duolink in situ pla blocking solution  (Santa Cruz Biotechnology)

 
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    Santa Cruz Biotechnology duolink in situ pla blocking solution
    Duolink In Situ Pla Blocking Solution, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/duolink in situ pla blocking solution/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
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    pla uhrf1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla uhrf1
    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of <t>UHRF1</t> expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
    Pla Uhrf1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth"

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-22-0182

    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
    Figure Legend Snippet: (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.

    Techniques Used: Labeling, Transfection, Luciferase, Activity Assay, Expressing, Positive Control, Derivative Assay

    The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.
    Figure Legend Snippet: The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.

    Techniques Used: Expressing

    (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).
    Figure Legend Snippet: (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).

    Techniques Used: Glo Assay, MTT Reduction Assay, Transduction, shRNA, Western Blot, Immunoprecipitation

    (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).
    Figure Legend Snippet: (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).

    Techniques Used: Expressing, Derivative Assay, Microarray, Isolation, Mutagenesis, Amplification

    (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).
    Figure Legend Snippet: (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).

    Techniques Used: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Luciferase, Transduction, shRNA, Imaging

    (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).
    Figure Legend Snippet: (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).

    Techniques Used: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Size-exclusion Chromatography, Filtration, Proximity Ligation Assay, Staining, Pull Down Assay, Binding Assay, Sequencing

    pla uhrf1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla uhrf1
    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of <t>UHRF1</t> expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
    Pla Uhrf1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pla uhrf1/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pla uhrf1 - by Bioz Stars, 2023-11
    86/100 stars

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    1) Product Images from "A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth"

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-22-0182

    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
    Figure Legend Snippet: (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.

    Techniques Used: Labeling, Transfection, Luciferase, Activity Assay, Expressing, Positive Control, Derivative Assay

    The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.
    Figure Legend Snippet: The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.

    Techniques Used: Expressing

    (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).
    Figure Legend Snippet: (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).

    Techniques Used: Glo Assay, MTT Reduction Assay, Transduction, shRNA, Western Blot, Immunoprecipitation

    (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).
    Figure Legend Snippet: (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).

    Techniques Used: Expressing, Derivative Assay, Microarray, Isolation, Mutagenesis, Amplification

    (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).
    Figure Legend Snippet: (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).

    Techniques Used: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Luciferase, Transduction, shRNA, Imaging

    (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).
    Figure Legend Snippet: (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).

    Techniques Used: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Size-exclusion Chromatography, Filtration, Proximity Ligation Assay, Staining, Pull Down Assay, Binding Assay, Sequencing

    duolink proximity ligation assay h441 cells  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology duolink proximity ligation assay h441 cells
    Duolink Proximity Ligation Assay H441 Cells, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla analysis  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla analysis
    (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the <t>indicated</t> <t>antibodies.</t> The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay <t>(PLA)</t> foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.
    Pla Analysis, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeting RBM10 deficiency in lung adenocarcinoma"

    Article Title: Targeting RBM10 deficiency in lung adenocarcinoma

    Journal: bioRxiv

    doi: 10.1101/2023.02.19.529108

    (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay (PLA) foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.
    Figure Legend Snippet: (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay (PLA) foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.

    Techniques Used: Fractionation, Western Blot, Molecular Weight, Expressing, Mass Spectrometry, Immunoprecipitation, Immunofluorescence, Microscopy, Proximity Ligation Assay

    pla probes incubation  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla probes incubation
    Pla Probes Incubation, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    exogenous pla  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology exogenous pla
    Detection of endogenous ADAR2 <t>and</t> <t>FMRP</t> interaction by Proximity Ligation Assay <t>(PLA)</t> in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.
    Exogenous Pla, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse"

    Article Title: Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

    Journal: RNA Biology

    doi: 10.1080/15476286.2017.1338232

    Detection of endogenous ADAR2 and FMRP interaction by Proximity Ligation Assay (PLA) in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.
    Figure Legend Snippet: Detection of endogenous ADAR2 and FMRP interaction by Proximity Ligation Assay (PLA) in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.

    Techniques Used: Proximity Ligation Assay

    pla probes  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla probes
    A . Melanoma cells were transfected <t>with</t> <t>HIF-2α</t> siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ <t>PLA</t> detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.
    Pla Probes, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2"

    Article Title: EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012265

    A . Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.
    Figure Legend Snippet: A . Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.

    Techniques Used: Transfection, Expressing, Western Blot, Confocal Microscopy, Positive Control, Immunoprecipitation, In Situ, Staining

    pla reaction  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology pla reaction
    Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay <t>(PLA)</t> shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth <t>Hormone</t> <t>Receptor,</t> where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.
    Pla Reaction, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells"

    Article Title: Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-15-15

    Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.
    Figure Legend Snippet: Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.

    Techniques Used: Expressing, Incubation, Staining, Negative Control, Imaging, Immunofluorescence, Marker, Proximity Ligation Assay, Positive Control

    pla  (Santa Cruz Biotechnology)

     
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    Pla, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    • duolink in situ pla blocking solution  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology duolink in situ pla blocking solution
    Duolink In Situ Pla Blocking Solution, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla uhrf1  (Santa Cruz Biotechnology)
    86
    Santa Cruz Biotechnology pla uhrf1
    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of <t>UHRF1</t> expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
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    duolink proximity ligation assay h441 cells  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology duolink proximity ligation assay h441 cells
    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of <t>UHRF1</t> expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.
    Duolink Proximity Ligation Assay H441 Cells, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla analysis  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology pla analysis
    (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the <t>indicated</t> <t>antibodies.</t> The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay <t>(PLA)</t> foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.
    Pla Analysis, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla probes incubation  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology pla probes incubation
    (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the <t>indicated</t> <t>antibodies.</t> The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay <t>(PLA)</t> foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.
    Pla Probes Incubation, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    exogenous pla  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology exogenous pla
    Detection of endogenous ADAR2 <t>and</t> <t>FMRP</t> interaction by Proximity Ligation Assay <t>(PLA)</t> in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.
    Exogenous Pla, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla probes  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology pla probes
    A . Melanoma cells were transfected <t>with</t> <t>HIF-2α</t> siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ <t>PLA</t> detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.
    Pla Probes, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla reaction  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology pla reaction
    Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay <t>(PLA)</t> shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth <t>Hormone</t> <t>Receptor,</t> where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.
    Pla Reaction, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pla  (Santa Cruz Biotechnology)
    86
    Santa Cruz Biotechnology pla
    Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay <t>(PLA)</t> shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth <t>Hormone</t> <t>Receptor,</t> where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.
    Pla, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: (A) A simplified schematic of the SHH signaling pathway, with positive regulators labeled in green and negative regulators labeled in red. GLIA: GLI activator form. (B) A schematic outlining the design of our siRNA-based screen. Light2 cells were seeded in 96-well plates, followed by transfection of pooled siRNAs (a mix of four distinct siRNA) from a siRNA library for 72h. The cells were then treated with the SMO agonist SAG in 0.5% serum for another 24h. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to that of TK-driven Renilla luciferase. (C) Data from a representative plate in the siRNA screen. The normalized luciferase activity of each sample was compared to that of a scramble siRNA control (siControl) treated sample. The siRNA targets whose reduction in expression attenuated luciferase activity more than 50% were identified as positive hits and labeled in red. siRNA targeting Gli2 was included as a positive control. Cells that were treated with control siRNA (siControl) without SAG induction (No SAG) were included as an additional control. Using the data from Cavalli et al [4], the expression level of (D)UHFR1 and (E)DNMT1 in patient derived MB tissue was assigned to a high or low expression group based on the median values of UHRF1 expression, and each group’s 5-year survival determined across the four major MB subgroups. (F) Sufu−/− iMEFs were transfected with pooled siRNA targeting the indicated prioritized genes for 96 h. The expression of the siRNA-target (Target) and the SHH-dependent target gene Gli1 were determined. These data were normalized to that from siControl treated cells and presented as relative gene expression. LHX5 was not detected in MEFs and two candidates encoding subunits of RNA polymerase II (Polr2d and Polr2j) were not pursued further. (G) Light2 cells were transfected with the indicated pooled siRNA for 2 days, followed by transfection of the indicated plasmids for another 2 days. Cells were then collected and GLI-driven Firefly luciferase activity determined and normalized to TK-driven Renilla luciferase activity. Normalized reporter activity was then compared to the control samples and presented as relative reporter activity.

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Labeling, Transfection, Luciferase, Activity Assay, Expressing, Positive Control, Derivative Assay

    The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: The expression of eight GLI regulator candidates is associated with poor SHH-MB patient outcome. Using outcome data from Cavalli et al [ 4 ], logrank tests of the 83 GLI regulator candidates identified in our siRNA-based screen were performed to determine if the expression level of each candidate was associated with patient outcome (5-year survival curve) across the four major subgroups of MB. Significant p values ( <0.05) are highlighted in green. Only candidate genes whose expression was significantly associated with poor SHH-MB patient outcome are shown.

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Expressing

    (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: (A) MSC4 cells were treated with 5-aza, 5-aza-dCR or GSK3484862 at the indicated concentrations for 72h. The number of viable cells was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated cells. (B) MSC4 cells were treated with the indicated concentrations of 5-aza for 48h. The number of viable cells was determined using an MTT reduction assay. (C) MSC4 cells were transduced with a control shRNA (shControl) or shRNA targeting Dnmt1 (shDnmt1) and 3 days later selected in puromycin for 4 days. The transduced MSCs was subsequently treated with DMSO or 2 μM 5-aza for 48h. Cellular viability was then determined using a CellTiter-Glo assay and normalized to results from DMSO treated shControl cells. (D) MSC4 cells were treated with the indicated concentrations of 5-aza for 24h followed by immunoblotting of the resultant lysates for the indicated proteins. A representative blot is shown (n=6). (E) The level of the indicated proteins in (D) was quantitated and normalized to that of GAPDH and then to results from DMSO treated samples to calculate relative protein levels (n=6). (F) MSC4 cells were treated with DMSO (D) or 200 nM 5-aza (A) for 24h, and GLI2 subsequently immunoprecipitated from the resultant lysates. A representative blot is shown (n=3). (G) The level of DNMT1 and UHRF1 in the GLI2-immunoprecipatation of the 5-aza lane (A) was quantitated and normalized to that of GLI2, and then normalized to results from the DMSO lane (D) to calculate the relative interaction intensity with GLI2 (n=3).

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Glo Assay, MTT Reduction Assay, Transduction, shRNA, Western Blot, Immunoprecipitation

    (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: (A) Meta-analysis comparing the expression of UHRF1 between normal brain tissue (NT) and patient derived MB tissue (MB) using the four indicated microarray datasets [42–45]. (B) The dependency on UHRF1 for patient-derived cancer cell line viability was compared between MB cell lines (MB) and non-MB cancer cell lines (Others) using the data from the DepMap project (Broad Institute). (C) Meta-analysis comparing the expression of UHRF1 among the four SHH subtypes (SHH α, β, γ and δ) and other subgroups of MB (Others), using microarray data from Cavalli et al [4]. (D) Normal mouse cerebella or SHH-MB tissue from Ptch1+/− mice were isolated and immunoblotted to determine the levels of GLI1, UHRF1 and GAPDH (n=3 mice per group). MB tissue from (E) ND2:SmoA1 or (F) patient-derived TRP53-mutant, MYCN-amplified MB xenograft (SJSHHMB-14-7196) mice were immunostained for UHRF1. Representative images are shown (n=3).

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Expressing, Derivative Assay, Microarray, Isolation, Mutagenesis, Amplification

    (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: (A and B) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Uhrf1 or Gli1. The number of viable cells were determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (C and D) The indicated SHH-MB sphere cultures were transfected with control or pooled siRNA targeting Uhrf1 for 4 days. The expression of Uhrf1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. (E) MSC4 cells were transfected with the indicated siRNA for 4 days and the resultant cell lysates immunoblotted for the indicated proteins. A representative blot is shown (n=3). (F) MSC4 cells expressing Firefly luciferase were transduced with a scramble shRNA control (shControl) or shRNA targeting Uhrf1 (shUhrf1) overnight. The next day, these transduced MSC4 cells were orthotopically implanted into the cerebella of CD1-Foxn1nu mice and tumor burden imaged 10 days later by IVIS. Representative IVIS imaging is shown (n=7 mice per group). (G) Bioluminescence quantification of all the mice in this cohort is shown (n=7 mice per group).

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Luciferase, Transduction, shRNA, Imaging

    (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).

    Journal: Molecular cancer research : MCR

    Article Title: A druggable UHRF1/DNMT1/GLI complex regulates Sonic hedgehog dependent tumor growth

    doi: 10.1158/1541-7786.MCR-22-0182

    Figure Lengend Snippet: (A) The indicated SHH-MB sphere cultures were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 or Gli1. The number of viable cells was determined 5 days later using an MTT reduction assay and the data normalized to results from siControl transfected cells. (B) MSC4 cells were transfected with a scramble siRNA control (siControl) or pooled siRNA targeting Dnmt1 for 4 days. The expression of Dnmt1 and Gli1 was then determined by RT-qPCR, initially normalized to the expression of Gapdh and then to the results from siControl transfected cells. MSC4 cells were fractionated into nuclear (N) and cytoplasmic (C) enriched fractions, and GLI1 (C) or GLI2 (D) subsequently immunoprecipitated from such lysates. The immunoprecipitates were analyzed by immunoblotting for the indicated proteins. GAPDH is a cytoplasmic loading control and HDAC1, a known interactor of GLI1 and GLI2 [25], is a nuclear loading control. Representative blots are shown (n=3). (E) The nuclear enriched fraction of MB4 tumor cells was fractionated over a size-exclusion chromatography column. The various fractions were analyzed by immunoblotting for the indicated proteins (Left), or subjected to immunoprecipitation and immunoblotting for the indicated proteins (Right). The elution peaks of the indicated protein standards used to calibrate this gel filtration column are indicated with an arrow above the immunoblot. A representative blot is shown (n=3). (F) MSC4 cells were seeded on cover slips and subsequently subjected to a Proximity Ligation Assay (PLA) to detect potential direct interactions between the indicated proteins (red) with DAPI co-stained (blue). No antibody and IgG controls were included. Representative images are shown (n=3). (G) MSC4 cell lysates were used in a DNA pull-down assay with beads coupled to oligonucleotides containing either a GLI-binding site (GLI) or a scrambled control (SC) sequence. The bead-enriched proteins were then immunoblotted for the indicated proteins. A representative blot is shown (n=3) (Left). The level of UHRF1 or DNMT1 in the GLI-binding site pulldown was quantitated and normalized to that of the control lane (SC) to calculate relative UHRF1 or DNMT1 levels (n=3) (Right).

    Article Snippet: The following antibodies were used for PLA: UHRF1 (Cat# sc-373750, Santa Cruz Biotechnology, RRID:AB_10947236), DNMT1 (Cat# 5032, Cell Signaling Technology, RRID:AB_10548197), GLI1 (prepared as described before [39]), GLI2 (Cat# AF3635, R&D Systems, RRID:AB_2111902), rabbit IgG (Cat# 12370, MilliporeSigma), mouse IgG (Cat# sc2025, Santa Cruz Biotechnology).

    Techniques: Transfection, MTT Reduction Assay, Expressing, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Size-exclusion Chromatography, Filtration, Proximity Ligation Assay, Staining, Pull Down Assay, Binding Assay, Sequencing

    (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay (PLA) foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.

    Journal: bioRxiv

    Article Title: Targeting RBM10 deficiency in lung adenocarcinoma

    doi: 10.1101/2023.02.19.529108

    Figure Lengend Snippet: (a) Biochemical fractionation of HCC827 cells into cytosolic (cyt), nuclear-soluble (nuc), and chromatin-bound (chr) fractions followed by immunoblotting with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (b) Venn diagram shows the intersection between RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr. HEK-293T cells expressing two main RBM10 isoforms fused to EGFP were subjected to GFP-trap followed by mass spectrometry to identify RBM10 interactome before and after MK1775 treatment. (c) Gene ontology analysis of RBM10 interactome in unchallenged cells as identified by GFP-trap followed by mass spectrometry analysis. (d) Heatmap summarizing RBM10-interacting proteins involved in cell cycle regulation, DNA damage response, and replication stress before and after MK1775 treatment. Colors represent relative protein intensity of RBM10-interacting proteins in untreated cells and cells treated with 300nM MK1775 for 4 hr across 3 replicates as identified by GFP-trap followed mass spectrometry. (e) HEK293T cells expressing EGFP-RBM10 or EGFP only were treated either with DMSO or 300nM MK1775 for 4 hr and subjected to GFP-trap followed by immunoblot analysis using the indicated antibodies. The positions of molecular weight markers are indicated to the right. (f) Immunoprecipitation of endogenous RBM10 in H1299 cells. Whole-cell lysates from H1299 cells were subjected to immunoprecipitation using RBM10 antibody or IgG and subjected to immunoblot analysis with the indicated antibodies. The positions of molecular weight markers are indicated to the right. (g,h) Representative immunofluorescence microscopy images of RBM10:PCNA (g) and RBM10:EdU-biotin (h) proximity ligation assay (PLA) foci in H1299 WT and H1299 RBM10-KO cells. Scale bar, 20μm.

    Article Snippet: Rabbit anti-RBM10 antibody (Sigma-Aldrich HPA034972, 1:2,000) and mouse anti-PCNA (Santa Cruz Bio cat. no. sc-56, 1:250) antibodies were incubated for 1 hr at 37°C prior to PLA analysis.

    Techniques: Fractionation, Western Blot, Molecular Weight, Expressing, Mass Spectrometry, Immunoprecipitation, Immunofluorescence, Microscopy, Proximity Ligation Assay

    Detection of endogenous ADAR2 and FMRP interaction by Proximity Ligation Assay (PLA) in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.

    Journal: RNA Biology

    Article Title: Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

    doi: 10.1080/15476286.2017.1338232

    Figure Lengend Snippet: Detection of endogenous ADAR2 and FMRP interaction by Proximity Ligation Assay (PLA) in mouse primary cortical neuron cultures. (A-B) PLA dots for ADAR2 and FMRP show manly a nuclear localization. (C) PLA dots for FMRP and FXR1P interaction are in the cytoplasm; (D) Without-primary-ADAR2 antibody processed samples do not show any dot in PLA experiment. Scale bars 5µm.

    Article Snippet: SC40), recognizing the HA tag for ADAR2 and the Myc tag for FMRP respectively, mouse anti α-synuclein (Santa Cruz cod. sc-12767), for exogenous PLA experiments on transfected HEK293T cells (Supplemental results).

    Techniques: Proximity Ligation Assay

    A . Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.

    Journal: PLoS ONE

    Article Title: EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

    doi: 10.1371/journal.pone.0012265

    Figure Lengend Snippet: A . Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to PPIA housekeeping gene of at least three independent experiments; bars , SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. B . HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.

    Article Snippet: Briefly, slides were blocked, incubated with antibodies directed against HIF-2α (1∶100; ep190b, Novus-Biologicals) and HIF-1α (1∶150; Santa cruz biotechnology) and thereafter incubated with PLA probes, which are secondary antibodies (anti-mouse and anti-rabbit) conjugated to unique oligonucleotides.

    Techniques: Transfection, Expressing, Western Blot, Confocal Microscopy, Positive Control, Immunoprecipitation, In Situ, Staining

    Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.

    Journal: BMC Cell Biology

    Article Title: Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells

    doi: 10.1186/1471-2121-15-15

    Figure Lengend Snippet: Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 ( F , all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.

    Article Snippet: The positive control was the PLA reaction between rabbit anti-Growth Hormone Receptor (GHR) clone H300 (1:200, Santa Cruz) and the mouse anti-GHR clone 3A12 (1:200, Sigma Aldrich), as these 2 different antibodies detect different epitopes of the GHR protein.

    Techniques: Expressing, Incubation, Staining, Negative Control, Imaging, Immunofluorescence, Marker, Proximity Ligation Assay, Positive Control