group) are summarized. **P

    Techniques Used: Staining

    Most TMZ-resistant rat glioma (C6-PL3) cells are SP cells. (A) The expression of BRCP1, MDR1, and β-actin in C6-PL3 and parental rat glioma (C6) cells was analyzed using western blotting. (B) C6-PL3 and C6 cells treated with or without 50 µM verapamil were labeled with the Hoechst 33342 and analyzed using flow cytometry. TMZ, temozolomide; SP, side population.
    Figure Legend Snippet: Most TMZ-resistant rat glioma (C6-PL3) cells are SP cells. (A) The expression of BRCP1, MDR1, and β-actin in C6-PL3 and parental rat glioma (C6) cells was analyzed using western blotting. (B) C6-PL3 and C6 cells treated with or without 50 µM verapamil were labeled with the Hoechst 33342 and analyzed using flow cytometry. TMZ, temozolomide; SP, side population.

    Techniques Used: Expressing, Western Blot, Labeling, Flow Cytometry, Cytometry

    Phase-contrast microscopy indicated morphological changes in (A and C) temozolomide-resistant rat glioma cells (C6-PL3) and (B and D) parental rat glioma cells (C6 cells). Representative images of (E) C6-PL3 cells and (F) C6 cells under F-actin staining. Images of F-actin were obtained at a magnification of ×400.
    Figure Legend Snippet: Phase-contrast microscopy indicated morphological changes in (A and C) temozolomide-resistant rat glioma cells (C6-PL3) and (B and D) parental rat glioma cells (C6 cells). Representative images of (E) C6-PL3 cells and (F) C6 cells under F-actin staining. Images of F-actin were obtained at a magnification of ×400.

    Techniques Used: Microscopy, Staining

    Verapamil increases TMZ-induced tumor sphere formation and apoptosis in TMZ-resistant rat glioma (C6-PL3) and parental rat glioma (C6) cells. (A) Tumor sphere formation in C6-PL3 and C6 cells was observed using phase-contrast microscopy after TMZ and verapamil treatment. (B and C) After treatment, apoptosis was detected using flow cytometry. The average apoptosis rate of TMZ treatment was 4.79%, while after adding the verapamil, the average apoptosis rate was 81.61%. ***P
    Figure Legend Snippet: Verapamil increases TMZ-induced tumor sphere formation and apoptosis in TMZ-resistant rat glioma (C6-PL3) and parental rat glioma (C6) cells. (A) Tumor sphere formation in C6-PL3 and C6 cells was observed using phase-contrast microscopy after TMZ and verapamil treatment. (B and C) After treatment, apoptosis was detected using flow cytometry. The average apoptosis rate of TMZ treatment was 4.79%, while after adding the verapamil, the average apoptosis rate was 81.61%. ***P

    Techniques Used: Microscopy, Flow Cytometry, Cytometry

    5) Product Images from "The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer"

    Article Title: The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2018.11.031

    Integrinβ1 rescues stalling of replication forks caused by MT1-MMP inhibition. A) % of replication forks. A total of 200 DNA fibers were counted in all groups. % of all fibers was calculated by the Student’s t test. B) Representative CldU and IdU stained DNA fibers. Red: stalled forks; Green: new origin; Red+Green: restarted forks after Hydroxy Urea.
    Figure Legend Snippet: Integrinβ1 rescues stalling of replication forks caused by MT1-MMP inhibition. A) % of replication forks. A total of 200 DNA fibers were counted in all groups. % of all fibers was calculated by the Student’s t test. B) Representative CldU and IdU stained DNA fibers. Red: stalled forks; Green: new origin; Red+Green: restarted forks after Hydroxy Urea.

    Techniques Used: Inhibition, Staining

    MT1-MMP affects ATM, ATR and replication fork stability. A) MDA-MB-231 cells expressing shGFP or shMT1-MMP (left), and MCF7 expressing active and catalytically dead MT1-MMP (right), showing phosphorylation levels of H2AX, Chk1, Chk2 and RPA32. B) Scheme of treatment. Red only fibers: stalled forks; Green only fibers: new origin; Red+Green: restarted forks after Hydroxy Urea. C) % of replication forks. A total of 200 DNA fibers were counted in all groups. % of all fibers was calculated by the Student’s t test. *,**,***p
    Figure Legend Snippet: MT1-MMP affects ATM, ATR and replication fork stability. A) MDA-MB-231 cells expressing shGFP or shMT1-MMP (left), and MCF7 expressing active and catalytically dead MT1-MMP (right), showing phosphorylation levels of H2AX, Chk1, Chk2 and RPA32. B) Scheme of treatment. Red only fibers: stalled forks; Green only fibers: new origin; Red+Green: restarted forks after Hydroxy Urea. C) % of replication forks. A total of 200 DNA fibers were counted in all groups. % of all fibers was calculated by the Student’s t test. *,**,***p

    Techniques Used: Multiple Displacement Amplification, Expressing

    MT1-MMP expression is elevated in breast cancer and inversely correlated to response to chemotherapy. A) Oncomine data comparing mRNA expression of MMP14 (gene name for MT1-MMP) between normal tissue and different subtypes of invasive breast carcinoma (TCGA breast data set). P values for all types compared to normal are
    Figure Legend Snippet: MT1-MMP expression is elevated in breast cancer and inversely correlated to response to chemotherapy. A) Oncomine data comparing mRNA expression of MMP14 (gene name for MT1-MMP) between normal tissue and different subtypes of invasive breast carcinoma (TCGA breast data set). P values for all types compared to normal are

    Techniques Used: Expressing

    Inhibition of MT1-MMP increases DNA DSBs. A) quantification of γH2AX foci in shGFP and shMT1-MMP MDA-MB-231 cells treated with 5Gy IR for the indicated times. B) quantification of γH2AX foci in MCF7 cells expressing an empty vector (pLM), the full length (FL) and the catalytically truncated (ΔCAT) MT1-MMP constructs, treated with 5Gy IR for the indicated times. C-D) Quantification of the tail moment at the indicated times after treatment with 8Gy IR. C - MDA-MB cells expressing shGFP and shMT1-MMP as shown in 2C; D – MCF7 expressing pLM, full length and catalytically truncated MT1-MMP mutant as shown in 2E. P values were calculated by the Student’s t test. Foci were counted in al least 50 cells. Tail moments were measured in at least 50 cells per sample.
    Figure Legend Snippet: Inhibition of MT1-MMP increases DNA DSBs. A) quantification of γH2AX foci in shGFP and shMT1-MMP MDA-MB-231 cells treated with 5Gy IR for the indicated times. B) quantification of γH2AX foci in MCF7 cells expressing an empty vector (pLM), the full length (FL) and the catalytically truncated (ΔCAT) MT1-MMP constructs, treated with 5Gy IR for the indicated times. C-D) Quantification of the tail moment at the indicated times after treatment with 8Gy IR. C - MDA-MB cells expressing shGFP and shMT1-MMP as shown in 2C; D – MCF7 expressing pLM, full length and catalytically truncated MT1-MMP mutant as shown in 2E. P values were calculated by the Student’s t test. Foci were counted in al least 50 cells. Tail moments were measured in at least 50 cells per sample.

    Techniques Used: Inhibition, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Construct, Mutagenesis

    MT1-MMP acts through integrinβ1 A) Quantification of γH2AX foci of MDA-MB-231 cells seeded in MT1-MMP degradable (D) and non-degradable (ND) matrixes for 24 hours. Foci were quantified from at least 50 cells. Data are the mean of two independent experiments. Bottom panel: western blotting to detect γH2AX, pChK1, pChK2, pRPA32 of cells embedded in the matrixes. B) Expression of integrinβ1 V37N (ITGB1) in MDA-MB-231 cells expressing shGFP or shMT1-MMP. pFAK Y297 and the markers in A are also shown. C) Clonogenic assay of the cells in B treated with the indicated IR doses or several doses of doxorubicin for 15 days (the duration of the clonogenic assay). (Left: * , ** , ***p
    Figure Legend Snippet: MT1-MMP acts through integrinβ1 A) Quantification of γH2AX foci of MDA-MB-231 cells seeded in MT1-MMP degradable (D) and non-degradable (ND) matrixes for 24 hours. Foci were quantified from at least 50 cells. Data are the mean of two independent experiments. Bottom panel: western blotting to detect γH2AX, pChK1, pChK2, pRPA32 of cells embedded in the matrixes. B) Expression of integrinβ1 V37N (ITGB1) in MDA-MB-231 cells expressing shGFP or shMT1-MMP. pFAK Y297 and the markers in A are also shown. C) Clonogenic assay of the cells in B treated with the indicated IR doses or several doses of doxorubicin for 15 days (the duration of the clonogenic assay). (Left: * , ** , ***p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, Clonogenic Assay

    Expression of MT1-MMP affects cell responses to genotoxic stresses. A) Expression of MT1-MMP in MDA-MB-231 (left), and other cell lines after removal of MDA-MB-231 and exposure of the film for a longer time (right). B) ER, PR, HER2 status of the cell lines in A (all cells are BRCA1WT). C) Knock down levels of MT1-MMP in MDA-MB-231. D) Clonogenic assay of the cells in C treated with the indicated IR doses or several doses of doxorubicin for 10 days (the duration of the clonogenic assay). * , ** , ***p
    Figure Legend Snippet: Expression of MT1-MMP affects cell responses to genotoxic stresses. A) Expression of MT1-MMP in MDA-MB-231 (left), and other cell lines after removal of MDA-MB-231 and exposure of the film for a longer time (right). B) ER, PR, HER2 status of the cell lines in A (all cells are BRCA1WT). C) Knock down levels of MT1-MMP in MDA-MB-231. D) Clonogenic assay of the cells in C treated with the indicated IR doses or several doses of doxorubicin for 10 days (the duration of the clonogenic assay). * , ** , ***p

    Techniques Used: Expressing, Multiple Displacement Amplification, Clonogenic Assay

    6) Product Images from "Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase"

    Article Title: Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00116-15

    Furin is a direct target of Notch1. (A) Schematic representation of the furin promoter. There are three putative CSL binding sequences (TTCCCAC), located at bp −3556 (B1), −1236 (B2), and +960 (B3). TSS, transcription start site. (B) Western
    Figure Legend Snippet: Furin is a direct target of Notch1. (A) Schematic representation of the furin promoter. There are three putative CSL binding sequences (TTCCCAC), located at bp −3556 (B1), −1236 (B2), and +960 (B3). TSS, transcription start site. (B) Western

    Techniques Used: Binding Assay, Western Blot

    Notch1 affects furin expression through binding to the furin promoter. (A) Schematic representation of luciferase reporter constructs containing the WT (TTCCCAC) or mutated (TCACAGC) binding site 2 sequence. The pGL2-SacI construct contains the sequence
    Figure Legend Snippet: Notch1 affects furin expression through binding to the furin promoter. (A) Schematic representation of luciferase reporter constructs containing the WT (TTCCCAC) or mutated (TCACAGC) binding site 2 sequence. The pGL2-SacI construct contains the sequence

    Techniques Used: Expressing, Binding Assay, Luciferase, Construct, Sequencing

    Inhibition of furin decreases Notch1 processing. (A) Expression of full-length (FL) and intracellular (NIC) Notch1 (probed with antibody mN1A), Notch-NIC (probed with an antibody that recognizes Notch1 NIC cleaved at Val-1744), MT1-MMP, ADAM10, and furin
    Figure Legend Snippet: Inhibition of furin decreases Notch1 processing. (A) Expression of full-length (FL) and intracellular (NIC) Notch1 (probed with antibody mN1A), Notch-NIC (probed with an antibody that recognizes Notch1 NIC cleaved at Val-1744), MT1-MMP, ADAM10, and furin

    Techniques Used: Inhibition, Expressing

    Notch1 induces MT1-MMP activation through furin. (A) Western blot analysis showing that the active fraction of MT1-MMP increases in NIC-expressing cells but decreases in furin knockdown cells. Experiments were carried out in three cell lines: SKMel2,
    Figure Legend Snippet: Notch1 induces MT1-MMP activation through furin. (A) Western blot analysis showing that the active fraction of MT1-MMP increases in NIC-expressing cells but decreases in furin knockdown cells. Experiments were carried out in three cell lines: SKMel2,

    Techniques Used: Activation Assay, Western Blot, Expressing

    Stimulation of endogenous Notch1. JAGGED1-dependent activation of endogenous Notch1 increases furin levels and promotes the processing of MT1-MMP and ADAM10 in two melanoma cell lines.
    Figure Legend Snippet: Stimulation of endogenous Notch1. JAGGED1-dependent activation of endogenous Notch1 increases furin levels and promotes the processing of MT1-MMP and ADAM10 in two melanoma cell lines.

    Techniques Used: Activation Assay

    Notch1 increases the active fraction of ADAM10 through furin. (A) Western blot analysis showing the expression of the propeptide and active forms of ADAM10 and the expression of Notch1-NIC in NIC-expressing SKMel2 (left) and V2387 (right) cells in the
    Figure Legend Snippet: Notch1 increases the active fraction of ADAM10 through furin. (A) Western blot analysis showing the expression of the propeptide and active forms of ADAM10 and the expression of Notch1-NIC in NIC-expressing SKMel2 (left) and V2387 (right) cells in the

    Techniques Used: Western Blot, Expressing

    Furin inhibition reduces migration and invasion by melanoma cells. (A) Expression levels of Notch1-NIC and furin in cells expressing active Notch1 and furin shRNA. The same cells were used for all panels. (B) Migration of the cells (determined by a scratch
    Figure Legend Snippet: Furin inhibition reduces migration and invasion by melanoma cells. (A) Expression levels of Notch1-NIC and furin in cells expressing active Notch1 and furin shRNA. The same cells were used for all panels. (B) Migration of the cells (determined by a scratch

    Techniques Used: Inhibition, Migration, Expressing, shRNA

    7) Product Images from "Synchronized targeting of Notch and ERBB signaling suppresses melanoma tumor growth through inhibition of Notch1 and ERBB3 *"

    Article Title: Synchronized targeting of Notch and ERBB signaling suppresses melanoma tumor growth through inhibition of Notch1 and ERBB3 *

    Journal: The Journal of investigative dermatology

    doi: 10.1016/j.jid.2015.11.006

    Inhibition of Notch1 and ERBB3 blocks tumor formation A) Growth of WM266-4 cells in SCID mice (n=8). Tumor volumes were normalized to 1 at time 0 (mean vol. 137 µM 3 ), time at which mice were given 50 mg/Kg lapatinib and 10 µMol/Kg DBZ thrice a week. (LAP vs DMSO, p=0.007 ; DBZ vs DMSO, p=0.002 ; DBZ+LAP vs DMSO, p=0.001; DBZ+LAP vs LAP, p=0.008 ; DBZ+LAP vs DBZ, p=0.05 , Student’s t test). B) Growth of WM266-4 cells expressing shRNA control (shGFP) or shRNAs against Notch1 and ERBB3, in SCID mice (n=10). (shGFP vs shNotch1, p
    Figure Legend Snippet: Inhibition of Notch1 and ERBB3 blocks tumor formation A) Growth of WM266-4 cells in SCID mice (n=8). Tumor volumes were normalized to 1 at time 0 (mean vol. 137 µM 3 ), time at which mice were given 50 mg/Kg lapatinib and 10 µMol/Kg DBZ thrice a week. (LAP vs DMSO, p=0.007 ; DBZ vs DMSO, p=0.002 ; DBZ+LAP vs DMSO, p=0.001; DBZ+LAP vs LAP, p=0.008 ; DBZ+LAP vs DBZ, p=0.05 , Student’s t test). B) Growth of WM266-4 cells expressing shRNA control (shGFP) or shRNAs against Notch1 and ERBB3, in SCID mice (n=10). (shGFP vs shNotch1, p

    Techniques Used: Inhibition, Mouse Assay, Expressing, shRNA

    Inhibition of Notch and ERBB inhibits AKT and NFκB activity A) Effects of lapatinib (10µM) on the phosphorylation levels of ERBB3, ERBB2, EGFR in WM266-4 cells. B) DBZ (10µM) and lapatinib (10µM) effects on Notch1 NIC , HEY1, phosphorylated AKT and ERK1/2 in WM266-4 cells. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. C) Expression levels of Notch1, ERBB3, HEY1, phosphorilated AKT and ERK in cells (WM266-4) expressing specific shRNAs against Notch1 and ERBB3. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. D–E) NFκB Reporter assay in cells treated with DBZ and lapatinib or expressing shGFP, shNotch1, shERBB3 or the two together. P values are calculated by the Student’s T test. Data are the average±SD between at least three independent experiments. F–G) Cytosolic and nuclear distribution of the NFκB elements p65 and p50 in DBZ/lapatinib treated cells ( F ) or in cells expressing shRNAs against Notch1 and ERBB3 ( G ).
    Figure Legend Snippet: Inhibition of Notch and ERBB inhibits AKT and NFκB activity A) Effects of lapatinib (10µM) on the phosphorylation levels of ERBB3, ERBB2, EGFR in WM266-4 cells. B) DBZ (10µM) and lapatinib (10µM) effects on Notch1 NIC , HEY1, phosphorylated AKT and ERK1/2 in WM266-4 cells. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. C) Expression levels of Notch1, ERBB3, HEY1, phosphorilated AKT and ERK in cells (WM266-4) expressing specific shRNAs against Notch1 and ERBB3. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. D–E) NFκB Reporter assay in cells treated with DBZ and lapatinib or expressing shGFP, shNotch1, shERBB3 or the two together. P values are calculated by the Student’s T test. Data are the average±SD between at least three independent experiments. F–G) Cytosolic and nuclear distribution of the NFκB elements p65 and p50 in DBZ/lapatinib treated cells ( F ) or in cells expressing shRNAs against Notch1 and ERBB3 ( G ).

    Techniques Used: Inhibition, Activity Assay, Western Blot, Expressing, Reporter Assay

    8) Product Images from "Acquisition of temozolomide resistance by the rat C6 glioma cell line increases cell migration and side population phenotype"

    Article Title: Acquisition of temozolomide resistance by the rat C6 glioma cell line increases cell migration and side population phenotype

    Journal: Oncology Reports

    doi: 10.3892/or.2019.7350

    TMZ-resistant rat glioma (C6-PL3) cells do not possess typical properties of cancer stem cells. (A) The ability of C6-PL3 cells and parental rat glioma (C6) cells to form tumor spheres was observed under phase-contrast microscopy. (B) Expression of Nestin, SOX2, BMI1, and β-actin in C6-PL3 and C6 cells was analyzed using western blotting. (C) Expression of nestin in C6-PL3 and C6 cells was analyzed using immunofluorescence. TMZ, temozolomide.
    Figure Legend Snippet: TMZ-resistant rat glioma (C6-PL3) cells do not possess typical properties of cancer stem cells. (A) The ability of C6-PL3 cells and parental rat glioma (C6) cells to form tumor spheres was observed under phase-contrast microscopy. (B) Expression of Nestin, SOX2, BMI1, and β-actin in C6-PL3 and C6 cells was analyzed using western blotting. (C) Expression of nestin in C6-PL3 and C6 cells was analyzed using immunofluorescence. TMZ, temozolomide.

    Techniques Used: Microscopy, Expressing, Western Blot, Immunofluorescence

    Drug resistance in parental rat glioma cells (C6 cells) induced promotion of cell invasion. (A) Transwell assays were carried out for TMZ-resistant rat glioma cells (C6-PL3) and C6 cells. Images of cells stained with Giemsa invading through Matrigel in a representative experiment are presented, and results of 15 different regions in three independent experiments (five regions per experiment) are summarized in (B) with an ×200 magnification. (C-H) Rats were allografted with C6-PL3 and C6 cells for 20 days. Brain slices were collected and stained. Representative images of brain slices collected from different rats are presented, with arrows indicating C6 glioma cells. Dotted lines indicate the boundary between tumor and normal brain tissue. (I) Quantitative results from both groups (three rats/group) are summarized. **P
    Figure Legend Snippet: Drug resistance in parental rat glioma cells (C6 cells) induced promotion of cell invasion. (A) Transwell assays were carried out for TMZ-resistant rat glioma cells (C6-PL3) and C6 cells. Images of cells stained with Giemsa invading through Matrigel in a representative experiment are presented, and results of 15 different regions in three independent experiments (five regions per experiment) are summarized in (B) with an ×200 magnification. (C-H) Rats were allografted with C6-PL3 and C6 cells for 20 days. Brain slices were collected and stained. Representative images of brain slices collected from different rats are presented, with arrows indicating C6 glioma cells. Dotted lines indicate the boundary between tumor and normal brain tissue. (I) Quantitative results from both groups (three rats/group) are summarized. **P

    Techniques Used: Staining

    Most TMZ-resistant rat glioma (C6-PL3) cells are SP cells. (A) The expression of BRCP1, MDR1, and β-actin in C6-PL3 and parental rat glioma (C6) cells was analyzed using western blotting. (B) C6-PL3 and C6 cells treated with or without 50 µM verapamil were labeled with the Hoechst 33342 and analyzed using flow cytometry. TMZ, temozolomide; SP, side population.
    Figure Legend Snippet: Most TMZ-resistant rat glioma (C6-PL3) cells are SP cells. (A) The expression of BRCP1, MDR1, and β-actin in C6-PL3 and parental rat glioma (C6) cells was analyzed using western blotting. (B) C6-PL3 and C6 cells treated with or without 50 µM verapamil were labeled with the Hoechst 33342 and analyzed using flow cytometry. TMZ, temozolomide; SP, side population.

    Techniques Used: Expressing, Western Blot, Labeling, Flow Cytometry, Cytometry

    Phase-contrast microscopy indicated morphological changes in (A and C) temozolomide-resistant rat glioma cells (C6-PL3) and (B and D) parental rat glioma cells (C6 cells). Representative images of (E) C6-PL3 cells and (F) C6 cells under F-actin staining. Images of F-actin were obtained at a magnification of ×400.
    Figure Legend Snippet: Phase-contrast microscopy indicated morphological changes in (A and C) temozolomide-resistant rat glioma cells (C6-PL3) and (B and D) parental rat glioma cells (C6 cells). Representative images of (E) C6-PL3 cells and (F) C6 cells under F-actin staining. Images of F-actin were obtained at a magnification of ×400.

    Techniques Used: Microscopy, Staining

    Verapamil increases TMZ-induced tumor sphere formation and apoptosis in TMZ-resistant rat glioma (C6-PL3) and parental rat glioma (C6) cells. (A) Tumor sphere formation in C6-PL3 and C6 cells was observed using phase-contrast microscopy after TMZ and verapamil treatment. (B and C) After treatment, apoptosis was detected using flow cytometry. The average apoptosis rate of TMZ treatment was 4.79%, while after adding the verapamil, the average apoptosis rate was 81.61%. ***P
    Figure Legend Snippet: Verapamil increases TMZ-induced tumor sphere formation and apoptosis in TMZ-resistant rat glioma (C6-PL3) and parental rat glioma (C6) cells. (A) Tumor sphere formation in C6-PL3 and C6 cells was observed using phase-contrast microscopy after TMZ and verapamil treatment. (B and C) After treatment, apoptosis was detected using flow cytometry. The average apoptosis rate of TMZ treatment was 4.79%, while after adding the verapamil, the average apoptosis rate was 81.61%. ***P

    Techniques Used: Microscopy, Flow Cytometry, Cytometry

    9) Product Images from "Synchronized targeting of Notch and ERBB signaling suppresses melanoma tumor growth through inhibition of Notch1 and ERBB3 *"

    Article Title: Synchronized targeting of Notch and ERBB signaling suppresses melanoma tumor growth through inhibition of Notch1 and ERBB3 *

    Journal: The Journal of investigative dermatology

    doi: 10.1016/j.jid.2015.11.006

    Inhibition of Notch1 and ERBB3 blocks tumor formation A) Growth of WM266-4 cells in SCID mice (n=8). Tumor volumes were normalized to 1 at time 0 (mean vol. 137 µM 3 ), time at which mice were given 50 mg/Kg lapatinib and 10 µMol/Kg DBZ thrice a week. (LAP vs DMSO, p=0.007 ; DBZ vs DMSO, p=0.002 ; DBZ+LAP vs DMSO, p=0.001; DBZ+LAP vs LAP, p=0.008 ; DBZ+LAP vs DBZ, p=0.05 , Student’s t test). B) Growth of WM266-4 cells expressing shRNA control (shGFP) or shRNAs against Notch1 and ERBB3, in SCID mice (n=10). (shGFP vs shNotch1, p
    Figure Legend Snippet: Inhibition of Notch1 and ERBB3 blocks tumor formation A) Growth of WM266-4 cells in SCID mice (n=8). Tumor volumes were normalized to 1 at time 0 (mean vol. 137 µM 3 ), time at which mice were given 50 mg/Kg lapatinib and 10 µMol/Kg DBZ thrice a week. (LAP vs DMSO, p=0.007 ; DBZ vs DMSO, p=0.002 ; DBZ+LAP vs DMSO, p=0.001; DBZ+LAP vs LAP, p=0.008 ; DBZ+LAP vs DBZ, p=0.05 , Student’s t test). B) Growth of WM266-4 cells expressing shRNA control (shGFP) or shRNAs against Notch1 and ERBB3, in SCID mice (n=10). (shGFP vs shNotch1, p

    Techniques Used: Inhibition, Mouse Assay, Expressing, shRNA

    Notch1 NIC and poshpo-ERBB3 are similarly expressed in mutated and wild type BRAF melanoma tumors A) Expression levels of Notch1 NIC (upper panel) and phoshporylated ERBB3 (lower panel) in five mutated and five wild type BRAF melanoma tumors. Cells treated with DBZ (10 µM) and lapatinib (10 µM) O/N (left panels) are used as positive controls for Notch1 NIC and phopho-ERBB3, respectively. GAPDH is used a loading control. B) Quantification of the bands in A normalized to their correspondent loading control. No statistical difference in expression of either Notch1 NIC or phospho-ERBB3 between mutated and wild type tumors was observed (student’s T test). C) Correlation between Notch1 NIC and phospho-ERBB3 among all samples in A.
    Figure Legend Snippet: Notch1 NIC and poshpo-ERBB3 are similarly expressed in mutated and wild type BRAF melanoma tumors A) Expression levels of Notch1 NIC (upper panel) and phoshporylated ERBB3 (lower panel) in five mutated and five wild type BRAF melanoma tumors. Cells treated with DBZ (10 µM) and lapatinib (10 µM) O/N (left panels) are used as positive controls for Notch1 NIC and phopho-ERBB3, respectively. GAPDH is used a loading control. B) Quantification of the bands in A normalized to their correspondent loading control. No statistical difference in expression of either Notch1 NIC or phospho-ERBB3 between mutated and wild type tumors was observed (student’s T test). C) Correlation between Notch1 NIC and phospho-ERBB3 among all samples in A.

    Techniques Used: Expressing

    Inhibition of Notch and ERBB inhibits AKT and NFκB activity A) Effects of lapatinib (10µM) on the phosphorylation levels of ERBB3, ERBB2, EGFR in WM266-4 cells. B) DBZ (10µM) and lapatinib (10µM) effects on Notch1 NIC , HEY1, phosphorylated AKT and ERK1/2 in WM266-4 cells. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. C) Expression levels of Notch1, ERBB3, HEY1, phosphorilated AKT and ERK in cells (WM266-4) expressing specific shRNAs against Notch1 and ERBB3. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. D–E) NFκB Reporter assay in cells treated with DBZ and lapatinib or expressing shGFP, shNotch1, shERBB3 or the two together. P values are calculated by the Student’s T test. Data are the average±SD between at least three independent experiments. F–G) Cytosolic and nuclear distribution of the NFκB elements p65 and p50 in DBZ/lapatinib treated cells ( F ) or in cells expressing shRNAs against Notch1 and ERBB3 ( G ).
    Figure Legend Snippet: Inhibition of Notch and ERBB inhibits AKT and NFκB activity A) Effects of lapatinib (10µM) on the phosphorylation levels of ERBB3, ERBB2, EGFR in WM266-4 cells. B) DBZ (10µM) and lapatinib (10µM) effects on Notch1 NIC , HEY1, phosphorylated AKT and ERK1/2 in WM266-4 cells. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. C) Expression levels of Notch1, ERBB3, HEY1, phosphorilated AKT and ERK in cells (WM266-4) expressing specific shRNAs against Notch1 and ERBB3. Numbers represent quantification of band intensity normalized to their respective loading control and are the average between three independent western blots. D–E) NFκB Reporter assay in cells treated with DBZ and lapatinib or expressing shGFP, shNotch1, shERBB3 or the two together. P values are calculated by the Student’s T test. Data are the average±SD between at least three independent experiments. F–G) Cytosolic and nuclear distribution of the NFκB elements p65 and p50 in DBZ/lapatinib treated cells ( F ) or in cells expressing shRNAs against Notch1 and ERBB3 ( G ).

    Techniques Used: Inhibition, Activity Assay, Western Blot, Expressing, Reporter Assay

    Notch1 NIC and phosphorilated ERBB3/ERBB2 correlate in melanoma A) Expression levels of phosphorylated and total ERBB receptors and of active Notch1 (Notch1 NIC ) in nine flash-frozen human metastatic melanomas. B) Correlation analysis between Notch1 NIC and phospho-ERBB3, phospho-ERBB2 and between phospho-ERBB3 and 2. C) Lack of correlation between the other components analyzed in A.
    Figure Legend Snippet: Notch1 NIC and phosphorilated ERBB3/ERBB2 correlate in melanoma A) Expression levels of phosphorylated and total ERBB receptors and of active Notch1 (Notch1 NIC ) in nine flash-frozen human metastatic melanomas. B) Correlation analysis between Notch1 NIC and phospho-ERBB3, phospho-ERBB2 and between phospho-ERBB3 and 2. C) Lack of correlation between the other components analyzed in A.

    Techniques Used: Expressing

    10) Product Images from "Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation *"

    Article Title: Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.516039

    Notch1 effects MT1-MMP-dependent melanoma cell growth. A , expression levels of MT1-MMP ( MT1 ) and Notch1 NIC in WM266-4 cells expressing shGFP or shMT1-MMP and transduced with active Notch1 ( NIC ). β- act , β-actin. B , relative growth at a
    Figure Legend Snippet: Notch1 effects MT1-MMP-dependent melanoma cell growth. A , expression levels of MT1-MMP ( MT1 ) and Notch1 NIC in WM266-4 cells expressing shGFP or shMT1-MMP and transduced with active Notch1 ( NIC ). β- act , β-actin. B , relative growth at a

    Techniques Used: Expressing, Transduction, Activated Clotting Time Assay

    Notch1 NIC and MT1-MMP expression correlate in melanoma. A , Western blotting on nine flash-frozen, human melanoma tumors showing expression of ADAM17 ( A-17 ), ADAM10 ( A-10 ), MT1-MMP ( MT1 ), and active Notch1 ( NIC ). β-Actin (β- Act ) was used
    Figure Legend Snippet: Notch1 NIC and MT1-MMP expression correlate in melanoma. A , Western blotting on nine flash-frozen, human melanoma tumors showing expression of ADAM17 ( A-17 ), ADAM10 ( A-10 ), MT1-MMP ( MT1 ), and active Notch1 ( NIC ). β-Actin (β- Act ) was used

    Techniques Used: Expressing, Western Blot, Activated Clotting Time Assay

    Active MT1-MMP promotes Notch1 cleavage independently of ADAM10 or -17. A , MT1-MMP ( MT1 ) protein expression in the syngeneic cell lines WM115 (primary melanoma) and WM2660-4 (metastatic melanoma). β- act , β-actin. B , schematic representation
    Figure Legend Snippet: Active MT1-MMP promotes Notch1 cleavage independently of ADAM10 or -17. A , MT1-MMP ( MT1 ) protein expression in the syngeneic cell lines WM115 (primary melanoma) and WM2660-4 (metastatic melanoma). β- act , β-actin. B , schematic representation

    Techniques Used: Expressing, Activated Clotting Time Assay

    MT1-MMP inhibition reduces Notch1 cleavage. A , expression of full-length Notch1 ( N1 ), Notch1 NIC ( NIC ), MT1-MMP ( MT1 ), and MMP2 in WM266-4 cells expressing shRNAs against GFP, MT1-MMP, MMP2, or both MT1-MMP and MMP2. The ratio between the Notch1 NIC band
    Figure Legend Snippet: MT1-MMP inhibition reduces Notch1 cleavage. A , expression of full-length Notch1 ( N1 ), Notch1 NIC ( NIC ), MT1-MMP ( MT1 ), and MMP2 in WM266-4 cells expressing shRNAs against GFP, MT1-MMP, MMP2, or both MT1-MMP and MMP2. The ratio between the Notch1 NIC band

    Techniques Used: Inhibition, Expressing

    11) Product Images from "The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer"

    Article Title: The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2018.11.031

    MT1-MMP expression is elevated in breast cancer and inversely correlated to response to chemotherapy. A) Oncomine data comparing mRNA expression of MMP14 (gene name for MT1-MMP) between normal tissue and different subtypes of invasive breast carcinoma (TCGA breast data set). P values for all types compared to normal are
    Figure Legend Snippet: MT1-MMP expression is elevated in breast cancer and inversely correlated to response to chemotherapy. A) Oncomine data comparing mRNA expression of MMP14 (gene name for MT1-MMP) between normal tissue and different subtypes of invasive breast carcinoma (TCGA breast data set). P values for all types compared to normal are

    Techniques Used: Expressing

    12) Product Images from "Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation *"

    Article Title: Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.516039

    Notch1 effects MT1-MMP-dependent melanoma cell growth. A , expression levels of MT1-MMP ( MT1 ) and Notch1 NIC in WM266-4 cells expressing shGFP or shMT1-MMP and transduced with active Notch1 ( NIC ). β- act , β-actin. B , relative growth at a
    Figure Legend Snippet: Notch1 effects MT1-MMP-dependent melanoma cell growth. A , expression levels of MT1-MMP ( MT1 ) and Notch1 NIC in WM266-4 cells expressing shGFP or shMT1-MMP and transduced with active Notch1 ( NIC ). β- act , β-actin. B , relative growth at a

    Techniques Used: Expressing, Transduction, Activated Clotting Time Assay

    Notch1 NIC and MT1-MMP expression correlate in melanoma. A , Western blotting on nine flash-frozen, human melanoma tumors showing expression of ADAM17 ( A-17 ), ADAM10 ( A-10 ), MT1-MMP ( MT1 ), and active Notch1 ( NIC ). β-Actin (β- Act ) was used
    Figure Legend Snippet: Notch1 NIC and MT1-MMP expression correlate in melanoma. A , Western blotting on nine flash-frozen, human melanoma tumors showing expression of ADAM17 ( A-17 ), ADAM10 ( A-10 ), MT1-MMP ( MT1 ), and active Notch1 ( NIC ). β-Actin (β- Act ) was used

    Techniques Used: Expressing, Western Blot, Activated Clotting Time Assay

    Active MT1-MMP promotes Notch1 cleavage independently of ADAM10 or -17. A , MT1-MMP ( MT1 ) protein expression in the syngeneic cell lines WM115 (primary melanoma) and WM2660-4 (metastatic melanoma). β- act , β-actin. B , schematic representation
    Figure Legend Snippet: Active MT1-MMP promotes Notch1 cleavage independently of ADAM10 or -17. A , MT1-MMP ( MT1 ) protein expression in the syngeneic cell lines WM115 (primary melanoma) and WM2660-4 (metastatic melanoma). β- act , β-actin. B , schematic representation

    Techniques Used: Expressing, Activated Clotting Time Assay

    MT1-MMP affects melanoma cell growth. A , relative growth of WM266-4 expressing a control shRNA ( shGFP ) or shMT1-MMP. B , relative growth of WM115 expressing a control vector ( pLM ) or either active ( MT1-FL ) or inactive ( MT1-Mut ) MT1-MMP. *, p
    Figure Legend Snippet: MT1-MMP affects melanoma cell growth. A , relative growth of WM266-4 expressing a control shRNA ( shGFP ) or shMT1-MMP. B , relative growth of WM115 expressing a control vector ( pLM ) or either active ( MT1-FL ) or inactive ( MT1-Mut ) MT1-MMP. *, p

    Techniques Used: Expressing, shRNA, Plasmid Preparation

    MT1-MMP inhibition reduces Notch1 cleavage. A , expression of full-length Notch1 ( N1 ), Notch1 NIC ( NIC ), MT1-MMP ( MT1 ), and MMP2 in WM266-4 cells expressing shRNAs against GFP, MT1-MMP, MMP2, or both MT1-MMP and MMP2. The ratio between the Notch1 NIC band
    Figure Legend Snippet: MT1-MMP inhibition reduces Notch1 cleavage. A , expression of full-length Notch1 ( N1 ), Notch1 NIC ( NIC ), MT1-MMP ( MT1 ), and MMP2 in WM266-4 cells expressing shRNAs against GFP, MT1-MMP, MMP2, or both MT1-MMP and MMP2. The ratio between the Notch1 NIC band

    Techniques Used: Inhibition, Expressing

    13) Product Images from "Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase"

    Article Title: Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00116-15

    Furin is a direct target of Notch1. (A) Schematic representation of the furin promoter. There are three putative CSL binding sequences (TTCCCAC), located at bp −3556 (B1), −1236 (B2), and +960 (B3). TSS, transcription start site. (B) Western
    Figure Legend Snippet: Furin is a direct target of Notch1. (A) Schematic representation of the furin promoter. There are three putative CSL binding sequences (TTCCCAC), located at bp −3556 (B1), −1236 (B2), and +960 (B3). TSS, transcription start site. (B) Western

    Techniques Used: Binding Assay, Western Blot

    Notch1 affects furin expression through binding to the furin promoter. (A) Schematic representation of luciferase reporter constructs containing the WT (TTCCCAC) or mutated (TCACAGC) binding site 2 sequence. The pGL2-SacI construct contains the sequence
    Figure Legend Snippet: Notch1 affects furin expression through binding to the furin promoter. (A) Schematic representation of luciferase reporter constructs containing the WT (TTCCCAC) or mutated (TCACAGC) binding site 2 sequence. The pGL2-SacI construct contains the sequence

    Techniques Used: Expressing, Binding Assay, Luciferase, Construct, Sequencing

    Inhibition of furin decreases Notch1 processing. (A) Expression of full-length (FL) and intracellular (NIC) Notch1 (probed with antibody mN1A), Notch-NIC (probed with an antibody that recognizes Notch1 NIC cleaved at Val-1744), MT1-MMP, ADAM10, and furin
    Figure Legend Snippet: Inhibition of furin decreases Notch1 processing. (A) Expression of full-length (FL) and intracellular (NIC) Notch1 (probed with antibody mN1A), Notch-NIC (probed with an antibody that recognizes Notch1 NIC cleaved at Val-1744), MT1-MMP, ADAM10, and furin

    Techniques Used: Inhibition, Expressing

    Notch1 induces MT1-MMP activation through furin. (A) Western blot analysis showing that the active fraction of MT1-MMP increases in NIC-expressing cells but decreases in furin knockdown cells. Experiments were carried out in three cell lines: SKMel2,
    Figure Legend Snippet: Notch1 induces MT1-MMP activation through furin. (A) Western blot analysis showing that the active fraction of MT1-MMP increases in NIC-expressing cells but decreases in furin knockdown cells. Experiments were carried out in three cell lines: SKMel2,

    Techniques Used: Activation Assay, Western Blot, Expressing

    Stimulation of endogenous Notch1. JAGGED1-dependent activation of endogenous Notch1 increases furin levels and promotes the processing of MT1-MMP and ADAM10 in two melanoma cell lines.
    Figure Legend Snippet: Stimulation of endogenous Notch1. JAGGED1-dependent activation of endogenous Notch1 increases furin levels and promotes the processing of MT1-MMP and ADAM10 in two melanoma cell lines.

    Techniques Used: Activation Assay

    Notch1 promotes MT1-MMP activity. (A) The active MT1-MMP fraction (55 kDa) is increased in Notch1-NIC-expressing cells. β-Actin is used as a loading control. (B) Notch1-NIC increases MT1-MMP activity in vitro . neg, negative control (buffer); pos,
    Figure Legend Snippet: Notch1 promotes MT1-MMP activity. (A) The active MT1-MMP fraction (55 kDa) is increased in Notch1-NIC-expressing cells. β-Actin is used as a loading control. (B) Notch1-NIC increases MT1-MMP activity in vitro . neg, negative control (buffer); pos,

    Techniques Used: Activity Assay, Expressing, In Vitro, Negative Control

    Notch1 increases the active fraction of ADAM10 through furin. (A) Western blot analysis showing the expression of the propeptide and active forms of ADAM10 and the expression of Notch1-NIC in NIC-expressing SKMel2 (left) and V2387 (right) cells in the
    Figure Legend Snippet: Notch1 increases the active fraction of ADAM10 through furin. (A) Western blot analysis showing the expression of the propeptide and active forms of ADAM10 and the expression of Notch1-NIC in NIC-expressing SKMel2 (left) and V2387 (right) cells in the

    Techniques Used: Western Blot, Expressing

    Notch1 promotes ADAM10 activity. (A) In SKMel2 and V2387 cells, the active ADAM10 fraction (60 kDa) is increased in Notch1-NIC-expressing cells. The level of ADAM17 does not change. The ratio of active to inactive ADAM10 normalized to the corresponding
    Figure Legend Snippet: Notch1 promotes ADAM10 activity. (A) In SKMel2 and V2387 cells, the active ADAM10 fraction (60 kDa) is increased in Notch1-NIC-expressing cells. The level of ADAM17 does not change. The ratio of active to inactive ADAM10 normalized to the corresponding

    Techniques Used: Activity Assay, Expressing

    Furin inhibition reduces migration and invasion by melanoma cells. (A) Expression levels of Notch1-NIC and furin in cells expressing active Notch1 and furin shRNA. The same cells were used for all panels. (B) Migration of the cells (determined by a scratch
    Figure Legend Snippet: Furin inhibition reduces migration and invasion by melanoma cells. (A) Expression levels of Notch1-NIC and furin in cells expressing active Notch1 and furin shRNA. The same cells were used for all panels. (B) Migration of the cells (determined by a scratch

    Techniques Used: Inhibition, Migration, Expressing, shRNA

    14) Product Images from "The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer"

    Article Title: The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA Damage Response that impacts chemo- and radiotherapy responses of breast cancer

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2018.11.031

    MT1-MMP acts through integrinβ1 A) Quantification of γH2AX foci of MDA-MB-231 cells seeded in MT1-MMP degradable (D) and non-degradable (ND) matrixes for 24 hours. Foci were quantified from at least 50 cells. Data are the mean of two independent experiments. Bottom panel: western blotting to detect γH2AX, pChK1, pChK2, pRPA32 of cells embedded in the matrixes. B) Expression of integrinβ1 V37N (ITGB1) in MDA-MB-231 cells expressing shGFP or shMT1-MMP. pFAK Y297 and the markers in A are also shown. C) Clonogenic assay of the cells in B treated with the indicated IR doses or several doses of doxorubicin for 15 days (the duration of the clonogenic assay). (Left: * , ** , ***p
    Figure Legend Snippet: MT1-MMP acts through integrinβ1 A) Quantification of γH2AX foci of MDA-MB-231 cells seeded in MT1-MMP degradable (D) and non-degradable (ND) matrixes for 24 hours. Foci were quantified from at least 50 cells. Data are the mean of two independent experiments. Bottom panel: western blotting to detect γH2AX, pChK1, pChK2, pRPA32 of cells embedded in the matrixes. B) Expression of integrinβ1 V37N (ITGB1) in MDA-MB-231 cells expressing shGFP or shMT1-MMP. pFAK Y297 and the markers in A are also shown. C) Clonogenic assay of the cells in B treated with the indicated IR doses or several doses of doxorubicin for 15 days (the duration of the clonogenic assay). (Left: * , ** , ***p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, Clonogenic Assay

    Expression of MT1-MMP affects cell responses to genotoxic stresses. A) Expression of MT1-MMP in MDA-MB-231 (left), and other cell lines after removal of MDA-MB-231 and exposure of the film for a longer time (right). B) ER, PR, HER2 status of the cell lines in A (all cells are BRCA1WT). C) Knock down levels of MT1-MMP in MDA-MB-231. D) Clonogenic assay of the cells in C treated with the indicated IR doses or several doses of doxorubicin for 10 days (the duration of the clonogenic assay). * , ** , ***p
    Figure Legend Snippet: Expression of MT1-MMP affects cell responses to genotoxic stresses. A) Expression of MT1-MMP in MDA-MB-231 (left), and other cell lines after removal of MDA-MB-231 and exposure of the film for a longer time (right). B) ER, PR, HER2 status of the cell lines in A (all cells are BRCA1WT). C) Knock down levels of MT1-MMP in MDA-MB-231. D) Clonogenic assay of the cells in C treated with the indicated IR doses or several doses of doxorubicin for 10 days (the duration of the clonogenic assay). * , ** , ***p

    Techniques Used: Expressing, Multiple Displacement Amplification, Clonogenic Assay

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    Article Snippet: .. Chemicals Anti-adiponectin receptor 1 (Abcam), anti–p-AMPK, anti-AKT (Cell Signaling), anti-β-actin, anti-AMPK, anti-caspase 3, anti-p-AKT, anti-Tau, anti-p-tau, anti-Aβ, anti-GFAP, anti-TNF-α, FITC-labelled goat-anti rabbit (Santa Cruz), anti-NeuN (Millipore), anti-iba-1 (Wako), insulin (Sigma), branched-PEI (Sigma), D-(+)- glucose solution (Sigma), DPX (Sigma), AdipoR1 shRNA plasmid (Qiagen), phosphatase inhibitor, protease inhibitor cocktail (GenDEPOT), a protein assay kit (Bio-Rad), skim milk (BD Difco), O.C.T. compound (Sakura), ECL solution (ATTO), and fluorescence mounting medium (Agilent Technologies) were purchased from the indicated manufacturers. .. Establishing the PEI-based shRNA-mediated AdipoR1 knockdown strategy To establish the PEI-based shRNA-mediated knockdown, we optimized the composition and condition of the PEI-shRNA mixture on the basis of a previous report.

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