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  • 97
    Name:
    N Myc Antibody
    Description:
    Members of the Myc Max Mad network function as transcriptional regulators with roles in various aspects of cell behavior including proliferation differentiation and apoptosis 1 These proteins share a common basic helix loop helix leucine zipper bHLH ZIP motif required for dimerization and DNA binding Max was originally discovered based on its ability to associate with c Myc and found to be required for the ability of Myc to bind DNA and activate transcription 2 Subsequently Max has been viewed as a central component of the transcriptional network forming homodimers as well as heterodimers with other members of the Myc and Mad families 1 The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior 1 The Mad family consists of four related proteins Mad1 Mad2 Mxi1 Mad3 and Mad4 and the more distantly related members of the bHLH ZIP family Mnt and Mga Like Myc the Mad proteins are tightly regulated with short half lives In general Mad family members interfere with Myc mediated processes such as proliferation transformation and prevention of apoptosis by inhibiting transcription 3 4 In humans the Myc family consists of 5 genes c Myc N Myc L Myc R Myc and B Myc While c Myc is expressed in many proliferating cells N Myc expression is very restricted with highest levels in during embryonic development and then in the adult during B cell development These expression patterns and results from targeted deletion of N Myc suggest that N Myc plays an important role in tissue development and differentiation 5 In addition amplification or overexpression of N Myc has been found in human neuroblastomas and is associated with rapid progression and poor prognosis 6 7
    Catalog Number:
    9405
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding lysine 351 of human N-Myc. Antibodies were purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human
    Applications:
    Western Blot
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti myc
    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with <t>anti-FLAG</t> ( α -globin) and <t>anti-MYC</t> ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated
    Members of the Myc Max Mad network function as transcriptional regulators with roles in various aspects of cell behavior including proliferation differentiation and apoptosis 1 These proteins share a common basic helix loop helix leucine zipper bHLH ZIP motif required for dimerization and DNA binding Max was originally discovered based on its ability to associate with c Myc and found to be required for the ability of Myc to bind DNA and activate transcription 2 Subsequently Max has been viewed as a central component of the transcriptional network forming homodimers as well as heterodimers with other members of the Myc and Mad families 1 The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior 1 The Mad family consists of four related proteins Mad1 Mad2 Mxi1 Mad3 and Mad4 and the more distantly related members of the bHLH ZIP family Mnt and Mga Like Myc the Mad proteins are tightly regulated with short half lives In general Mad family members interfere with Myc mediated processes such as proliferation transformation and prevention of apoptosis by inhibiting transcription 3 4 In humans the Myc family consists of 5 genes c Myc N Myc L Myc R Myc and B Myc While c Myc is expressed in many proliferating cells N Myc expression is very restricted with highest levels in during embryonic development and then in the adult during B cell development These expression patterns and results from targeted deletion of N Myc suggest that N Myc plays an important role in tissue development and differentiation 5 In addition amplification or overexpression of N Myc has been found in human neuroblastomas and is associated with rapid progression and poor prognosis 6 7
    https://www.bioz.com/result/anti myc/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti myc - by Bioz Stars, 2021-07
    97/100 stars

    Images

    1) Product Images from "Neuronal hemoglobin affects dopaminergic cells' response to stress"

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.458

    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated

    Techniques Used: Western Blot, Fractionation, Immunofluorescence

    AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated
    Figure Legend Snippet: AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Infection

    Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)
    Figure Legend Snippet: Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)

    Techniques Used: Western Blot, Expressing, FACS

    Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated

    Techniques Used: Western Blot, Cell Fractionation, Immunofluorescence, Expressing

    2) Product Images from "Identification of MYC as an antinecroptotic protein that stifles RIPK1–RIPK3 complex formation"

    Article Title: Identification of MYC as an antinecroptotic protein that stifles RIPK1–RIPK3 complex formation

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

    doi: 10.1073/pnas.2000979117

    MYC interacts with RIPK3 and disrupts the interaction between RIPK1 and RIPK3. ( A ) MYC prevents RIPK3 from interacting with RIPK1. HT-29 cells stably expressing FLAG-MYC or empty vector were immunoprecipitated with an anti-RIPK3 antibody. ( B ) MYC prevents the interaction between RIPK1 and RIPK3 in vitro. Recombinant GST-MYC purified using a wheat germ system was added to the recombinant MBP-RIPK1 RHIM domain (496–583) and His-MBP-RIPK3 (388–514) as indicated, followed by Ni 2+ -pulldown analysis. RIPK3-bound RIPK1 was detected using anti-RIPK1 antibodies. ( C ) The N terminus of MYC is responsible for suppressing the RIPK1 and RIPK3 interaction. Bacterially purified MBP-MYC (1–144) was added to the RHIM domains of RIPK1 and RIPK3. The interaction of RIPK1 and RIPK3 was determined as described above. ( D and E ) MYC ablation increases the levels of the RIPK1 and RIPK3 proteins. The levels of the RIPK1, RIPK3, and pMLKL proteins in HT-29 ( D ) and HeLa/RIPK3 cells ( E ) were analyzed using immunoblotting after MYC knockdown and/or treatment with 30 μM Nec-1 or 3 μM GSK′872. ( F and G ) RIPK1 and RIPK3 form a complex in MYC-depleted HT-29 cells. HT-29 cells transfected with the indicated siRNAs were treated with DMSO, GSK′963, or Nec-1. After chemical treatment, an immunoprecipitation assay was carried out using an anti-RIPK3 antibody. ( H ) MYC depletion induces the formation of small and inactive necrosome complexes. HT-29 cells were transfected or treated with the indicated siRNA or stimuli, respectively. The cell lysates were fractionated according to molecular size by gel-filtration chromatography and examined by immunoblotting. ( I ) MYC induces the accumulation of RIPK1 and RIPK3 proteins in the soluble fraction. HT-29 cells were transfected with MYC siRNA and treated with TBZ for 4 h. Cells were then fractioned into lysis buffer soluble (Sol.) and insoluble (Insol.) fractions, followed by immunoblot analysis. The phosphorylated form of RIPK3 (p-RIPK3) is indicated by the arrowhead, and the asterisks indicate p-MLKL.
    Figure Legend Snippet: MYC interacts with RIPK3 and disrupts the interaction between RIPK1 and RIPK3. ( A ) MYC prevents RIPK3 from interacting with RIPK1. HT-29 cells stably expressing FLAG-MYC or empty vector were immunoprecipitated with an anti-RIPK3 antibody. ( B ) MYC prevents the interaction between RIPK1 and RIPK3 in vitro. Recombinant GST-MYC purified using a wheat germ system was added to the recombinant MBP-RIPK1 RHIM domain (496–583) and His-MBP-RIPK3 (388–514) as indicated, followed by Ni 2+ -pulldown analysis. RIPK3-bound RIPK1 was detected using anti-RIPK1 antibodies. ( C ) The N terminus of MYC is responsible for suppressing the RIPK1 and RIPK3 interaction. Bacterially purified MBP-MYC (1–144) was added to the RHIM domains of RIPK1 and RIPK3. The interaction of RIPK1 and RIPK3 was determined as described above. ( D and E ) MYC ablation increases the levels of the RIPK1 and RIPK3 proteins. The levels of the RIPK1, RIPK3, and pMLKL proteins in HT-29 ( D ) and HeLa/RIPK3 cells ( E ) were analyzed using immunoblotting after MYC knockdown and/or treatment with 30 μM Nec-1 or 3 μM GSK′872. ( F and G ) RIPK1 and RIPK3 form a complex in MYC-depleted HT-29 cells. HT-29 cells transfected with the indicated siRNAs were treated with DMSO, GSK′963, or Nec-1. After chemical treatment, an immunoprecipitation assay was carried out using an anti-RIPK3 antibody. ( H ) MYC depletion induces the formation of small and inactive necrosome complexes. HT-29 cells were transfected or treated with the indicated siRNA or stimuli, respectively. The cell lysates were fractionated according to molecular size by gel-filtration chromatography and examined by immunoblotting. ( I ) MYC induces the accumulation of RIPK1 and RIPK3 proteins in the soluble fraction. HT-29 cells were transfected with MYC siRNA and treated with TBZ for 4 h. Cells were then fractioned into lysis buffer soluble (Sol.) and insoluble (Insol.) fractions, followed by immunoblot analysis. The phosphorylated form of RIPK3 (p-RIPK3) is indicated by the arrowhead, and the asterisks indicate p-MLKL.

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Purification, Transfection, Filtration, Chromatography, Lysis

    Both full-length MYC and MYC-nick are responsible for suppressing necroptosis. ( A ) The levels of MYC and MYC-nick in the cytoplasm and nucleus were determined by performing an immunoblot analysis using anti-MYC antibodies (#5605, Cell Signaling). The asterisks indicate a MYC variant. ( B and C ) Expression of FLAG-tagged MYC, MYC-nick, and MYC Δ290–300 in HT-29/MYC KO cells. HT-29/MYC KO cells were reconstituted with lentiviral MYC expression vectors, followed by immunoblot analyses. ( D ) Levels of both the MYC and MYC-nick proteins were decreased in response to TBZ stimulation in HT-29 cells. ( E ) Analysis of the viability of HT-29 cells/MYC KO cells reconstituted with MYC and MYC-nick after treatment with TBZ in the absence or presence of GSK′963. Data are the means ± SD, n = 3, with ** P
    Figure Legend Snippet: Both full-length MYC and MYC-nick are responsible for suppressing necroptosis. ( A ) The levels of MYC and MYC-nick in the cytoplasm and nucleus were determined by performing an immunoblot analysis using anti-MYC antibodies (#5605, Cell Signaling). The asterisks indicate a MYC variant. ( B and C ) Expression of FLAG-tagged MYC, MYC-nick, and MYC Δ290–300 in HT-29/MYC KO cells. HT-29/MYC KO cells were reconstituted with lentiviral MYC expression vectors, followed by immunoblot analyses. ( D ) Levels of both the MYC and MYC-nick proteins were decreased in response to TBZ stimulation in HT-29 cells. ( E ) Analysis of the viability of HT-29 cells/MYC KO cells reconstituted with MYC and MYC-nick after treatment with TBZ in the absence or presence of GSK′963. Data are the means ± SD, n = 3, with ** P

    Techniques Used: Variant Assay, Expressing

    Down-regulation of MYC sensitizes acute myeloid leukemia cells to necroptosis induced by birinapant plus the caspase inhibitor and increases the antileukemia activity in xenograft models. ( A ) The effects of birinapant (Bi) on the levels of cIAP1 in Molm13 cells. ( B ) Immunoblot analysis of various leukemia cell lines. ( C ) Each leukemia cell line was treated with the indicated concentrations of birinapant alone or birinapant plus 20 μM zVAD for 24 h, followed by a FACS analysis to measure cell viability. ( D – F ) Necroptosis and necrosome analysis of Molm13 cells stably expressing shMYC and treated with 25 nM birinapant plus 1 μM emricasan. GSK′963 (RIPK1 inhibitor) was administered to cells at 100 nM to inhibit RIPK1-dependent necroptosis. After treatment, the cells were examined using a FACS analysis to determine the percentage of cell death ( D ), using an immunoblot analysis to determine the phosphorylation of RIPK3 and MLKL ( E ), and using immunoprecipitation to analyze the necrosome ( F ). ( G – J ) MYC depletion suppresses tumor growth after treatment with birinapant (Bir) and emricasan (Emri) in vivo. A total of 5 × 10 5 Molm13 cells were implanted subcutaneously into the flank of 6-wk-old nude mice. After 6 d, the mice were treated with Bir (2 mg/kg) plus Emri (1 mg/kg) as indicated by intraperitoneal injection for 2 wk, and tumor growth is shown in G . Data are the means ± SEM, n = 7 per treatment group, with * P
    Figure Legend Snippet: Down-regulation of MYC sensitizes acute myeloid leukemia cells to necroptosis induced by birinapant plus the caspase inhibitor and increases the antileukemia activity in xenograft models. ( A ) The effects of birinapant (Bi) on the levels of cIAP1 in Molm13 cells. ( B ) Immunoblot analysis of various leukemia cell lines. ( C ) Each leukemia cell line was treated with the indicated concentrations of birinapant alone or birinapant plus 20 μM zVAD for 24 h, followed by a FACS analysis to measure cell viability. ( D – F ) Necroptosis and necrosome analysis of Molm13 cells stably expressing shMYC and treated with 25 nM birinapant plus 1 μM emricasan. GSK′963 (RIPK1 inhibitor) was administered to cells at 100 nM to inhibit RIPK1-dependent necroptosis. After treatment, the cells were examined using a FACS analysis to determine the percentage of cell death ( D ), using an immunoblot analysis to determine the phosphorylation of RIPK3 and MLKL ( E ), and using immunoprecipitation to analyze the necrosome ( F ). ( G – J ) MYC depletion suppresses tumor growth after treatment with birinapant (Bir) and emricasan (Emri) in vivo. A total of 5 × 10 5 Molm13 cells were implanted subcutaneously into the flank of 6-wk-old nude mice. After 6 d, the mice were treated with Bir (2 mg/kg) plus Emri (1 mg/kg) as indicated by intraperitoneal injection for 2 wk, and tumor growth is shown in G . Data are the means ± SEM, n = 7 per treatment group, with * P

    Techniques Used: Activity Assay, FACS, Stable Transfection, Expressing, Immunoprecipitation, In Vivo, Mouse Assay, Injection

    MYC negatively regulates the formation of the RIPK3-containing necrosome. ( A ) MYC depletion promotes necrosome formation upon TBZ stimulation. HT-29 cells transfected with siNT or siMYC were treated with TB or TBZ. After treatment, the cell lysates were immunoprecipitated with an anticaspase-8 antibody and analyzed using immunoblotting. The asterisk indicates RIPK3. ( B ) MYC deletion facilitates necrosome formation. MYC WT and KO HT-29 cells were treated with TBZ for the indicated times, followed by immunoprecipitation with an RIPK3 antibody. ( C ) MYC depletion does not regulate TBZ-induced RIPK1-dependent complex IIb formation in HeLa cells. HeLa cells were transfected with siMYC and treated with TBZ for 3 h. The complex IIb was isolated by immunoprecipitation of caspase-8. ( D ) HT-29 cells were transfected with the indicated siRNA and treated with TBZ for 3 h. The cell lysates were fractionated according to molecular size by gel-filtration chromatography and examined using immunoblotting.
    Figure Legend Snippet: MYC negatively regulates the formation of the RIPK3-containing necrosome. ( A ) MYC depletion promotes necrosome formation upon TBZ stimulation. HT-29 cells transfected with siNT or siMYC were treated with TB or TBZ. After treatment, the cell lysates were immunoprecipitated with an anticaspase-8 antibody and analyzed using immunoblotting. The asterisk indicates RIPK3. ( B ) MYC deletion facilitates necrosome formation. MYC WT and KO HT-29 cells were treated with TBZ for the indicated times, followed by immunoprecipitation with an RIPK3 antibody. ( C ) MYC depletion does not regulate TBZ-induced RIPK1-dependent complex IIb formation in HeLa cells. HeLa cells were transfected with siMYC and treated with TBZ for 3 h. The complex IIb was isolated by immunoprecipitation of caspase-8. ( D ) HT-29 cells were transfected with the indicated siRNA and treated with TBZ for 3 h. The cell lysates were fractionated according to molecular size by gel-filtration chromatography and examined using immunoblotting.

    Techniques Used: Transfection, Immunoprecipitation, Isolation, Filtration, Chromatography

    3) Product Images from "Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade"

    Article Title: Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122373

    The MTOR/EIF4EBP1/YY1/MYC/SLC2A1 signaling mediated the chemopreventive effect of combined resveratrol and silymarin product on tumor growth: Total tumor size (A) and gross view of representative HCCs (B) with or without treatment of resveratrol (Res) and silymarin (Sily). Arrows indicate tumors. (C and D) Western blot analysis revealed a lower expression level of MTOR signal cascade in the treated cells and mice group (six smallest tumor adjacent tissues) than the untreated cells and mice group (six biggest tumor adjacent tissues). Quantitative results by coexpression of pre-S2 mutant and X proteins were relative to the untreated control cells and mice.
    Figure Legend Snippet: The MTOR/EIF4EBP1/YY1/MYC/SLC2A1 signaling mediated the chemopreventive effect of combined resveratrol and silymarin product on tumor growth: Total tumor size (A) and gross view of representative HCCs (B) with or without treatment of resveratrol (Res) and silymarin (Sily). Arrows indicate tumors. (C and D) Western blot analysis revealed a lower expression level of MTOR signal cascade in the treated cells and mice group (six smallest tumor adjacent tissues) than the untreated cells and mice group (six biggest tumor adjacent tissues). Quantitative results by coexpression of pre-S2 mutant and X proteins were relative to the untreated control cells and mice.

    Techniques Used: Western Blot, Expressing, Mouse Assay, Mutagenesis

    MTOR, YY1, MYC, and SLC2A1 signals were chronologically activated in pre-S2 mutant transgenic livers and HCCs: (A) Western blot analysis of the indicated biomarkers in different ages of pre-S2 mutant and non-transgenic livers, as well as paired nontumorous livers and tumors. Six livers were used in each group except the tumor stage due to small tumor size and low tumor formation rate. (B) Quantitative results were normalized by age-matched control livers.
    Figure Legend Snippet: MTOR, YY1, MYC, and SLC2A1 signals were chronologically activated in pre-S2 mutant transgenic livers and HCCs: (A) Western blot analysis of the indicated biomarkers in different ages of pre-S2 mutant and non-transgenic livers, as well as paired nontumorous livers and tumors. Six livers were used in each group except the tumor stage due to small tumor size and low tumor formation rate. (B) Quantitative results were normalized by age-matched control livers.

    Techniques Used: Mutagenesis, Transgenic Assay, Western Blot

    Activation of MTOR/YY1/MYC/SLC2A1 signaling by pre-S2 mutant was mediated by EIF4EBP1 and validated in human HBV-related HCCs: (A) Quantitative results of Western blots of pEIF4EBP1 and pRPS6KB1 were normalized by age-matched control livers. (B) The effect of EIF4EBP1 and RPS6KB1 siRNAs on MTOR signaling activation was determined by Western blot assay in HuH-7 cells. (C) Western blot analysis revealed enhanced expression of the indicated biomarkers in HBV-related HCCs at a level comparable to or even higher than that in the paired nontumorous livers. (D) The data were quantified and statistically analyzed.
    Figure Legend Snippet: Activation of MTOR/YY1/MYC/SLC2A1 signaling by pre-S2 mutant was mediated by EIF4EBP1 and validated in human HBV-related HCCs: (A) Quantitative results of Western blots of pEIF4EBP1 and pRPS6KB1 were normalized by age-matched control livers. (B) The effect of EIF4EBP1 and RPS6KB1 siRNAs on MTOR signaling activation was determined by Western blot assay in HuH-7 cells. (C) Western blot analysis revealed enhanced expression of the indicated biomarkers in HBV-related HCCs at a level comparable to or even higher than that in the paired nontumorous livers. (D) The data were quantified and statistically analyzed.

    Techniques Used: Activation Assay, Mutagenesis, Western Blot, Expressing

    Pre-S2 mutant activated MTOR/YY1/MYC/SLC2A1 signaling cascade to promote SLC2A1 translocation, aerobic glycolysis, and growth advantages in HuH-7 cells: (A and B) HuH-7 cells were transfected with pre-S2 mutant or control plasmid (Ctrl). After 24 hours (h), cells were left untreated or treated with rapamycin (Rapa), YY1 siRNA (si YY1 ) and MYC siRNA (si MYC ) for another 24 hours, and analyzed by Western blot for the indicated biomarkers. (C) Western blots of the whole cell lysate fraction (CL) and the plasma membrane fraction (PM). SLC2A1 translocation represented the level of SLC2A1 in the PM fraction. (D) Confocal microscopy and IF staining of HA (green), SLC2A1 (red), and DAPI (blue). Arrows indicate the peripheral expression of SLC2A1. Original magnification, ×80. Scale bar, 20 μm. (E) For functional in vitro assays, HuH-7 cells transfected with pre-S2 mutant or control plasmid with or without further treatment were subjected to glucose uptake (a), lactate production (b), and cell proliferation (c) assays 48 hours after transfection. Data in each experiment were presented as relative values to the untreated control cells. All the transfection experiments were performed in triplicate and repeated at least three times independently.
    Figure Legend Snippet: Pre-S2 mutant activated MTOR/YY1/MYC/SLC2A1 signaling cascade to promote SLC2A1 translocation, aerobic glycolysis, and growth advantages in HuH-7 cells: (A and B) HuH-7 cells were transfected with pre-S2 mutant or control plasmid (Ctrl). After 24 hours (h), cells were left untreated or treated with rapamycin (Rapa), YY1 siRNA (si YY1 ) and MYC siRNA (si MYC ) for another 24 hours, and analyzed by Western blot for the indicated biomarkers. (C) Western blots of the whole cell lysate fraction (CL) and the plasma membrane fraction (PM). SLC2A1 translocation represented the level of SLC2A1 in the PM fraction. (D) Confocal microscopy and IF staining of HA (green), SLC2A1 (red), and DAPI (blue). Arrows indicate the peripheral expression of SLC2A1. Original magnification, ×80. Scale bar, 20 μm. (E) For functional in vitro assays, HuH-7 cells transfected with pre-S2 mutant or control plasmid with or without further treatment were subjected to glucose uptake (a), lactate production (b), and cell proliferation (c) assays 48 hours after transfection. Data in each experiment were presented as relative values to the untreated control cells. All the transfection experiments were performed in triplicate and repeated at least three times independently.

    Techniques Used: Mutagenesis, Translocation Assay, Transfection, Plasmid Preparation, Western Blot, Confocal Microscopy, Staining, Expressing, Functional Assay, In Vitro

    Schematic model for the upregulation of aerobic glycolysis by pre-S2 mutant in HBV tumorigenesis: Through the induction of ER stress-dependent VEGFA/AKT signaling, pre-S2 mutant activates MTOR signal, which then increases YY1 expression through the EIF4EBP1-mediated translational control. Upon the activation of MYC and SLC2A1 signals by YY1 at the advanced stage of HCC tumorigenesis, hepatocytes may undergo a metabolic switch toward an increase in aerobic glycolysis. The combined effects of aerobic glycolysis and cell cycle progression induced by MYC and SLC2A1 may contribute to growth advantages of hepatocytes and HCC development.
    Figure Legend Snippet: Schematic model for the upregulation of aerobic glycolysis by pre-S2 mutant in HBV tumorigenesis: Through the induction of ER stress-dependent VEGFA/AKT signaling, pre-S2 mutant activates MTOR signal, which then increases YY1 expression through the EIF4EBP1-mediated translational control. Upon the activation of MYC and SLC2A1 signals by YY1 at the advanced stage of HCC tumorigenesis, hepatocytes may undergo a metabolic switch toward an increase in aerobic glycolysis. The combined effects of aerobic glycolysis and cell cycle progression induced by MYC and SLC2A1 may contribute to growth advantages of hepatocytes and HCC development.

    Techniques Used: Mutagenesis, Expressing, Activation Assay

    4) Product Images from "Long noncoding RNA EZR-AS1 promotes tumor growth and metastasis by modulating Wnt/β-catenin pathway in breast cancer"

    Article Title: Long noncoding RNA EZR-AS1 promotes tumor growth and metastasis by modulating Wnt/β-catenin pathway in breast cancer

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6461

    EZR-AS1 protects β-catenin from degradation and activates the Wnt/β-catenin pathway in BC cells. (A) RNA immunoprecipitation assays indicated that EZR-AS1 was enriched by anti-β-catenin in MCF7 and MDA-MB-231 cells. (B) An RNA pull-down assay indicated that β-catenin was precipitated by biotin-labeled EZR-AS1. (C) EZR-AS1 overexpression prevented β-catenin degradation in MCF7 cells. (D) Western blotting was performed to assess the effect of EZR-AS1 knockdown on nuclear β-catenin, MYC, SOX4 and Cyclin D1 expression in MCF7 and MDA-MB-231 cells. (E) Correlation between EZR-AS1 and MYC or SOX4 in BC tissues. *P
    Figure Legend Snippet: EZR-AS1 protects β-catenin from degradation and activates the Wnt/β-catenin pathway in BC cells. (A) RNA immunoprecipitation assays indicated that EZR-AS1 was enriched by anti-β-catenin in MCF7 and MDA-MB-231 cells. (B) An RNA pull-down assay indicated that β-catenin was precipitated by biotin-labeled EZR-AS1. (C) EZR-AS1 overexpression prevented β-catenin degradation in MCF7 cells. (D) Western blotting was performed to assess the effect of EZR-AS1 knockdown on nuclear β-catenin, MYC, SOX4 and Cyclin D1 expression in MCF7 and MDA-MB-231 cells. (E) Correlation between EZR-AS1 and MYC or SOX4 in BC tissues. *P

    Techniques Used: Immunoprecipitation, Multiple Displacement Amplification, Pull Down Assay, Labeling, Over Expression, Western Blot, Expressing

    5) Product Images from "Phosphomimetic Substitution of Heterogeneous Nuclear Ribonucleoprotein A1 at Serine 199 Abolishes AKT-dependent Internal Ribosome Entry Site-transacting Factor (ITAF) Function via Effects on Strand Annealing and Results in Mammalian Target of Rapamycin Complex 1 (mTORC1) Inhibitor Sensitivity"

    Article Title: Phosphomimetic Substitution of Heterogeneous Nuclear Ribonucleoprotein A1 at Serine 199 Abolishes AKT-dependent Internal Ribosome Entry Site-transacting Factor (ITAF) Function via Effects on Strand Annealing and Results in Mammalian Target of Rapamycin Complex 1 (mTORC1) Inhibitor Sensitivity

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.205096

    Conditionally active AKT regulates rapamycin hypersensitivity and cyclin D1 and c-MYC IRES activities. A , immunoblot analysis of LN229 and MEF cells expressing the myr-AKT-MER fusion protein or EV control transfectants. The indicated cells were treated
    Figure Legend Snippet: Conditionally active AKT regulates rapamycin hypersensitivity and cyclin D1 and c-MYC IRES activities. A , immunoblot analysis of LN229 and MEF cells expressing the myr-AKT-MER fusion protein or EV control transfectants. The indicated cells were treated

    Techniques Used: Expressing

    6) Product Images from "Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation"

    Article Title: Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202000799

    HIPK2 regulates spastin via proteasomal degradation through K554 polyubiquitination. (A) Representative Western blot (WB) of HeLa cells transfected as in Fig 1A and lysed 96 h posttransfection and 8 h after treatment with 20 μM MG132 or its solvent DMSO. MDM2 stabilization has shown as MG132 positive control. Statistical differences, ANOVA test. (B) HeLa cells were transfected as in Fig 1A and harvested 96 h posttransfection and 8 h after treatment with 20 μM MG132 or DMSO. Total cell extract (TCE) was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub ladder was normalized by the intensity of spastin band in IP and reported relative to siCtr DMSO–treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) NSC34 cells were transfected as in Fig 1B and harvested 5 d posttransfection and 8 h after treatment with 20 μM MG132. TCE was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a specific band. The intensity of spastin-Ub ladder was calculated and reported as in Fig 2B . Statistical differences, ANOVA test. (D) HeLa Ctr-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing HA-tagged Ub-WT (Ub-HA) or its derivative KoUb-HA (i.e., Ub with all lysines mutated in arginines) and treated 24 h posttransfection with 20 μM MG132 or DMSO for 8 h. TCEs were analysed as in Fig 2B . IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin band in IP and reported relative to the correspondent DMSO-treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (E) Spastin amino acid sequence encompassing the K554 is reported for indicated organisms. Fly = Drosophila melanogaster ; worm = Caenorhabditis elegans . (F) HIPK2-Cas9 HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-WT or spastin-K554R in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE were analysed by WB and immunoprecipitated with anti-Flag Ab (mouse Ab by Origene Technologies). IPs were analysed by WB with anti-HA and anti-Flag Ab (rabbit Ab by Sigma-Aldrich). The arrows indicate the position of the unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin bands in IP and reported as mean ± SD (n = 4). Statistical difference, unpaired t test. (G) HIPK2-Cas9 and Ctr-Cas9 HeLa cells were transfected with vectors expressing spastin-WT or spastin-K554R in combination with peGFP vector at 10:1 molar ratio and analysed by WB 24 h posttransfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to correspondent Ctr-Cas9 control cells. Statistical differences, ANOVA test. Source data are available for this figure.
    Figure Legend Snippet: HIPK2 regulates spastin via proteasomal degradation through K554 polyubiquitination. (A) Representative Western blot (WB) of HeLa cells transfected as in Fig 1A and lysed 96 h posttransfection and 8 h after treatment with 20 μM MG132 or its solvent DMSO. MDM2 stabilization has shown as MG132 positive control. Statistical differences, ANOVA test. (B) HeLa cells were transfected as in Fig 1A and harvested 96 h posttransfection and 8 h after treatment with 20 μM MG132 or DMSO. Total cell extract (TCE) was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub ladder was normalized by the intensity of spastin band in IP and reported relative to siCtr DMSO–treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) NSC34 cells were transfected as in Fig 1B and harvested 5 d posttransfection and 8 h after treatment with 20 μM MG132. TCE was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a specific band. The intensity of spastin-Ub ladder was calculated and reported as in Fig 2B . Statistical differences, ANOVA test. (D) HeLa Ctr-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing HA-tagged Ub-WT (Ub-HA) or its derivative KoUb-HA (i.e., Ub with all lysines mutated in arginines) and treated 24 h posttransfection with 20 μM MG132 or DMSO for 8 h. TCEs were analysed as in Fig 2B . IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin band in IP and reported relative to the correspondent DMSO-treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (E) Spastin amino acid sequence encompassing the K554 is reported for indicated organisms. Fly = Drosophila melanogaster ; worm = Caenorhabditis elegans . (F) HIPK2-Cas9 HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-WT or spastin-K554R in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE were analysed by WB and immunoprecipitated with anti-Flag Ab (mouse Ab by Origene Technologies). IPs were analysed by WB with anti-HA and anti-Flag Ab (rabbit Ab by Sigma-Aldrich). The arrows indicate the position of the unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin bands in IP and reported as mean ± SD (n = 4). Statistical difference, unpaired t test. (G) HIPK2-Cas9 and Ctr-Cas9 HeLa cells were transfected with vectors expressing spastin-WT or spastin-K554R in combination with peGFP vector at 10:1 molar ratio and analysed by WB 24 h posttransfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to correspondent Ctr-Cas9 control cells. Statistical differences, ANOVA test. Source data are available for this figure.

    Techniques Used: Western Blot, Transfection, Positive Control, Immunoprecipitation, Expressing, Sequencing, Plasmid Preparation

    Kinase activity of HIPK2 regulates spastin protein levels. (A) Representative Western blot (WB) of HeLa (right panels) and NSC34 cells (left panels) transfected with vectors expressing HA-tagged HIPK2-WT, its derivative -K228A mutant or HA-alone (empty). Statistical differences, Anova test. (B) Representative WB of HeLa cells transfected with vectors expressing flag-myc spastin-WT or indicated spastin mutants in combination with peGFP vector at 10:1 molar ratio and lysed at indicated time post transfection. GFP expression was used as internal control for transfection efficiency. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to spastin-WT for each time point. Statistical differences, ANOVA test. (C) HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-S268A or spastin-WT, treated with 25 μg/ml cycloeximide 36 h posttransfection and analysed by WB at indicated times after treatment. Note that to minimize differences in spastin levels at the time 0, cells were transfected with different doses of the expressing vectors, that is, 1 μg of spastin-S268A–expressing vector and 0.5 μg of spastin-WT–expressing vectors. Representative WB is shown. The levels of spastin-Flag bands relative to those of GAPDH were measured at each time point and reported as mean ± SEM of four different independent experiments in the right panel. Statistical differences were calculated and reported for each time point, unpaired t test. Source data are available for this figure.
    Figure Legend Snippet: Kinase activity of HIPK2 regulates spastin protein levels. (A) Representative Western blot (WB) of HeLa (right panels) and NSC34 cells (left panels) transfected with vectors expressing HA-tagged HIPK2-WT, its derivative -K228A mutant or HA-alone (empty). Statistical differences, Anova test. (B) Representative WB of HeLa cells transfected with vectors expressing flag-myc spastin-WT or indicated spastin mutants in combination with peGFP vector at 10:1 molar ratio and lysed at indicated time post transfection. GFP expression was used as internal control for transfection efficiency. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to spastin-WT for each time point. Statistical differences, ANOVA test. (C) HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-S268A or spastin-WT, treated with 25 μg/ml cycloeximide 36 h posttransfection and analysed by WB at indicated times after treatment. Note that to minimize differences in spastin levels at the time 0, cells were transfected with different doses of the expressing vectors, that is, 1 μg of spastin-S268A–expressing vector and 0.5 μg of spastin-WT–expressing vectors. Representative WB is shown. The levels of spastin-Flag bands relative to those of GAPDH were measured at each time point and reported as mean ± SEM of four different independent experiments in the right panel. Statistical differences were calculated and reported for each time point, unpaired t test. Source data are available for this figure.

    Techniques Used: Activity Assay, Western Blot, Transfection, Expressing, Mutagenesis, Plasmid Preparation

    Spastin is K48-polyubiquitinated. (A) Data quantification of spastin/loading control levels by Western blot (WB) on total cell extract (TCE), relative to data shown in Fig 2B . Quantification was performed as in Fig 2A . Bars are mean ± SD of three independent experiments; Statistical differences, ANOVA test. (B) As in Fig 2D , HeLa cells were transfected with vector expressing Ub-HA and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-spastin mouse monocolonal Ab, sp3G11/1. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. (C, D) HeLa HIPK2-Cas9 and Ctr-Cas9 cells were transfected with vectors expressing HA-Ub or its derivative K48-Ub-HA (i.e., Ub with only K48, the other lysines are mutated in arginines) and treated 24 h post transfection with 20 μM MG132 or DMSO for 8 h. TCE were analysed by WB and IP as in Fig 2D . In (C), representative WB of three independent experiment is shown. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. In (D), relative spastin-Ub-HA levels were quantified and reported as in Fig 2C . Statistical differences, unpaired t test. (E) As in Fig 2F , HeLa cells were transfected with vectors expressing flag-myc tagged spastin in combination with Ub-HA and 24 h post transfection cells were treated with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-Flag mouse monoclonal Ab. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-Flag Abs. The arrow indicates the position of the unmodified spastin.
    Figure Legend Snippet: Spastin is K48-polyubiquitinated. (A) Data quantification of spastin/loading control levels by Western blot (WB) on total cell extract (TCE), relative to data shown in Fig 2B . Quantification was performed as in Fig 2A . Bars are mean ± SD of three independent experiments; Statistical differences, ANOVA test. (B) As in Fig 2D , HeLa cells were transfected with vector expressing Ub-HA and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-spastin mouse monocolonal Ab, sp3G11/1. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. (C, D) HeLa HIPK2-Cas9 and Ctr-Cas9 cells were transfected with vectors expressing HA-Ub or its derivative K48-Ub-HA (i.e., Ub with only K48, the other lysines are mutated in arginines) and treated 24 h post transfection with 20 μM MG132 or DMSO for 8 h. TCE were analysed by WB and IP as in Fig 2D . In (C), representative WB of three independent experiment is shown. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. In (D), relative spastin-Ub-HA levels were quantified and reported as in Fig 2C . Statistical differences, unpaired t test. (E) As in Fig 2F , HeLa cells were transfected with vectors expressing flag-myc tagged spastin in combination with Ub-HA and 24 h post transfection cells were treated with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-Flag mouse monoclonal Ab. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-Flag Abs. The arrow indicates the position of the unmodified spastin.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation

    Phosphorylation in S268 prevents spastin polyubiquitination and degradation. (A, B) HeLa Ctr-Cas9 (A) and HIPK2-Cas9 (B) cells were transfected with vectors expressing indicated flag-myc–tagged spastin-WT or its derivative mutants in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. Total cell extracts (TCEs) were analysed by Western blot (WB) and IP as in Fig 2F . Samples were processed in parallel and analysed on the same blot to make comparison between HIPK2 proficient and null cells. The arrows indicate the position of unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin-Flag bands in IP and reported as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) HeLa Crt-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing indicated flag-myc–tagged spastin WT or its derivative S268D in combination with peGFP vector as in Fig 2G and analysed by WB 24 h post transfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to Ctr-Cas9 control cells. Statistical differences, Anova test. (D) Representative Co-IP showing that spastin-S268D preferentially binds CAND1. HeLa cells were co-transfected with the plasmid expressing MYC-CAND1 in combination with empty vector or vectors expressing spastin-S268A or spastin-S268D. Cells were collected 24 h posttransfection. TCE were analysed by WB or immunoprecipitated with anti-Flag or anti-CAND1 Abs and analysed as indicated. The asterisk indicates an aspecific band. TCE and IP samples were loaded on the same gel and processed on the same filter. Blots were vertically cropped to show appropriate expositions. Full blots are shown in the source data F4 file. (E) Co-IP showing that spastin interaction with CAND1 is stronger in HeLa Ctr-Cas9 cells compared with HIPK2-Cas9 cells. TCE from Ctr-Cas9 and HIPK2-Cas9 cells were analysed by WB or immunoprecipitated with anti-spastin Ab and analysed with indicated Abs. IgG immunoglobulins were used as IP negative control. Band intensities of co-immunoprecipitated CAND1 were normalized by band intensities of spastin immunoprecipitated and their relative values are reported as mean ± SD (n = 3). Statistical difference, unpaired t test. Source data are available for this figure.
    Figure Legend Snippet: Phosphorylation in S268 prevents spastin polyubiquitination and degradation. (A, B) HeLa Ctr-Cas9 (A) and HIPK2-Cas9 (B) cells were transfected with vectors expressing indicated flag-myc–tagged spastin-WT or its derivative mutants in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. Total cell extracts (TCEs) were analysed by Western blot (WB) and IP as in Fig 2F . Samples were processed in parallel and analysed on the same blot to make comparison between HIPK2 proficient and null cells. The arrows indicate the position of unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin-Flag bands in IP and reported as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) HeLa Crt-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing indicated flag-myc–tagged spastin WT or its derivative S268D in combination with peGFP vector as in Fig 2G and analysed by WB 24 h post transfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to Ctr-Cas9 control cells. Statistical differences, Anova test. (D) Representative Co-IP showing that spastin-S268D preferentially binds CAND1. HeLa cells were co-transfected with the plasmid expressing MYC-CAND1 in combination with empty vector or vectors expressing spastin-S268A or spastin-S268D. Cells were collected 24 h posttransfection. TCE were analysed by WB or immunoprecipitated with anti-Flag or anti-CAND1 Abs and analysed as indicated. The asterisk indicates an aspecific band. TCE and IP samples were loaded on the same gel and processed on the same filter. Blots were vertically cropped to show appropriate expositions. Full blots are shown in the source data F4 file. (E) Co-IP showing that spastin interaction with CAND1 is stronger in HeLa Ctr-Cas9 cells compared with HIPK2-Cas9 cells. TCE from Ctr-Cas9 and HIPK2-Cas9 cells were analysed by WB or immunoprecipitated with anti-spastin Ab and analysed with indicated Abs. IgG immunoglobulins were used as IP negative control. Band intensities of co-immunoprecipitated CAND1 were normalized by band intensities of spastin immunoprecipitated and their relative values are reported as mean ± SD (n = 3). Statistical difference, unpaired t test. Source data are available for this figure.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control

    7) Product Images from "Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells"

    Article Title: Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells

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

    doi: 10.1016/j.neo.2018.08.013

    DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P
    Figure Legend Snippet: DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P

    Techniques Used: Stable Transfection, Flow Cytometry, Cytometry, Mutagenesis, Transformation Assay, Immunofluorescence, Microscopy, Quantitation Assay, Software

    DNMT3A -R882 mutants reduce ROS production and overrode the ROS-mediated apoptosis in the presence of an oxidizing agent. (A) Stably transduced U937 cells incubated with H2DCF-DA for 30 minutes at 37°C incubator; fluorescent oxidized DCF (green) and DAPI (blue) were photographed with fluorescence microscopy (left panel, original magnification: ×1000). All the images were taken with same contrast and exposure time. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry (right upper panel) and percentage of ROS induction are shown (right lower panel). (B) Stably transduced HL-60 cells stained with H2DCF-DA; staining cells were photographed with phase-contrast microscopy (left upper panel) and fluorescence microscopy (left lower panel); original magnification: ×100. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry and percentage of ROS induction (right panel) are shown. (C) Inhibition of cell survival following 200 μM TBHP treatment for 24 hours. (D) After treatment of 200 μM TBHP for 24 hours, cells were harvested and labeled with FITC-Annexin V as described in methods. Flow cytometry was used to detect Annexin V–positive cells. (E) Apoptosis triggering by TBHP treatment; transformed U937 cells were incubated with 1 mM TBHP for 2 hours followed by a 2-hour incubation without TBHP. Cell lysates were prepared, and apoptosis inducing proteins including MYC and P-MYC expressions were checked by immunoblot. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted; * P
    Figure Legend Snippet: DNMT3A -R882 mutants reduce ROS production and overrode the ROS-mediated apoptosis in the presence of an oxidizing agent. (A) Stably transduced U937 cells incubated with H2DCF-DA for 30 minutes at 37°C incubator; fluorescent oxidized DCF (green) and DAPI (blue) were photographed with fluorescence microscopy (left panel, original magnification: ×1000). All the images were taken with same contrast and exposure time. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry (right upper panel) and percentage of ROS induction are shown (right lower panel). (B) Stably transduced HL-60 cells stained with H2DCF-DA; staining cells were photographed with phase-contrast microscopy (left upper panel) and fluorescence microscopy (left lower panel); original magnification: ×100. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry and percentage of ROS induction (right panel) are shown. (C) Inhibition of cell survival following 200 μM TBHP treatment for 24 hours. (D) After treatment of 200 μM TBHP for 24 hours, cells were harvested and labeled with FITC-Annexin V as described in methods. Flow cytometry was used to detect Annexin V–positive cells. (E) Apoptosis triggering by TBHP treatment; transformed U937 cells were incubated with 1 mM TBHP for 2 hours followed by a 2-hour incubation without TBHP. Cell lysates were prepared, and apoptosis inducing proteins including MYC and P-MYC expressions were checked by immunoblot. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted; * P

    Techniques Used: Stable Transfection, Incubation, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Staining, Inhibition, Labeling, Transformation Assay

    DNMT3A -R882 mutants augmented PRDX2 expression in myeloid leukemia cells. ( A and B) Immunoblot showing overexpression of DNMT3A -mutant into U937 (A) and HL-60 (B) cells augmented protein expression of PRDX2 with increasing MYC and P-MYC expression. β-Actin was used as a control for equal loading. (C) Cytoplasmic localization of PRDX2 in U937 cells transduced with EV-, WT-, and mutant- DNMT3A ; immunostained with anti-PRDX2 (red) and DAPI (blue) (original magnification: ×1000). All the images were taken with same contrast and exposure time. (D) Co-immunoprecipitation data showing FLAG-tagged WT- and mutant DNMT3A both interacted with PRDX2 in 293T cells. Cell extracts were incubated with DNAse prior to immunoprecipitation (Supplementary Fig. S1 for effectiveness of DNAse treatment).
    Figure Legend Snippet: DNMT3A -R882 mutants augmented PRDX2 expression in myeloid leukemia cells. ( A and B) Immunoblot showing overexpression of DNMT3A -mutant into U937 (A) and HL-60 (B) cells augmented protein expression of PRDX2 with increasing MYC and P-MYC expression. β-Actin was used as a control for equal loading. (C) Cytoplasmic localization of PRDX2 in U937 cells transduced with EV-, WT-, and mutant- DNMT3A ; immunostained with anti-PRDX2 (red) and DAPI (blue) (original magnification: ×1000). All the images were taken with same contrast and exposure time. (D) Co-immunoprecipitation data showing FLAG-tagged WT- and mutant DNMT3A both interacted with PRDX2 in 293T cells. Cell extracts were incubated with DNAse prior to immunoprecipitation (Supplementary Fig. S1 for effectiveness of DNAse treatment).

    Techniques Used: Expressing, Over Expression, Mutagenesis, Transduction, Immunoprecipitation, Incubation

    PRDX2 blocks apoptosis of myeloid leukemia cells. (A) Silenced PRDX2 using two independent shRNA and scrambled (shLuc) K562 cells were treated with 300 nM ABT-263 or without drug for 72 hours, and cell proliferation was measured by trypan blue exclusion method. (B) Colony formation ability was assayed in Methocult medium after being stably silenced of PRDX2 in K562 cells. After 7 days, colonies were photographed and counted manually; original magnification: ×100. (C) PRDX 2-silenced and scrambled K562 cells treated with 300 nM ABT-263 for 72 hours. Apoptosis cells were analyzed using Annexin V and PI staining by flow cytometric analysis. Representative flow cytometry analysis (left panel) and the % of Annexin V and PI-positive cells are shown (right panel). (D) Immunoblot data showing the PRDX2 silenced efficiency in K562 cells and protein expression of P-MYC and MYC after knock down of PRDX2 in K562 cells. β-Actin was used as a control for equal loading. (E) Proliferation of U937 cells transduced with WT- or R882H and knocked down of PRDX2 from transduced cells. (F) Apoptosis analysis of R882H-expressed U937 cells knocked down with shLuc or shPRDX2 and treatment of 300 nM ABT-263 for 72 hours. (G) Immunoblot data showing the PRDX2 silenced efficiency in U937 cells transduced with R882H and protein expression of MYC and CDK1. β-Actin was used as a control for equal loading. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted. * P
    Figure Legend Snippet: PRDX2 blocks apoptosis of myeloid leukemia cells. (A) Silenced PRDX2 using two independent shRNA and scrambled (shLuc) K562 cells were treated with 300 nM ABT-263 or without drug for 72 hours, and cell proliferation was measured by trypan blue exclusion method. (B) Colony formation ability was assayed in Methocult medium after being stably silenced of PRDX2 in K562 cells. After 7 days, colonies were photographed and counted manually; original magnification: ×100. (C) PRDX 2-silenced and scrambled K562 cells treated with 300 nM ABT-263 for 72 hours. Apoptosis cells were analyzed using Annexin V and PI staining by flow cytometric analysis. Representative flow cytometry analysis (left panel) and the % of Annexin V and PI-positive cells are shown (right panel). (D) Immunoblot data showing the PRDX2 silenced efficiency in K562 cells and protein expression of P-MYC and MYC after knock down of PRDX2 in K562 cells. β-Actin was used as a control for equal loading. (E) Proliferation of U937 cells transduced with WT- or R882H and knocked down of PRDX2 from transduced cells. (F) Apoptosis analysis of R882H-expressed U937 cells knocked down with shLuc or shPRDX2 and treatment of 300 nM ABT-263 for 72 hours. (G) Immunoblot data showing the PRDX2 silenced efficiency in U937 cells transduced with R882H and protein expression of MYC and CDK1. β-Actin was used as a control for equal loading. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted. * P

    Techniques Used: shRNA, Stable Transfection, Staining, Flow Cytometry, Cytometry, Expressing, Transduction

    8) Product Images from "AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells"

    Article Title: AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells

    Journal: Cell Research

    doi: 10.1038/s41422-020-00460-y

    AXL binds to SARS-CoV-2 S and facilitates SARS-CoV-2 entry into host cells. a AXL facilitates SARS-CoV-2 virus pseudotype infection as potently as ACE2. HEK293T cells were transfected with MYC-tagged ACE2, AXL, LDLR or EGFR, infected with the GFP-labeled SARS-CoV-2 virus pseudotype, and visualized by microscopy at 24 h post infection. The scale bar indicates 250 μm. b The fluorescence intensities in a were quantitated as indicated. c – e AXL specifically facilitates SARS-CoV-2 virus pseudotype infection. c HEK293T cells were transfected with MYC-tagged AXL, MER or FGFR, and expression was evaluated by western blotting assay with antibodies recognizing the MYC epitope tag. d The cells were infected with the GFP-labeled SARS-CoV-2 virus pseudotype and visualized by microscopy at 24 h post infection. The scale bar indicates 250 μm. e The fluorescence intensities in d were quantitated as indicated. AXL binds to SARS-CoV-2 S and internalizes it in cooperation with endocytosis-related proteins. f AXL facilitates SARS-CoV-2 virus pseudotype binding to the cell surface. HEK293T cells were transfected with MYC-tagged AXL, ACE2, FGFR or MER and infected with a SARS-CoV-2 virus pseudotype for 2 h. The cells were fixed and subjected to immunofluorescence with an anti-FLAG antibody against the indicated proteins (red), an anti-SARS-CoV-2 S antibody (green) and DAPI (blue) and visualized by confocal microscopy. The white arrowheads indicate the virus. The scale bar indicates 15 μm. g AXL facilitates SARS-CoV-2 virus pseudotype entry into host cells, utilizing the host endocytosis system. H1299 cells were infected with a SARS-CoV-2 virus pseudotype for 4 h. The cells were fixed; subjected to immunofluorescence with antibodies against AXL (gray), SARS-CoV-2 S (green) and the indicated endocytosis-related proteins (red) and with DAPI (blue); and visualized by confocal microscopy. The scale bar indicates 15 μm. h AXL promotes SARS-CoV-2 virus pseudotype production in host cells. HEK293T cells were transfected with MYC-tagged AXL and infected with the GFP-labeled SARS-CoV-2 virus pseudotype for 24 h. The cells were fixed and subjected to immunofluorescence with an anti-MYC antibody against AXL (red) and DAPI (blue) and visualized by confocal microscopy. The scale bar indicates 20 μm. i The AXL NTD is required for binding with SARS-CoV-2 S. MYC-tagged wild-type or truncated AXL plasmids were transfected with FLAG-tagged SARS-CoV-2 S; co-IP assays were performed using an anti-MYC antibody, and epitope-tagged proteins were detected using western blotting assay. j The AXL NTD is required for SARS-CoV-2 virus pseudotype infection. HEK293T cells were transfected with full-length AXL or its N-terminal deletion mutant, infected with the GFP-labeled SARS-CoV-2 virus pseudotype and visualized by microscopy. The scale bar indicates 250 μm. k The fluorescence intensities in j were quantitated as indicated. The data shown are representative results from three independent experiments ( a – k , n = 3). The data are shown as the means ± SEM from three independent experiments. P values were calculated using two-way ANOVA (* P
    Figure Legend Snippet: AXL binds to SARS-CoV-2 S and facilitates SARS-CoV-2 entry into host cells. a AXL facilitates SARS-CoV-2 virus pseudotype infection as potently as ACE2. HEK293T cells were transfected with MYC-tagged ACE2, AXL, LDLR or EGFR, infected with the GFP-labeled SARS-CoV-2 virus pseudotype, and visualized by microscopy at 24 h post infection. The scale bar indicates 250 μm. b The fluorescence intensities in a were quantitated as indicated. c – e AXL specifically facilitates SARS-CoV-2 virus pseudotype infection. c HEK293T cells were transfected with MYC-tagged AXL, MER or FGFR, and expression was evaluated by western blotting assay with antibodies recognizing the MYC epitope tag. d The cells were infected with the GFP-labeled SARS-CoV-2 virus pseudotype and visualized by microscopy at 24 h post infection. The scale bar indicates 250 μm. e The fluorescence intensities in d were quantitated as indicated. AXL binds to SARS-CoV-2 S and internalizes it in cooperation with endocytosis-related proteins. f AXL facilitates SARS-CoV-2 virus pseudotype binding to the cell surface. HEK293T cells were transfected with MYC-tagged AXL, ACE2, FGFR or MER and infected with a SARS-CoV-2 virus pseudotype for 2 h. The cells were fixed and subjected to immunofluorescence with an anti-FLAG antibody against the indicated proteins (red), an anti-SARS-CoV-2 S antibody (green) and DAPI (blue) and visualized by confocal microscopy. The white arrowheads indicate the virus. The scale bar indicates 15 μm. g AXL facilitates SARS-CoV-2 virus pseudotype entry into host cells, utilizing the host endocytosis system. H1299 cells were infected with a SARS-CoV-2 virus pseudotype for 4 h. The cells were fixed; subjected to immunofluorescence with antibodies against AXL (gray), SARS-CoV-2 S (green) and the indicated endocytosis-related proteins (red) and with DAPI (blue); and visualized by confocal microscopy. The scale bar indicates 15 μm. h AXL promotes SARS-CoV-2 virus pseudotype production in host cells. HEK293T cells were transfected with MYC-tagged AXL and infected with the GFP-labeled SARS-CoV-2 virus pseudotype for 24 h. The cells were fixed and subjected to immunofluorescence with an anti-MYC antibody against AXL (red) and DAPI (blue) and visualized by confocal microscopy. The scale bar indicates 20 μm. i The AXL NTD is required for binding with SARS-CoV-2 S. MYC-tagged wild-type or truncated AXL plasmids were transfected with FLAG-tagged SARS-CoV-2 S; co-IP assays were performed using an anti-MYC antibody, and epitope-tagged proteins were detected using western blotting assay. j The AXL NTD is required for SARS-CoV-2 virus pseudotype infection. HEK293T cells were transfected with full-length AXL or its N-terminal deletion mutant, infected with the GFP-labeled SARS-CoV-2 virus pseudotype and visualized by microscopy. The scale bar indicates 250 μm. k The fluorescence intensities in j were quantitated as indicated. The data shown are representative results from three independent experiments ( a – k , n = 3). The data are shown as the means ± SEM from three independent experiments. P values were calculated using two-way ANOVA (* P

    Techniques Used: Infection, Transfection, Labeling, Microscopy, Fluorescence, Expressing, Western Blot, Binding Assay, Immunofluorescence, Confocal Microscopy, Co-Immunoprecipitation Assay, Mutagenesis

    The SARS-CoV-2 S glycoprotein interacts with host AXL. a Validation of the interaction between SARS-CoV-2 S and ACE2, AXL, LDLR or EGFR. HEK293T cells were transfected with FLAG-tagged SARS-CoV-2 S and MYC-tagged ACE2, AXL, LDLR or EGFR for 24 h. The cells were lysed, and the lysates were incubated with FLAG-M2 resin; 5% lysate was used as the input control. Blots with antibodies recognizing the FLAG- or MYC-epitope tags are shown. b Co-localization assay of SARS-CoV-2 S and ACE2, AXL, LDLR or EGFR. HEK293T cells were transfected with the indicated constructs and subjected to immunofluorescence with an anti-FLAG antibody against SARS-CoV-2 S (red), an anti-MYC antibody against candidate receptors (green) and DAPI (blue) and visualized by microscopy. The scale bar indicates 15 μm. c In vitro pull-down assay of SARS-CoV-2 S and AXL. FLAG-tagged SARS-CoV-2 S and His-tagged AXL (amino acids 1–449) were expressed in HEK293T cells, affinity-purified, eluted and co-incubated for 1 h. Blots with antibodies recognizing the FLAG- or His-epitope tags are shown. d In vitro binding assay of SARS-CoV-2 S NTD and AXL. His-tagged SARS-CoV-2 S NTD and FLAG-tagged AXL were expressed in 293F cells, affinity-purified and eluted. The KD between His-tagged SARS-CoV-2 S NTD and FLAG-tagged AXL was measured using a BLI quantification assay. e Endogenous AXL interacts with the NTD of SARS-CoV-2 S. The in vitro-purified His-tagged SARS-CoV-2 S S1 domain, NTD and RBD were incubated with H1299 cell lysate and Ni-NTA resin; 5% lysate was used as the input control. Blots with antibodies recognizing endogenous AXL or His-epitope tags are shown. AXL is highly expressed in the H1299 and BEAS-2B cell lines. The expression of ACE2, AXL, LDLR and EGFR was examined in the HEK293T, H1299 and BEAS-2B cell lines by western blotting assay ( f ) and RT-qPCR ( g ). h AXL is highly expressed in human lung tissue. Human lung tissue sections were immunostained with antibodies against ACE2 or AXL (green) and with DAPI (blue) and visualized by confocal microscopy. The scale bars indicate 500 μm. AXL expression levels in pulmonary ( i , j ) and bronchial cells ( k , l ) were evaluated using the human cell landscape at the single-cell level. Gene expression for each cell type was visualized using tSNE ( i , k ) and violin plots ( j , l ). The data shown are representative results from three independent experiments ( a – h , n = 3). The data are shown as the means ± SEM from three independent experiments.
    Figure Legend Snippet: The SARS-CoV-2 S glycoprotein interacts with host AXL. a Validation of the interaction between SARS-CoV-2 S and ACE2, AXL, LDLR or EGFR. HEK293T cells were transfected with FLAG-tagged SARS-CoV-2 S and MYC-tagged ACE2, AXL, LDLR or EGFR for 24 h. The cells were lysed, and the lysates were incubated with FLAG-M2 resin; 5% lysate was used as the input control. Blots with antibodies recognizing the FLAG- or MYC-epitope tags are shown. b Co-localization assay of SARS-CoV-2 S and ACE2, AXL, LDLR or EGFR. HEK293T cells were transfected with the indicated constructs and subjected to immunofluorescence with an anti-FLAG antibody against SARS-CoV-2 S (red), an anti-MYC antibody against candidate receptors (green) and DAPI (blue) and visualized by microscopy. The scale bar indicates 15 μm. c In vitro pull-down assay of SARS-CoV-2 S and AXL. FLAG-tagged SARS-CoV-2 S and His-tagged AXL (amino acids 1–449) were expressed in HEK293T cells, affinity-purified, eluted and co-incubated for 1 h. Blots with antibodies recognizing the FLAG- or His-epitope tags are shown. d In vitro binding assay of SARS-CoV-2 S NTD and AXL. His-tagged SARS-CoV-2 S NTD and FLAG-tagged AXL were expressed in 293F cells, affinity-purified and eluted. The KD between His-tagged SARS-CoV-2 S NTD and FLAG-tagged AXL was measured using a BLI quantification assay. e Endogenous AXL interacts with the NTD of SARS-CoV-2 S. The in vitro-purified His-tagged SARS-CoV-2 S S1 domain, NTD and RBD were incubated with H1299 cell lysate and Ni-NTA resin; 5% lysate was used as the input control. Blots with antibodies recognizing endogenous AXL or His-epitope tags are shown. AXL is highly expressed in the H1299 and BEAS-2B cell lines. The expression of ACE2, AXL, LDLR and EGFR was examined in the HEK293T, H1299 and BEAS-2B cell lines by western blotting assay ( f ) and RT-qPCR ( g ). h AXL is highly expressed in human lung tissue. Human lung tissue sections were immunostained with antibodies against ACE2 or AXL (green) and with DAPI (blue) and visualized by confocal microscopy. The scale bars indicate 500 μm. AXL expression levels in pulmonary ( i , j ) and bronchial cells ( k , l ) were evaluated using the human cell landscape at the single-cell level. Gene expression for each cell type was visualized using tSNE ( i , k ) and violin plots ( j , l ). The data shown are representative results from three independent experiments ( a – h , n = 3). The data are shown as the means ± SEM from three independent experiments.

    Techniques Used: Transfection, Incubation, Construct, Immunofluorescence, Microscopy, In Vitro, Pull Down Assay, Affinity Purification, Binding Assay, Purification, Expressing, Western Blot, Quantitative RT-PCR, Confocal Microscopy

    9) Product Images from "p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1"

    Article Title: p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201701049

    Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P
    Figure Legend Snippet: Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P

    Techniques Used: Activity Assay, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection

    p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.
    Figure Legend Snippet: p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.

    Techniques Used: Recombinant, In Vitro, Incubation, Co-Immunoprecipitation Assay, Western Blot

    10) Product Images from "Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma"

    Article Title: Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aal2668

    Induction of apoptosis by CMLD010509 ( A ) Apoptosis assay measured by caspase-3 and caspase-7 activation (Caspase-Glo 3/7) at 3 and 24 hours in NCI-H929 and MM1S cells treated with three concentrations of CMLD010509 versus vehicle control. ( B ) Immunoblots for cleavage of both PARP and caspase-3 in response to three concentrations of CMLD010509 in NCI-H929 and MM1S cells at 3 and 24 hours. ( C ) Immunoblots for MYC and MCL-1 in response to three concentrations of CMLD010509 in NCI-H929 and MM1S cells at 3 and 24 hours.
    Figure Legend Snippet: Induction of apoptosis by CMLD010509 ( A ) Apoptosis assay measured by caspase-3 and caspase-7 activation (Caspase-Glo 3/7) at 3 and 24 hours in NCI-H929 and MM1S cells treated with three concentrations of CMLD010509 versus vehicle control. ( B ) Immunoblots for cleavage of both PARP and caspase-3 in response to three concentrations of CMLD010509 in NCI-H929 and MM1S cells at 3 and 24 hours. ( C ) Immunoblots for MYC and MCL-1 in response to three concentrations of CMLD010509 in NCI-H929 and MM1S cells at 3 and 24 hours.

    Techniques Used: Apoptosis Assay, Activation Assay, Western Blot

    11) Product Images from "Actin-like protein 6A/MYC/CDK2 axis confers high proliferative activity in triple-negative breast cancer"

    Article Title: Actin-like protein 6A/MYC/CDK2 axis confers high proliferative activity in triple-negative breast cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-021-01856-3

    ACTL6A enhances CDK2 transcriptional activity by increasing the enrichment of MYC and KAT5 on its promoters. a A Venn diagram among MYC related genes, ACTL6A related genes, and cell cycle related genes. b Real-time PCR analysis of the expression of 9 genes in SUM159PT cells with altered ACTL6A expression. c Western blot analysis of CDK2 in indicated cells. d Luciferase activity of CDK2 reporter were detected in the indicated cells. e Schematic illustration of the PCR fragments of the human CDK2 gene promoters (upper panel). Chromatin immunoprecipitation (ChIP) assays were performed in SUM159PT-ACTL6A-Flag cells using antibodies against Flag and MYC to identify the occupancy on CDK2 gene promoters. Immunoglobulin G (IgG) was used as a negative control. f Enrichment of MYC on p2 fragment of CDK2 promoter. g IP assay revealed that ACTL6A formed complex with MYC and KAT5 in SUM159PT-ACTL6A cells. h Silencing of ACTL6A abrogated interaction between MYC and KAT5. (I) Enrichment of KAT5 on p2 fragment of CDK2 promoter. j Appearance of acetylated histone H3K14ac and histone H4 (K12ac, K16ac, K5ac and K8ac) on p2 fragment of CDK2 promote in the indicated cells. Each bar represents the mean ± S.D. of three independent experiments. Two-tailed Student’s t test was used. * P
    Figure Legend Snippet: ACTL6A enhances CDK2 transcriptional activity by increasing the enrichment of MYC and KAT5 on its promoters. a A Venn diagram among MYC related genes, ACTL6A related genes, and cell cycle related genes. b Real-time PCR analysis of the expression of 9 genes in SUM159PT cells with altered ACTL6A expression. c Western blot analysis of CDK2 in indicated cells. d Luciferase activity of CDK2 reporter were detected in the indicated cells. e Schematic illustration of the PCR fragments of the human CDK2 gene promoters (upper panel). Chromatin immunoprecipitation (ChIP) assays were performed in SUM159PT-ACTL6A-Flag cells using antibodies against Flag and MYC to identify the occupancy on CDK2 gene promoters. Immunoglobulin G (IgG) was used as a negative control. f Enrichment of MYC on p2 fragment of CDK2 promoter. g IP assay revealed that ACTL6A formed complex with MYC and KAT5 in SUM159PT-ACTL6A cells. h Silencing of ACTL6A abrogated interaction between MYC and KAT5. (I) Enrichment of KAT5 on p2 fragment of CDK2 promoter. j Appearance of acetylated histone H3K14ac and histone H4 (K12ac, K16ac, K5ac and K8ac) on p2 fragment of CDK2 promote in the indicated cells. Each bar represents the mean ± S.D. of three independent experiments. Two-tailed Student’s t test was used. * P

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Luciferase, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Two Tailed Test

    12) Product Images from "InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment"

    Article Title: InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25624

    MYC , one of the most influential genes, accounts for the drug-induced change in gene expression (a, b) Immunoblot analysis of MYC under (a) 16-h treatment and (b) 6-h treatment of HT-29 cells with the indicated compounds. mTOR was used as a loading control. Blot intensities of MYC relative to those of mTOR (n=3 independent experiments, mean ± SD) are shown (b, below). The drug concentrations were the same with the description in Supplementary Table 1 . (c, d) Immunoblot analysis of MYC upon treatment with MYC siRNAs. RPS3 and β-actin were used as a loading control. (e, f) Cell growth assay after treatment with MYC siRNAs. ON-TARGETplus SMART pool siRNA was used (in c, e) and Silencer Select Pre-designed siRNAs were used (in d, f). (g) Hierarchical clustering analysis of indicated conditions using DEGs of MYC siRNA. (h) Enrichment plot using MYC siRNA-increased gene sets. Running enrichment score ( top portion, green curve ) and the statistics were calculated from the order of gene sets based on the gene expression change ( bottom ) upon treatment with U-0126. GEM, gemcitabine; MTX, methotrexate; ETP, etoposide; 6-MP, 6-mercaptopurine; MMC, mitomycin C; TOP, topotecan; DOX, doxorubicin; MIT, mitoxantrone.
    Figure Legend Snippet: MYC , one of the most influential genes, accounts for the drug-induced change in gene expression (a, b) Immunoblot analysis of MYC under (a) 16-h treatment and (b) 6-h treatment of HT-29 cells with the indicated compounds. mTOR was used as a loading control. Blot intensities of MYC relative to those of mTOR (n=3 independent experiments, mean ± SD) are shown (b, below). The drug concentrations were the same with the description in Supplementary Table 1 . (c, d) Immunoblot analysis of MYC upon treatment with MYC siRNAs. RPS3 and β-actin were used as a loading control. (e, f) Cell growth assay after treatment with MYC siRNAs. ON-TARGETplus SMART pool siRNA was used (in c, e) and Silencer Select Pre-designed siRNAs were used (in d, f). (g) Hierarchical clustering analysis of indicated conditions using DEGs of MYC siRNA. (h) Enrichment plot using MYC siRNA-increased gene sets. Running enrichment score ( top portion, green curve ) and the statistics were calculated from the order of gene sets based on the gene expression change ( bottom ) upon treatment with U-0126. GEM, gemcitabine; MTX, methotrexate; ETP, etoposide; 6-MP, 6-mercaptopurine; MMC, mitomycin C; TOP, topotecan; DOX, doxorubicin; MIT, mitoxantrone.

    Techniques Used: Expressing, Growth Assay

    13) Product Images from "InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment"

    Article Title: InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25624

    MYC , one of the most influential genes, accounts for the drug-induced change in gene expression (a, b) . (c, d) Immunoblot analysis of MYC upon treatment with MYC siRNAs. RPS3 and β-actin were used as a loading control. (e, f) Cell growth assay after treatment with MYC siRNAs. ON-TARGETplus SMART pool siRNA was used (in c, e) and Silencer Select Pre-designed siRNAs were used (in d, f). (g) Hierarchical clustering analysis of indicated conditions using DEGs of MYC siRNA. (h) Enrichment plot using MYC siRNA-increased gene sets. Running enrichment score ( top portion, green curve ) and the statistics were calculated from the order of gene sets based on the gene expression change ( bottom ) upon treatment with U-0126. GEM, gemcitabine; MTX, methotrexate; ETP, etoposide; 6-MP, 6-mercaptopurine; MMC, mitomycin C; TOP, topotecan; DOX, doxorubicin; MIT, mitoxantrone.
    Figure Legend Snippet: MYC , one of the most influential genes, accounts for the drug-induced change in gene expression (a, b) . (c, d) Immunoblot analysis of MYC upon treatment with MYC siRNAs. RPS3 and β-actin were used as a loading control. (e, f) Cell growth assay after treatment with MYC siRNAs. ON-TARGETplus SMART pool siRNA was used (in c, e) and Silencer Select Pre-designed siRNAs were used (in d, f). (g) Hierarchical clustering analysis of indicated conditions using DEGs of MYC siRNA. (h) Enrichment plot using MYC siRNA-increased gene sets. Running enrichment score ( top portion, green curve ) and the statistics were calculated from the order of gene sets based on the gene expression change ( bottom ) upon treatment with U-0126. GEM, gemcitabine; MTX, methotrexate; ETP, etoposide; 6-MP, 6-mercaptopurine; MMC, mitomycin C; TOP, topotecan; DOX, doxorubicin; MIT, mitoxantrone.

    Techniques Used: Expressing, Growth Assay

    14) Product Images from "p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1"

    Article Title: p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201701049

    Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P
    Figure Legend Snippet: Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P

    Techniques Used: Activity Assay, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection

    p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.
    Figure Legend Snippet: p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.

    Techniques Used: Recombinant, In Vitro, Incubation, Co-Immunoprecipitation Assay, Western Blot

    15) Product Images from "TIMELESS contributes to the progression of breast cancer through activation of MYC"

    Article Title: TIMELESS contributes to the progression of breast cancer through activation of MYC

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-017-0838-1

    MYC is the downstream effector molecule of TIM. a Gene set enrichment analysis showing that TIM expression positively correlated with MYC-regulated gene signatures in The Cancer Genome Atlas ( TCGA ) dataset. b Western blot analysis of protein levels of MYC in MCF-7 and T47D with TIM overexpression or knockdown. c , d Real-time PCR analysis of the mRNA levels of MYC in MCF-7 and T47D with TIM overexpression ( c ) or knockdown ( d ). e Luciferase analysis indicating the transactivity of MYC in MCF-7 and T47D with TIM overexpression or knockdown. * P
    Figure Legend Snippet: MYC is the downstream effector molecule of TIM. a Gene set enrichment analysis showing that TIM expression positively correlated with MYC-regulated gene signatures in The Cancer Genome Atlas ( TCGA ) dataset. b Western blot analysis of protein levels of MYC in MCF-7 and T47D with TIM overexpression or knockdown. c , d Real-time PCR analysis of the mRNA levels of MYC in MCF-7 and T47D with TIM overexpression ( c ) or knockdown ( d ). e Luciferase analysis indicating the transactivity of MYC in MCF-7 and T47D with TIM overexpression or knockdown. * P

    Techniques Used: Expressing, Western Blot, Over Expression, Real-time Polymerase Chain Reaction, Luciferase

    Inhibition of MYC abrogates the phenotype caused by TIM overexpression. a Mammosphere formation analysis of cells with indicated treatment ( left panel ) and quantification of mammosphere number ( right panel ). * P
    Figure Legend Snippet: Inhibition of MYC abrogates the phenotype caused by TIM overexpression. a Mammosphere formation analysis of cells with indicated treatment ( left panel ) and quantification of mammosphere number ( right panel ). * P

    Techniques Used: Inhibition, Over Expression

    16) Product Images from "Targeted inhibition of KDM6 histone demethylases eradicates tumor-initiating cells via enhancer reprogramming in colorectal cancer"

    Article Title: Targeted inhibition of KDM6 histone demethylases eradicates tumor-initiating cells via enhancer reprogramming in colorectal cancer

    Journal: Theranostics

    doi: 10.7150/thno.47081

    GSK-J4 strongly represses TIC properties in CRC. ( A ) Sphere formation assay in HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. Scale bar represents 100 µm. ( B ) Left panel: representative results of ALDH activity assay in HCT116 cells treated with or without GSK-J4 as determined by flow cytometry. A group of mock cells incubated with both activated ALDH reagent and its inhibitor DEAB was introduced as background control. Cells in Gate P2 were defined as ALDH positive cells. Right panel: quantification of the percentages of ALDH positive cells from flow cytometry analysis in HCT116 and HT29 cells. ( C ) Sphere formation assay in ALDH-positive HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. Scale bar represents 100 µm. ( D ) Flow cytometry analysis of CD24 + CD44 + cells (upper-right quadrant) with or without GSK-J4 treatment for 48 h. ( E ) qRT-PCR analysis of the expression of indicated genes in HCT116 cells treated with or without 15 µm GSK-J4 and in HT29 cells treated with or without 20 µm GSK-J4 for 48 h. ( F ) Western blotting results showing the expression of total β-catenin, AXIN2 and MYC in HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. β-actin was used as a loading control. * p
    Figure Legend Snippet: GSK-J4 strongly represses TIC properties in CRC. ( A ) Sphere formation assay in HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. Scale bar represents 100 µm. ( B ) Left panel: representative results of ALDH activity assay in HCT116 cells treated with or without GSK-J4 as determined by flow cytometry. A group of mock cells incubated with both activated ALDH reagent and its inhibitor DEAB was introduced as background control. Cells in Gate P2 were defined as ALDH positive cells. Right panel: quantification of the percentages of ALDH positive cells from flow cytometry analysis in HCT116 and HT29 cells. ( C ) Sphere formation assay in ALDH-positive HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. Scale bar represents 100 µm. ( D ) Flow cytometry analysis of CD24 + CD44 + cells (upper-right quadrant) with or without GSK-J4 treatment for 48 h. ( E ) qRT-PCR analysis of the expression of indicated genes in HCT116 cells treated with or without 15 µm GSK-J4 and in HT29 cells treated with or without 20 µm GSK-J4 for 48 h. ( F ) Western blotting results showing the expression of total β-catenin, AXIN2 and MYC in HCT116 and HT29 cells treated with or without GSK-J4 at indicated doses. β-actin was used as a loading control. * p

    Techniques Used: Tube Formation Assay, Activity Assay, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Western Blot

    17) Product Images from "Neuronal hemoglobin affects dopaminergic cells' response to stress"

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.458

    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated

    Techniques Used: Western Blot, Fractionation, Immunofluorescence

    AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated
    Figure Legend Snippet: AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Infection

    Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)
    Figure Legend Snippet: Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)

    Techniques Used: Western Blot, Expressing, FACS

    Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated

    Techniques Used: Western Blot, Cell Fractionation, Immunofluorescence, Expressing

    18) Product Images from "Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin, et al. Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin"

    Article Title: Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin, et al. Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin

    Journal: Aging Cell

    doi: 10.1111/acel.12732

    Smurf2 regulates the stability of lamin A and progerin through the lysosomal proteolysis. (a) Western blot analysis of lamin A/C levels and protein turnover in Smurf2 KO MEF s. CHX —cycloheximide. (b) Western blot analysis showing the effects of Smurf2 on the levels of Flag–lamin A and endogenous lamin A/C in CHX ‐treated and untreated HEK ‐293T cells. The decrease in lamin A/C in Smurf2 overexpressing cells was detected using two different antibodies for lamin A/C, recognizing these proteins at two distinct epitopes. (c) Western blot analysis showing that Smurf2‐mediated degradation of lamin A can be rescued by the inhibition of the lysosomal degradation pathway. HEK ‐293T cells were co‐transfected with Flag–lamin A and Myc‐Smurf2, or with an empty Myc vector. Twenty‐four hours later, cells were treated with either proteasome inhibitor MG 132 ( MG ; 2.5 μ m ), lysosomal inhibitor chloroquine ( CQ ; 20 μ m ), or with a combination of both for additional 4 hr. Cells were then lysed in RIPA buffer, and cell extracts analyzed in Western blot with the indicated antibodies. Inhibition of the proteasomal degradation pathway was verified with anti‐poly‐ubiquitin‐Lys48 (poly‐Ub‐K48) antibody. Note that Smurf2 predominantly affected the stability of lamin A. (d) Repetition of the experiments described in (c) with gradually increased concentrations of Myc‐Smurf2 (0; 2; 4 μg), and sample sonication. (e) Western blot analysis showing that Smurf2‐mediated degradation of progerin can be rescued by the inhibition of the lysosomal breakdown pathway
    Figure Legend Snippet: Smurf2 regulates the stability of lamin A and progerin through the lysosomal proteolysis. (a) Western blot analysis of lamin A/C levels and protein turnover in Smurf2 KO MEF s. CHX —cycloheximide. (b) Western blot analysis showing the effects of Smurf2 on the levels of Flag–lamin A and endogenous lamin A/C in CHX ‐treated and untreated HEK ‐293T cells. The decrease in lamin A/C in Smurf2 overexpressing cells was detected using two different antibodies for lamin A/C, recognizing these proteins at two distinct epitopes. (c) Western blot analysis showing that Smurf2‐mediated degradation of lamin A can be rescued by the inhibition of the lysosomal degradation pathway. HEK ‐293T cells were co‐transfected with Flag–lamin A and Myc‐Smurf2, or with an empty Myc vector. Twenty‐four hours later, cells were treated with either proteasome inhibitor MG 132 ( MG ; 2.5 μ m ), lysosomal inhibitor chloroquine ( CQ ; 20 μ m ), or with a combination of both for additional 4 hr. Cells were then lysed in RIPA buffer, and cell extracts analyzed in Western blot with the indicated antibodies. Inhibition of the proteasomal degradation pathway was verified with anti‐poly‐ubiquitin‐Lys48 (poly‐Ub‐K48) antibody. Note that Smurf2 predominantly affected the stability of lamin A. (d) Repetition of the experiments described in (c) with gradually increased concentrations of Myc‐Smurf2 (0; 2; 4 μg), and sample sonication. (e) Western blot analysis showing that Smurf2‐mediated degradation of progerin can be rescued by the inhibition of the lysosomal breakdown pathway

    Techniques Used: Western Blot, Inhibition, Transfection, Plasmid Preparation, Sonication

    Smurf2 physically interacts with lamin A and its dominant mutant form progerin. (a) Schematic diagram of the structure of lamin A, lamin C, and progerin ( LA Δ50). Progerin retains its C‐terminal CAAX motif that is stably farnesylated. NLS —nuclear localization signal. (b) Immunohistochemistry ( IHC ) analysis of Smurf2 expression and biodistribution in a panel of human normal tissues ( FDA 999m TMA ). IHC staining of both Smurf2 and A‐lamins in liver tissues is also shown. (c) Confocal images showing co‐localization of GFP –Smurf2 with mCherry‐lamin A and Flag–progerin expressed in human HEK ‐293T cells. Bars, 5 μm. (d) Co‐localization of GFP –Smurf2 with lamin A/C and progerin in normal and HGPS HDF cells. Bars, 10 μm. (e) Co‐ IP analysis of endogenous Smurf2 and lamin A/C interaction in HEK ‐293T cells. WCL , whole cell lysate. (f) In vitro binding assay showing a direct interaction between purified GST –Smurf2 and Flag–lamin A. (g) In vitro binding assay showing a direct interaction between Flag–progerin and GST –Smurf2. (h) Proximity ligation assay ( PLA ) assay indicating sites of direct protein–protein interaction of Flag–lamin A and Myc‐Smurf2 in HEK ‐293T cell nuclei (red signal). Cells transfected with Flag–lamin A and empty Myc vector served as a control. Bars, 5 μm. (i) Quantification of lamin A–Smurf2 PLA analysis. **** P
    Figure Legend Snippet: Smurf2 physically interacts with lamin A and its dominant mutant form progerin. (a) Schematic diagram of the structure of lamin A, lamin C, and progerin ( LA Δ50). Progerin retains its C‐terminal CAAX motif that is stably farnesylated. NLS —nuclear localization signal. (b) Immunohistochemistry ( IHC ) analysis of Smurf2 expression and biodistribution in a panel of human normal tissues ( FDA 999m TMA ). IHC staining of both Smurf2 and A‐lamins in liver tissues is also shown. (c) Confocal images showing co‐localization of GFP –Smurf2 with mCherry‐lamin A and Flag–progerin expressed in human HEK ‐293T cells. Bars, 5 μm. (d) Co‐localization of GFP –Smurf2 with lamin A/C and progerin in normal and HGPS HDF cells. Bars, 10 μm. (e) Co‐ IP analysis of endogenous Smurf2 and lamin A/C interaction in HEK ‐293T cells. WCL , whole cell lysate. (f) In vitro binding assay showing a direct interaction between purified GST –Smurf2 and Flag–lamin A. (g) In vitro binding assay showing a direct interaction between Flag–progerin and GST –Smurf2. (h) Proximity ligation assay ( PLA ) assay indicating sites of direct protein–protein interaction of Flag–lamin A and Myc‐Smurf2 in HEK ‐293T cell nuclei (red signal). Cells transfected with Flag–lamin A and empty Myc vector served as a control. Bars, 5 μm. (i) Quantification of lamin A–Smurf2 PLA analysis. **** P

    Techniques Used: Mutagenesis, Stable Transfection, Immunohistochemistry, Expressing, Staining, Co-Immunoprecipitation Assay, In Vitro, Binding Assay, Purification, Proximity Ligation Assay, Transfection, Plasmid Preparation

    19) Product Images from "Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability"

    Article Title: Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201708289

    T‐025 exhibited a board range of anti‐proliferative effect in a panel of cancer cell lines IC 50 values of T‐025 in 240 cell lines. Each gray circle indicates a single cell line sorted according to its original organ/disease type. Correlation between T‐025 sensitivity and MYC amplification status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red bar indicate cell lines with amplified MYC . Correlation between T‐025 sensitivity and CLK2 expression status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red, gray, or blue bar indicate cell lines with high, medium, or low CLK2. Data information: In (B and C), a Mann–Whitney test was performed.
    Figure Legend Snippet: T‐025 exhibited a board range of anti‐proliferative effect in a panel of cancer cell lines IC 50 values of T‐025 in 240 cell lines. Each gray circle indicates a single cell line sorted according to its original organ/disease type. Correlation between T‐025 sensitivity and MYC amplification status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red bar indicate cell lines with amplified MYC . Correlation between T‐025 sensitivity and CLK2 expression status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red, gray, or blue bar indicate cell lines with high, medium, or low CLK2. Data information: In (B and C), a Mann–Whitney test was performed.

    Techniques Used: Amplification, Expressing, MANN-WHITNEY

    T‐025 exerted anti‐tumor activity in MYC‐driven breast cancers Anti‐tumor efficacy of T‐025 in a MMTV‐ MYC allograft model. T‐025 was administered twice daily on 2 days per week (red arrow). Tumor volume (A) and body weight (B) during the treatment cycle are shown. Data are shown as means ± standard errors of the means ( n = 8). Kaplan–Meier survival curve of breast cancer patients categorized as an ER‐positive, HER2 negative, and high‐proliferative subpopulation. Patients were divided into two groups by MYC ‐amplified status and expression level of CLK2. Median survival time of each group was calculated by Prism, and a log rank test was performed. Data information: In (A and B), an unpaired Student's t ‐test or an unpaired Student's t ‐test with Welch's correction was performed.
    Figure Legend Snippet: T‐025 exerted anti‐tumor activity in MYC‐driven breast cancers Anti‐tumor efficacy of T‐025 in a MMTV‐ MYC allograft model. T‐025 was administered twice daily on 2 days per week (red arrow). Tumor volume (A) and body weight (B) during the treatment cycle are shown. Data are shown as means ± standard errors of the means ( n = 8). Kaplan–Meier survival curve of breast cancer patients categorized as an ER‐positive, HER2 negative, and high‐proliferative subpopulation. Patients were divided into two groups by MYC ‐amplified status and expression level of CLK2. Median survival time of each group was calculated by Prism, and a log rank test was performed. Data information: In (A and B), an unpaired Student's t ‐test or an unpaired Student's t ‐test with Welch's correction was performed.

    Techniques Used: Activity Assay, Amplification, Expressing

    The CLK inhibitors T3 and T‐025 yielded similar profiles Chemical structure of T3. The enzymatic assay results of T3 and T‐025 against the CLK and DYRK family kinases. Comparison of SEs caused by T‐025 and T3. Correlation between the IC 50 values of T‐025 and T3 in a panel of 60 cancer cell lines. Each circle indicates a single cell line. The r 2 value was calculated using Prism. Correlation between growth suppressive sensitivity to T3 and the protein level of CLK2 in cell lines in solid cancer cell lines. The definition of CLK2 protein expression was same as Fig 4 A. A Mann–Whitney test was performed. Dose–response growth inhibition curve of T3 in MYC‐inducible SK‐MEL‐28 cells. Cells pretreated with Dox for 48 h were additionally incubated with T‐025 for 72 h. Data are shown as the means ± s.d. of three independent experiments ( n = 3). IC 50 values and 95% CI were determined by using Prism. Data information: The chemical structure of T3, the result of enzymatic assay of T3, and AS events modulated by T3 in HCT116 are cited from a previous article (Funnell et al , 2017 ).
    Figure Legend Snippet: The CLK inhibitors T3 and T‐025 yielded similar profiles Chemical structure of T3. The enzymatic assay results of T3 and T‐025 against the CLK and DYRK family kinases. Comparison of SEs caused by T‐025 and T3. Correlation between the IC 50 values of T‐025 and T3 in a panel of 60 cancer cell lines. Each circle indicates a single cell line. The r 2 value was calculated using Prism. Correlation between growth suppressive sensitivity to T3 and the protein level of CLK2 in cell lines in solid cancer cell lines. The definition of CLK2 protein expression was same as Fig 4 A. A Mann–Whitney test was performed. Dose–response growth inhibition curve of T3 in MYC‐inducible SK‐MEL‐28 cells. Cells pretreated with Dox for 48 h were additionally incubated with T‐025 for 72 h. Data are shown as the means ± s.d. of three independent experiments ( n = 3). IC 50 values and 95% CI were determined by using Prism. Data information: The chemical structure of T3, the result of enzymatic assay of T3, and AS events modulated by T3 in HCT116 are cited from a previous article (Funnell et al , 2017 ).

    Techniques Used: Enzymatic Assay, Expressing, MANN-WHITNEY, Inhibition, Incubation

    Additional analysis of Oncopanel based on the original organ type IC 50 values of the cancer cell lines with both high CLK2 and amplified MYC were compared with that without these biomarkers. A Mann–Whitney test was performed.
    Figure Legend Snippet: Additional analysis of Oncopanel based on the original organ type IC 50 values of the cancer cell lines with both high CLK2 and amplified MYC were compared with that without these biomarkers. A Mann–Whitney test was performed.

    Techniques Used: Amplification, MANN-WHITNEY

    Additional analysis of Oncopanel Analysis flow of the tested cell lines. IC 50 value, CLK2 expression, and MYC status of 19 hematological cancer cell lines are shown. Correlation between T‐025 sensitivity and MYC status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC. Correlation between T‐025 sensitivity and MYC family gene status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and MYC family gene status in the solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and CLK2 expression in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and blue, gray, or red bar indicates cell lines with high, medium, or low CLK2. Data information: In (C–F), a Mann–Whitney test was performed.
    Figure Legend Snippet: Additional analysis of Oncopanel Analysis flow of the tested cell lines. IC 50 value, CLK2 expression, and MYC status of 19 hematological cancer cell lines are shown. Correlation between T‐025 sensitivity and MYC status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC. Correlation between T‐025 sensitivity and MYC family gene status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and MYC family gene status in the solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and CLK2 expression in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and blue, gray, or red bar indicates cell lines with high, medium, or low CLK2. Data information: In (C–F), a Mann–Whitney test was performed.

    Techniques Used: Flow Cytometry, Expressing, MANN-WHITNEY

    20) Product Images from "Characterization of a Novel DWD Protein that Participates in Heat Stress Response in Arabidopsis"

    Article Title: Characterization of a Novel DWD Protein that Participates in Heat Stress Response in Arabidopsis

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2014.0224

    Interaction patterns of various proteins with HTD1. (A) Direct interaction between HTD1 and the adaptor of the CRL4 complex based on yeast two-hybrid assays. Assays were performed with HTD1 protein as prey and DDB1a as bait to check their interactions. Empty vector (B42) and GFP proteins (B42-GFP) were used as negative controls. (B) Co-immunoprecipitation (Co-IP) assay of HTD1 with DDB1b and various HSP proteins. Transgenic plants overexpressing FLAG-DDB1b or FLAG-DDB1b/HTD1-MYC were used for these assays. The immunoblot used anti-RPN6 as a loading control. Total, 5% of the crude extracts used for Co-IP assays. (C) Interaction pattern between HTD1 and HSP90-1 in yeast two hybrid assay.
    Figure Legend Snippet: Interaction patterns of various proteins with HTD1. (A) Direct interaction between HTD1 and the adaptor of the CRL4 complex based on yeast two-hybrid assays. Assays were performed with HTD1 protein as prey and DDB1a as bait to check their interactions. Empty vector (B42) and GFP proteins (B42-GFP) were used as negative controls. (B) Co-immunoprecipitation (Co-IP) assay of HTD1 with DDB1b and various HSP proteins. Transgenic plants overexpressing FLAG-DDB1b or FLAG-DDB1b/HTD1-MYC were used for these assays. The immunoblot used anti-RPN6 as a loading control. Total, 5% of the crude extracts used for Co-IP assays. (C) Interaction pattern between HTD1 and HSP90-1 in yeast two hybrid assay.

    Techniques Used: Plasmid Preparation, Co-Immunoprecipitation Assay, Transgenic Assay, Y2H Assay

    21) Product Images from "DWA1 and DWA2, Two Arabidopsis DWD Protein Components of CUL4-Based E3 Ligases, Act Together as Negative Regulators in ABA Signal Transduction [C] DWD Protein Components of CUL4-Based E3 Ligases, Act Together as Negative Regulators in ABA Signal Transduction [C] [W]"

    Article Title: DWA1 and DWA2, Two Arabidopsis DWD Protein Components of CUL4-Based E3 Ligases, Act Together as Negative Regulators in ABA Signal Transduction [C] DWD Protein Components of CUL4-Based E3 Ligases, Act Together as Negative Regulators in ABA Signal Transduction [C] [W]

    Journal: The Plant Cell

    doi: 10.1105/tpc.109.073783

    Co-IP of ABI5 and Components of CUL4-DDB1-DWAs. (A) Association of DWAs with ABI5. Seedlings were grown in the presence of 5 μM ABA for 5 d after stratification. After protein extraction, the extracts were coimmunoprecipitated with α-MYC or α-ABI5. The arrowheads indicate two predominant forms of ABI5 protein. Two images from an immunoblot with anti-ABI5 are shown after short or long exposure to film (Fuji). Control, FLAG-tagged DDB1b transgenic plants; MYC-DWA1 or 2, FLAG-tagged DDB1b with MYC-tagged DWA1 or DWA2 transgenic plants; Ws, Wassilewskija. Total, 5% of the crude extracts used for co-IP assays. (B) Association of FLAG-CUL4 and ABI5. Seedlings were grown in the presence of 5 μM ABA for 5 d after stratification. After protein extraction, the extracts were coimmunoprecipitated with α-ABI5. The extracts of FLAG-CUL4 coimmunoprecipitated with α-MYC were used as a negative control to exclude the possibility of nonspecifically bound FLAG-CUL4. The arrowheads on the anti-FLAG and anti-ABI5 panels represent the positions of FLAG-CUL4 and ABI5 proteins, respectively. (C) MYC2 does not detectably associate with DWA proteins. Five-day-old seedlings treated with 5 μM ABA were used for co-IP with the anti-MYC antibody (which recognizes the MYC tag but not MYC2). The arrowheads represent endogenous MYC2 protein. To show clearly the existence of MYC2 induced by ABA, the extracts without ABA were loaded on the same gel. Control, FLAG-tagged DDB1b transgenic plants; MYC-DWA1 or 2, FLAG-tagged DDB1b with MYC-tagged DWA1 or DWA2 transgenic plants. Total, 5% of the crude extracts used for co-IP assays.
    Figure Legend Snippet: Co-IP of ABI5 and Components of CUL4-DDB1-DWAs. (A) Association of DWAs with ABI5. Seedlings were grown in the presence of 5 μM ABA for 5 d after stratification. After protein extraction, the extracts were coimmunoprecipitated with α-MYC or α-ABI5. The arrowheads indicate two predominant forms of ABI5 protein. Two images from an immunoblot with anti-ABI5 are shown after short or long exposure to film (Fuji). Control, FLAG-tagged DDB1b transgenic plants; MYC-DWA1 or 2, FLAG-tagged DDB1b with MYC-tagged DWA1 or DWA2 transgenic plants; Ws, Wassilewskija. Total, 5% of the crude extracts used for co-IP assays. (B) Association of FLAG-CUL4 and ABI5. Seedlings were grown in the presence of 5 μM ABA for 5 d after stratification. After protein extraction, the extracts were coimmunoprecipitated with α-ABI5. The extracts of FLAG-CUL4 coimmunoprecipitated with α-MYC were used as a negative control to exclude the possibility of nonspecifically bound FLAG-CUL4. The arrowheads on the anti-FLAG and anti-ABI5 panels represent the positions of FLAG-CUL4 and ABI5 proteins, respectively. (C) MYC2 does not detectably associate with DWA proteins. Five-day-old seedlings treated with 5 μM ABA were used for co-IP with the anti-MYC antibody (which recognizes the MYC tag but not MYC2). The arrowheads represent endogenous MYC2 protein. To show clearly the existence of MYC2 induced by ABA, the extracts without ABA were loaded on the same gel. Control, FLAG-tagged DDB1b transgenic plants; MYC-DWA1 or 2, FLAG-tagged DDB1b with MYC-tagged DWA1 or DWA2 transgenic plants. Total, 5% of the crude extracts used for co-IP assays.

    Techniques Used: Co-Immunoprecipitation Assay, Protein Extraction, Transgenic Assay, Negative Control

    22) Product Images from "Telomerase Reverse Transcriptase Contains a BH3-Like Motif and Interacts with BCL-2 Family Members"

    Article Title: Telomerase Reverse Transcriptase Contains a BH3-Like Motif and Interacts with BCL-2 Family Members

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0206

    Modulation of BCL-2 family protein complexes by hTERT (A) Co-IP of BAX and BCL-xL in hTERT-overexpressing HEK 293T/17 cells. Cells were co-transfected with FLAG-BCL-xL and MYC-hTERT, and then lysed in either Triton X-100 or CHAPS buffer. FLAG-BCL-xL was immunoprecipitated with anti-FLAG beads, followed by western blot analysis with the indicated antibodies. No significant differences in BAX/BCL-xL ratios were observed after hTERT overexpression. (B) Co-IP of BAX and BCL-xL with BAD and hTERT overexpression. The cells were lysed with detergent containing Triton X-100. BAD can disrupt BAX/BCL-xL heterodimers, but hTERT did not affect the BAD-dependent BAX/BCL-xL complex ratios. (C) Co-IP experiment of BAK and MCL-1 with hTERT overexpression. (D) Co-IP analysis of BCL-xL and BECN1 with hTERT overexpression. The BECN1/BCL-xL interaction is not altered by hTERT overexpression.
    Figure Legend Snippet: Modulation of BCL-2 family protein complexes by hTERT (A) Co-IP of BAX and BCL-xL in hTERT-overexpressing HEK 293T/17 cells. Cells were co-transfected with FLAG-BCL-xL and MYC-hTERT, and then lysed in either Triton X-100 or CHAPS buffer. FLAG-BCL-xL was immunoprecipitated with anti-FLAG beads, followed by western blot analysis with the indicated antibodies. No significant differences in BAX/BCL-xL ratios were observed after hTERT overexpression. (B) Co-IP of BAX and BCL-xL with BAD and hTERT overexpression. The cells were lysed with detergent containing Triton X-100. BAD can disrupt BAX/BCL-xL heterodimers, but hTERT did not affect the BAD-dependent BAX/BCL-xL complex ratios. (C) Co-IP experiment of BAK and MCL-1 with hTERT overexpression. (D) Co-IP analysis of BCL-xL and BECN1 with hTERT overexpression. The BECN1/BCL-xL interaction is not altered by hTERT overexpression.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Over Expression

    23) Product Images from "Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells"

    Article Title: Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells

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

    doi: 10.1016/j.neo.2018.08.013

    DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P
    Figure Legend Snippet: DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P

    Techniques Used: Stable Transfection, Flow Cytometry, Cytometry, Mutagenesis, Transformation Assay, Immunofluorescence, Microscopy, Quantitation Assay, Software

    DNMT3A -R882 mutants reduce ROS production and overrode the ROS-mediated apoptosis in the presence of an oxidizing agent. (A) Stably transduced U937 cells incubated with H2DCF-DA for 30 minutes at 37°C incubator; fluorescent oxidized DCF (green) and DAPI (blue) were photographed with fluorescence microscopy (left panel, original magnification: ×1000). All the images were taken with same contrast and exposure time. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry (right upper panel) and percentage of ROS induction are shown (right lower panel). (B) Stably transduced HL-60 cells stained with H2DCF-DA; staining cells were photographed with phase-contrast microscopy (left upper panel) and fluorescence microscopy (left lower panel); original magnification: ×100. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry and percentage of ROS induction (right panel) are shown. (C) Inhibition of cell survival following 200 μM TBHP treatment for 24 hours. (D) After treatment of 200 μM TBHP for 24 hours, cells were harvested and labeled with FITC-Annexin V as described in methods. Flow cytometry was used to detect Annexin V–positive cells. (E) Apoptosis triggering by TBHP treatment; transformed U937 cells were incubated with 1 mM TBHP for 2 hours followed by a 2-hour incubation without TBHP. Cell lysates were prepared, and apoptosis inducing proteins including MYC and P-MYC expressions were checked by immunoblot. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted; * P
    Figure Legend Snippet: DNMT3A -R882 mutants reduce ROS production and overrode the ROS-mediated apoptosis in the presence of an oxidizing agent. (A) Stably transduced U937 cells incubated with H2DCF-DA for 30 minutes at 37°C incubator; fluorescent oxidized DCF (green) and DAPI (blue) were photographed with fluorescence microscopy (left panel, original magnification: ×1000). All the images were taken with same contrast and exposure time. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry (right upper panel) and percentage of ROS induction are shown (right lower panel). (B) Stably transduced HL-60 cells stained with H2DCF-DA; staining cells were photographed with phase-contrast microscopy (left upper panel) and fluorescence microscopy (left lower panel); original magnification: ×100. Flow cytometry detecting the generation of fluorescent oxidized DCF; representative flow cytometry and percentage of ROS induction (right panel) are shown. (C) Inhibition of cell survival following 200 μM TBHP treatment for 24 hours. (D) After treatment of 200 μM TBHP for 24 hours, cells were harvested and labeled with FITC-Annexin V as described in methods. Flow cytometry was used to detect Annexin V–positive cells. (E) Apoptosis triggering by TBHP treatment; transformed U937 cells were incubated with 1 mM TBHP for 2 hours followed by a 2-hour incubation without TBHP. Cell lysates were prepared, and apoptosis inducing proteins including MYC and P-MYC expressions were checked by immunoblot. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted; * P

    Techniques Used: Stable Transfection, Incubation, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Staining, Inhibition, Labeling, Transformation Assay

    DNMT3A -R882 mutants augmented PRDX2 expression in myeloid leukemia cells. ( A and B) Immunoblot showing overexpression of DNMT3A -mutant into U937 (A) and HL-60 (B) cells augmented protein expression of PRDX2 with increasing MYC and P-MYC expression. β-Actin was used as a control for equal loading. (C) Cytoplasmic localization of PRDX2 in U937 cells transduced with EV-, WT-, and mutant- DNMT3A ; immunostained with anti-PRDX2 (red) and DAPI (blue) (original magnification: ×1000). All the images were taken with same contrast and exposure time. (D) Co-immunoprecipitation data showing FLAG-tagged WT- and mutant DNMT3A both interacted with PRDX2 in 293T cells. Cell extracts were incubated with DNAse prior to immunoprecipitation (Supplementary Fig. S1 for effectiveness of DNAse treatment).
    Figure Legend Snippet: DNMT3A -R882 mutants augmented PRDX2 expression in myeloid leukemia cells. ( A and B) Immunoblot showing overexpression of DNMT3A -mutant into U937 (A) and HL-60 (B) cells augmented protein expression of PRDX2 with increasing MYC and P-MYC expression. β-Actin was used as a control for equal loading. (C) Cytoplasmic localization of PRDX2 in U937 cells transduced with EV-, WT-, and mutant- DNMT3A ; immunostained with anti-PRDX2 (red) and DAPI (blue) (original magnification: ×1000). All the images were taken with same contrast and exposure time. (D) Co-immunoprecipitation data showing FLAG-tagged WT- and mutant DNMT3A both interacted with PRDX2 in 293T cells. Cell extracts were incubated with DNAse prior to immunoprecipitation (Supplementary Fig. S1 for effectiveness of DNAse treatment).

    Techniques Used: Expressing, Over Expression, Mutagenesis, Transduction, Immunoprecipitation, Incubation

    PRDX2 blocks apoptosis of myeloid leukemia cells. (A) Silenced PRDX2 using two independent shRNA and scrambled (shLuc) K562 cells were treated with 300 nM ABT-263 or without drug for 72 hours, and cell proliferation was measured by trypan blue exclusion method. (B) Colony formation ability was assayed in Methocult medium after being stably silenced of PRDX2 in K562 cells. After 7 days, colonies were photographed and counted manually; original magnification: ×100. (C) PRDX 2-silenced and scrambled K562 cells treated with 300 nM ABT-263 for 72 hours. Apoptosis cells were analyzed using Annexin V and PI staining by flow cytometric analysis. Representative flow cytometry analysis (left panel) and the % of Annexin V and PI-positive cells are shown (right panel). (D) Immunoblot data showing the PRDX2 silenced efficiency in K562 cells and protein expression of P-MYC and MYC after knock down of PRDX2 in K562 cells. β-Actin was used as a control for equal loading. (E) Proliferation of U937 cells transduced with WT- or R882H and knocked down of PRDX2 from transduced cells. (F) Apoptosis analysis of R882H-expressed U937 cells knocked down with shLuc or shPRDX2 and treatment of 300 nM ABT-263 for 72 hours. (G) Immunoblot data showing the PRDX2 silenced efficiency in U937 cells transduced with R882H and protein expression of MYC and CDK1. β-Actin was used as a control for equal loading. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted. * P
    Figure Legend Snippet: PRDX2 blocks apoptosis of myeloid leukemia cells. (A) Silenced PRDX2 using two independent shRNA and scrambled (shLuc) K562 cells were treated with 300 nM ABT-263 or without drug for 72 hours, and cell proliferation was measured by trypan blue exclusion method. (B) Colony formation ability was assayed in Methocult medium after being stably silenced of PRDX2 in K562 cells. After 7 days, colonies were photographed and counted manually; original magnification: ×100. (C) PRDX 2-silenced and scrambled K562 cells treated with 300 nM ABT-263 for 72 hours. Apoptosis cells were analyzed using Annexin V and PI staining by flow cytometric analysis. Representative flow cytometry analysis (left panel) and the % of Annexin V and PI-positive cells are shown (right panel). (D) Immunoblot data showing the PRDX2 silenced efficiency in K562 cells and protein expression of P-MYC and MYC after knock down of PRDX2 in K562 cells. β-Actin was used as a control for equal loading. (E) Proliferation of U937 cells transduced with WT- or R882H and knocked down of PRDX2 from transduced cells. (F) Apoptosis analysis of R882H-expressed U937 cells knocked down with shLuc or shPRDX2 and treatment of 300 nM ABT-263 for 72 hours. (G) Immunoblot data showing the PRDX2 silenced efficiency in U937 cells transduced with R882H and protein expression of MYC and CDK1. β-Actin was used as a control for equal loading. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted. * P

    Techniques Used: shRNA, Stable Transfection, Staining, Flow Cytometry, Cytometry, Expressing, Transduction

    24) Product Images from "p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1"

    Article Title: p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201701049

    Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P
    Figure Legend Snippet: Phosphorylation by p38α MAPK reduces ULK1 kinase activity and disrupts the ULK1–ATG13 complex. (A) Expression of p38α MAPK reduces ULK1 kinase activity. MYC-ULK1 with different amounts of FLAG–p38α MAPK was cotransfected into HEK293 cells for 24 h. The activity of immunoprecipitated MYC-ULK1 was measured by in vitro kinase assay using MBP as the substrate. The bottom graph shows relative change of [ 32 P]-MBP signal. (B) LPS treatment reduces ULK1 kinase activity. BV2 cells were treated with LPS (1 µg/ml) for the indicated time. Endogenous ULK1 was immunoprecipitated and analyzed by in vitro kinase assay. (C) p38α MAPK disrupts the ULK1–ATG13 complex and reduces ATG13 phosphorylation. ATG13 and ULK1 were coexpressed with or without coexpression of p38α MAPK in HEK293 for 24 h. The whole-cellular lysates (200 µg) were immunoprecipitated with an anti-MYC antibody, and the precipitates were blotted with an anti-HA or anti-FLAG antibody (top). The levels of phosphorylated ATG13 (S318) were determined using a phospho-S318–specific antibody (bottom). (D) SB203580 blocks p38α MAPK–induced ULK1–ATG13 complex disruption. BV2 cells were treated as described in C with the presence of SB203580 (10 µM). Co-IP was performed by incubating whole-cell lysates (200 µg) with an anti-HA antibody. WB, Western blot. (E) SB203580 blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were treated with LPS (1 µg/ml) with or without SB203580 (10 µM) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ULK1 antibody. (F) Knockdown of p38α MAPK blocks LPS (1 µg/ml)-induced disruption of the endogenous ULK1–ATG13 complex. BV2 cells were transfected with scramble or p38α MAPK–specific siRNA for 48 h and treated with LPS (1 µg/ml) for 2 h. Co-IP was performed by incubating whole-cell lysates (400 µg) with an anti-ATG13 antibody. The left panel shows the effect of knockdown. IB, immunoblot. *, P

    Techniques Used: Activity Assay, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection

    p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.
    Figure Legend Snippet: p38α MAPK interacts with ULK1. (A) Recombinant p38α MAPK and ULK1 interact with each other in vitro. Recombinant p38α MAPK (50 ng) was incubated with recombinant ULK1 (50 ng) in IP buffer at 4°C for 2 h. Pulldown assays were performed by using an anti–p38α MAPK antibody and blotted with an anti-ULK1 antibody. (B) Expressed p38α MAPK and ULK1 interact with each other. FLAG-p38α MAPK and/or MYC-ULK1 were overexpressed in HEK293 cells (bottom). Co-IP was performed by incubating whole-cell lysates (200 µg) with either an anti-MYC or an anti-FLAG antibody (top and middle). WB, Western blot. (C) p38α MAPK interacts with the ULK1 C terminus. HA-tagged WT ULK1 or ULK1 fragments were coexpressed with FLAG–p38α MAPK in HEK293 cells. The level of FLAG–p38α MAPK coimmunoprecipitated with HA-ULK1 from 200 µg cell lysates was analyzed with an anti-FLAG antibody. CTD, C-terminal domain. Asterisks denote HA-ULK1 fragments. (D and E) Endogenous p38α MAPK and ULK1 interact with each other in HEK293 (D) and BV2 cells (E). Co-IP was performed by incubating whole-cell lysates (400 µg) with anti-ULK1 antibody, and the precipitates were blotted with anti-p38 antibody.

    Techniques Used: Recombinant, In Vitro, Incubation, Co-Immunoprecipitation Assay, Western Blot

    25) Product Images from "S-acylation of a geminivirus C4 protein is essential for regulating the CLAVATA pathway in symptom determination"

    Article Title: S-acylation of a geminivirus C4 protein is essential for regulating the CLAVATA pathway in symptom determination

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery228

    S-acylation of C4 is important for its interaction with CLV1. (A) Detection of the interaction between C4 (fused to the binding domain, BD) and the intracellular domain (704AA-967AA) of CLV1 (CLV1C; fused to the activation domain, AD) in a yeast two-hybrid assay. Protein interactions were tested using a stringent (SD/–Leu/–Trp/–His) selection medium containing 3-amino-1,2,4-triazole (3AT). (B) The in vivo interaction between C4 and CLV1 was measured using bimolecular fluorescence complementation assays. The combinations with empty vectors were used as negative controls. The YFP signals and merged signals (yellow for YFP, red for chloroplast autofluorescence, and gray for bright field) are shown. Scale bars are 10 µm. (C) Detection of the interaction between C4-MYC and CLV1-GFP using co-immunoprecipitation (Co-IP). The wild-type (WT) or mutant form of pCanG-C4-MYC (C8 mutated from a cysteine to a serine) was co-transformed with 35S::CLV1-GFP or 35S::GFP (control). After 48 h, the transformed protoplasts were collected for Co-IP using GFP-Trap resin. (D) Measurement of the interaction between C4-YFP-FLAG 3 His 6 and CLV1-MYC using Co-IP. The wild-type or mutant form of C4-YFP-FLAG 3 His 6 was co-expressed with pCanG-CLV1-MYC or pCanG-MYC (control). Co-IP was performed using an anti-MYC antibody with Protein A resins. * indicates the unspecific signal from the antibody. The results in this figure are representative of independent experiments.
    Figure Legend Snippet: S-acylation of C4 is important for its interaction with CLV1. (A) Detection of the interaction between C4 (fused to the binding domain, BD) and the intracellular domain (704AA-967AA) of CLV1 (CLV1C; fused to the activation domain, AD) in a yeast two-hybrid assay. Protein interactions were tested using a stringent (SD/–Leu/–Trp/–His) selection medium containing 3-amino-1,2,4-triazole (3AT). (B) The in vivo interaction between C4 and CLV1 was measured using bimolecular fluorescence complementation assays. The combinations with empty vectors were used as negative controls. The YFP signals and merged signals (yellow for YFP, red for chloroplast autofluorescence, and gray for bright field) are shown. Scale bars are 10 µm. (C) Detection of the interaction between C4-MYC and CLV1-GFP using co-immunoprecipitation (Co-IP). The wild-type (WT) or mutant form of pCanG-C4-MYC (C8 mutated from a cysteine to a serine) was co-transformed with 35S::CLV1-GFP or 35S::GFP (control). After 48 h, the transformed protoplasts were collected for Co-IP using GFP-Trap resin. (D) Measurement of the interaction between C4-YFP-FLAG 3 His 6 and CLV1-MYC using Co-IP. The wild-type or mutant form of C4-YFP-FLAG 3 His 6 was co-expressed with pCanG-CLV1-MYC or pCanG-MYC (control). Co-IP was performed using an anti-MYC antibody with Protein A resins. * indicates the unspecific signal from the antibody. The results in this figure are representative of independent experiments.

    Techniques Used: Binding Assay, Activation Assay, Y2H Assay, Selection, In Vivo, Fluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Transformation Assay

    26) Product Images from "Wild-type p53 binds to MYC promoter G-quadruplex"

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    Journal: Bioscience Reports

    doi: 10.1042/BSR20160232

    Wtp53 represses MYC promoter activity and binds to MYC promoter in vivo ( A ) Influence of DNA topology on wtp53-driven repression of MYC promoter. Luciferase assay showing stronger repression of MYC promoter in supercoiled pGL4-MYCII plasmid by wtp53 in contrast to linear plasmid pGL4-MYCII/BamHI. Supercoiled pGL4.17 and linear pGL4.17/BamHI were used as control vectors. Mean values of relative luciferase assay (normalized on Renilla luciferase) from three independent experiments were plotted on the graph. ( B ) ChIP showing wtp53 binding to MYC promoter which contains a G-quadruplex motif. DNA fragments were immunoprecipitated using DO1 antibody against p53 in Hwtp53 (top, lane 3) and HCT116 (p53+/+) (top, lane 6) cells, negative control ChIP without Ab (lanes 2 and 5), positive input control (1/15 input for ChIP, lanes 1 and 4). The same procedure was performed in p53 null cell lines H1299 and HCT116 (p53−/−). ( C ) Wtp53 mediated down-regulation of MYC at the protein level and activation of BAX and CDKN1A was analysed in Hwtp53 cells compared with H1299 without p53 expression. Western blots presenting the protein levels of p53, MYC, CDKN1A, β-Actin and BAX. Wtp53 mediated down-regulation of MYC at the protein level was analysed in HCT116 (p53+/+) compared with HCT116 (p53−/−).
    Figure Legend Snippet: Wtp53 represses MYC promoter activity and binds to MYC promoter in vivo ( A ) Influence of DNA topology on wtp53-driven repression of MYC promoter. Luciferase assay showing stronger repression of MYC promoter in supercoiled pGL4-MYCII plasmid by wtp53 in contrast to linear plasmid pGL4-MYCII/BamHI. Supercoiled pGL4.17 and linear pGL4.17/BamHI were used as control vectors. Mean values of relative luciferase assay (normalized on Renilla luciferase) from three independent experiments were plotted on the graph. ( B ) ChIP showing wtp53 binding to MYC promoter which contains a G-quadruplex motif. DNA fragments were immunoprecipitated using DO1 antibody against p53 in Hwtp53 (top, lane 3) and HCT116 (p53+/+) (top, lane 6) cells, negative control ChIP without Ab (lanes 2 and 5), positive input control (1/15 input for ChIP, lanes 1 and 4). The same procedure was performed in p53 null cell lines H1299 and HCT116 (p53−/−). ( C ) Wtp53 mediated down-regulation of MYC at the protein level and activation of BAX and CDKN1A was analysed in Hwtp53 cells compared with H1299 without p53 expression. Western blots presenting the protein levels of p53, MYC, CDKN1A, β-Actin and BAX. Wtp53 mediated down-regulation of MYC at the protein level was analysed in HCT116 (p53+/+) compared with HCT116 (p53−/−).

    Techniques Used: Activity Assay, In Vivo, Luciferase, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Activation Assay, Expressing, Western Blot

    27) Product Images from "Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability"

    Article Title: Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201708289

    T‐025 exhibited a board range of anti‐proliferative effect in a panel of cancer cell lines IC 50 values of T‐025 in 240 cell lines. Each gray circle indicates a single cell line sorted according to its original organ/disease type. Correlation between T‐025 sensitivity and MYC amplification status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red bar indicate cell lines with amplified MYC . Correlation between T‐025 sensitivity and CLK2 expression status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red, gray, or blue bar indicate cell lines with high, medium, or low CLK2. Data information: In (B and C), a Mann–Whitney test was performed.
    Figure Legend Snippet: T‐025 exhibited a board range of anti‐proliferative effect in a panel of cancer cell lines IC 50 values of T‐025 in 240 cell lines. Each gray circle indicates a single cell line sorted according to its original organ/disease type. Correlation between T‐025 sensitivity and MYC amplification status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red bar indicate cell lines with amplified MYC . Correlation between T‐025 sensitivity and CLK2 expression status in solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and red, gray, or blue bar indicate cell lines with high, medium, or low CLK2. Data information: In (B and C), a Mann–Whitney test was performed.

    Techniques Used: Amplification, Expressing, MANN-WHITNEY

    T‐025 exerted anti‐tumor activity in MYC‐driven breast cancers A, B Anti‐tumor efficacy of T‐025 in a MMTV‐ MYC allograft model. T‐025 was administered twice daily on 2 days per week (red arrow). Tumor volume (A) and body weight (B) during the treatment cycle are shown. Data are shown as means ± standard errors of the means ( n = 8). C Kaplan–Meier survival curve of breast cancer patients categorized as an ER‐positive, HER2 negative, and high‐proliferative subpopulation. Patients were divided into two groups by MYC ‐amplified status and expression level of CLK2. Median survival time of each group was calculated by Prism, and a log rank test was performed. Data information: In (A and B), an unpaired Student's t ‐test or an unpaired Student's t ‐test with Welch's correction was performed.
    Figure Legend Snippet: T‐025 exerted anti‐tumor activity in MYC‐driven breast cancers A, B Anti‐tumor efficacy of T‐025 in a MMTV‐ MYC allograft model. T‐025 was administered twice daily on 2 days per week (red arrow). Tumor volume (A) and body weight (B) during the treatment cycle are shown. Data are shown as means ± standard errors of the means ( n = 8). C Kaplan–Meier survival curve of breast cancer patients categorized as an ER‐positive, HER2 negative, and high‐proliferative subpopulation. Patients were divided into two groups by MYC ‐amplified status and expression level of CLK2. Median survival time of each group was calculated by Prism, and a log rank test was performed. Data information: In (A and B), an unpaired Student's t ‐test or an unpaired Student's t ‐test with Welch's correction was performed.

    Techniques Used: Activity Assay, Amplification, Expressing

    Additional analysis of Oncopanel Analysis flow of the tested cell lines. IC 50 value, CLK2 expression, and MYC status of 19 hematological cancer cell lines are shown. Correlation between T‐025 sensitivity and MYC status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC. Correlation between T‐025 sensitivity and MYC family gene status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and MYC family gene status in the solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and CLK2 expression in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and blue, gray, or red bar indicates cell lines with high, medium, or low CLK2. Data information: In (C–F), a Mann–Whitney test was performed.
    Figure Legend Snippet: Additional analysis of Oncopanel Analysis flow of the tested cell lines. IC 50 value, CLK2 expression, and MYC status of 19 hematological cancer cell lines are shown. Correlation between T‐025 sensitivity and MYC status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC. Correlation between T‐025 sensitivity and MYC family gene status in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and MYC family gene status in the solid cancer cell lines ( n = 150). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T‐025 sensitivity and CLK2 expression in the hematological cancer cell lines ( n = 19). Each bar indicates a single cell line, and blue, gray, or red bar indicates cell lines with high, medium, or low CLK2. Data information: In (C–F), a Mann–Whitney test was performed.

    Techniques Used: Flow Cytometry, Expressing, MANN-WHITNEY

    28) Product Images from "The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification"

    Article Title: The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008583

    TMEM98 prevents MYRF self-cleavage and binds to the C-terminal part of MYRF. (A) Co-immunoprecipitation experiment where HEK293T cells were transiently transfected with the indicated epitope-tagged expression constructs and immunoprecipitated with anti-V5.The two MYRF constructs were either full-length (MYRF) or lacked exon 19 (MYRF Δ19). Western blot analysis of the inputs (left) and immunoprecipitated fractions (right) probed with anti-MYC (Cell Signaling Technology, 2276), anti-FLAG (Cell Signaling Technology, 2368) and anti-V5 antibodies are shown. Anti-tubulin antibody was used to probe the input Western as a loading control. The Western of the input samples shows that MYRF cleaves when transfected alone but remains largely intact when co-transfected with TMEM98-V5. The Western of the immunoprecipitated fractions shows that intact MYC-MYRF-FLAG and the C-terminal part tagged with FLAG are co-immunoprecipitated with TMEM98-V5 indicating that TMEM98 interacts with the C-terminal part of MYRF. Uncropped Western blot images are shown in S9 Fig . (B-D) ARPE-19 cells were transiently transfected with TMEM98-V5 and/or MYC-MYRF-FLAG and immunostained with anti-V5 (magenta), anti-MYC (Cell Signaling Technology, 2278) (red) and anti-FLAG (Biolegend, 637302) (green) antibodies as indicated. DAPI staining is in blue. (C) When transfected alone MYC-MYRF-FLAG cleaves and the N-terminal part tagged with MYC translocates to the nucleus whilst the C-terminal part tagged with FLAG is membrane-bound. (D) When MYC-MYRF-FLAG is co-transfected with TMEM98-V5 it remains intact and colocalises with TMEM98-V5 in the membrane. Scale bars represent 20 μm.
    Figure Legend Snippet: TMEM98 prevents MYRF self-cleavage and binds to the C-terminal part of MYRF. (A) Co-immunoprecipitation experiment where HEK293T cells were transiently transfected with the indicated epitope-tagged expression constructs and immunoprecipitated with anti-V5.The two MYRF constructs were either full-length (MYRF) or lacked exon 19 (MYRF Δ19). Western blot analysis of the inputs (left) and immunoprecipitated fractions (right) probed with anti-MYC (Cell Signaling Technology, 2276), anti-FLAG (Cell Signaling Technology, 2368) and anti-V5 antibodies are shown. Anti-tubulin antibody was used to probe the input Western as a loading control. The Western of the input samples shows that MYRF cleaves when transfected alone but remains largely intact when co-transfected with TMEM98-V5. The Western of the immunoprecipitated fractions shows that intact MYC-MYRF-FLAG and the C-terminal part tagged with FLAG are co-immunoprecipitated with TMEM98-V5 indicating that TMEM98 interacts with the C-terminal part of MYRF. Uncropped Western blot images are shown in S9 Fig . (B-D) ARPE-19 cells were transiently transfected with TMEM98-V5 and/or MYC-MYRF-FLAG and immunostained with anti-V5 (magenta), anti-MYC (Cell Signaling Technology, 2278) (red) and anti-FLAG (Biolegend, 637302) (green) antibodies as indicated. DAPI staining is in blue. (C) When transfected alone MYC-MYRF-FLAG cleaves and the N-terminal part tagged with MYC translocates to the nucleus whilst the C-terminal part tagged with FLAG is membrane-bound. (D) When MYC-MYRF-FLAG is co-transfected with TMEM98-V5 it remains intact and colocalises with TMEM98-V5 in the membrane. Scale bars represent 20 μm.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Western Blot, Staining

    29) Product Images from "RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling"

    Article Title: RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1166

    Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.
    Figure Legend Snippet: Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.

    Techniques Used: Mutagenesis, Expressing, Transfection, Construct, Western Blot, Fractionation, Marker

    30) Product Images from "Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation"

    Article Title: Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1474995

    PRDX1 is negatively involved in autophagy activation through inhibiting BECN1 ubiquitination. ( A ) HEK293T cells were transfected with Mock, Flag-TRAF6 wild type (WT), Flag-TRAF6 truncated mutants, or MYC-BECN1 as indicated. At 38 h post transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then subjected to IB assay using anti-Flag or anti-MYC antibody. ( B ) HEK293T cells were transfected with Mock, Flag-BECN1, or MYC-PRDX1 as indicated. At 38 h post transfection, transfected cells were extracted and cell lysates were subjected to immunoprecipitation with anti-MYC antibody followed by IB using anti-Flag or anti-MYC antibody. ( C ) Model showing of how PRDX1-TRAF6 interaction inhibits the ubiquitination of BECN1. See text for details. ( D ) HEK293T cells were transfected with Mock, Flag-BECN1, HA-Ub, MYC-TRAF6, or different concentrations of MYC-PRDX1 as indicated. At 38 h post transfection, transfected cells were extracted and cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by IB using anti-Flag or anti-HA antibody. ( E ) Control (Ctrl) and PRDX1 KD THP-1 cells were treated with or without LPS (200 ng/ml) in the presence or absence of pepstatin A (10 μM) and 3-MA (5 mM) for 6 h as indicated. These cells were lysed and subjected to SDS-PAGE followed by immunoblotting with LC3-I/-II or GAPDH antibody. Band intensity was quantified using ImageJ software. Quantitative data were calculated from 3 independent experiments. Data are shown as mean ±SEM. ( F ) Control (Ctrl) and PRDX1 KD THP-1 cells were treated with or without LPS (200 ng/ml) in the presence or absence of pepstatin A (10 μM) and 3-MA (5 mM) for 6 h as indicated, and then fixed. Immunofluorescence assay was performed with anti-LC3 antibody. Digital images were captured with confocal microscopy and the number of LC3-puncta was scored. Quantification represents the mean ±SEM of puncta per cell ( n = 5) from 3 independent experiments. Scale bar: 10 μm.
    Figure Legend Snippet: PRDX1 is negatively involved in autophagy activation through inhibiting BECN1 ubiquitination. ( A ) HEK293T cells were transfected with Mock, Flag-TRAF6 wild type (WT), Flag-TRAF6 truncated mutants, or MYC-BECN1 as indicated. At 38 h post transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then subjected to IB assay using anti-Flag or anti-MYC antibody. ( B ) HEK293T cells were transfected with Mock, Flag-BECN1, or MYC-PRDX1 as indicated. At 38 h post transfection, transfected cells were extracted and cell lysates were subjected to immunoprecipitation with anti-MYC antibody followed by IB using anti-Flag or anti-MYC antibody. ( C ) Model showing of how PRDX1-TRAF6 interaction inhibits the ubiquitination of BECN1. See text for details. ( D ) HEK293T cells were transfected with Mock, Flag-BECN1, HA-Ub, MYC-TRAF6, or different concentrations of MYC-PRDX1 as indicated. At 38 h post transfection, transfected cells were extracted and cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by IB using anti-Flag or anti-HA antibody. ( E ) Control (Ctrl) and PRDX1 KD THP-1 cells were treated with or without LPS (200 ng/ml) in the presence or absence of pepstatin A (10 μM) and 3-MA (5 mM) for 6 h as indicated. These cells were lysed and subjected to SDS-PAGE followed by immunoblotting with LC3-I/-II or GAPDH antibody. Band intensity was quantified using ImageJ software. Quantitative data were calculated from 3 independent experiments. Data are shown as mean ±SEM. ( F ) Control (Ctrl) and PRDX1 KD THP-1 cells were treated with or without LPS (200 ng/ml) in the presence or absence of pepstatin A (10 μM) and 3-MA (5 mM) for 6 h as indicated, and then fixed. Immunofluorescence assay was performed with anti-LC3 antibody. Digital images were captured with confocal microscopy and the number of LC3-puncta was scored. Quantification represents the mean ±SEM of puncta per cell ( n = 5) from 3 independent experiments. Scale bar: 10 μm.

    Techniques Used: Activation Assay, Transfection, Immunoprecipitation, SDS Page, Software, Immunofluorescence, Confocal Microscopy

    31) Product Images from "Pairing your Sox: Identification of Sox11 partner proteins and interaction domains in the developing neural plate"

    Article Title: Pairing your Sox: Identification of Sox11 partner proteins and interaction domains in the developing neural plate

    Journal: bioRxiv

    doi: 10.1101/2020.04.23.057919

    mSOX11 interacts with mNGN1, but xSox11 does not. A-B Immunoprecipitation (IP) of xSox11-FLAG (A), mouse SOX11-FLAG (mSOX11-FLAG, B), with mouse NGN1-MYC (mNGN1-MYC) using either FLAG (red) or MYC (blue) antibodies in HEK293 cells. Samples were analyzed using western blot (WB) with either anti-FLAG-HRP or anti-MYC-HRP. Inputs, actin, and IgG (IP Control) were run as control.
    Figure Legend Snippet: mSOX11 interacts with mNGN1, but xSox11 does not. A-B Immunoprecipitation (IP) of xSox11-FLAG (A), mouse SOX11-FLAG (mSOX11-FLAG, B), with mouse NGN1-MYC (mNGN1-MYC) using either FLAG (red) or MYC (blue) antibodies in HEK293 cells. Samples were analyzed using western blot (WB) with either anti-FLAG-HRP or anti-MYC-HRP. Inputs, actin, and IgG (IP Control) were run as control.

    Techniques Used: Immunoprecipitation, Western Blot

    Sox11 N-terminus is essential for protein-protein interactions A Schematic of xSox11domains including HMG domain (yellow), transactivation domain (orange) and FLAG tag (red) along with xSox11 deletion constructs. ΔN46-xSox11-FLAG that removes 46 amino acids upstream of the HMG domain, ΔHMG-xSox11-FLAG that removes the 72 amino acid HMG domain, and the ΔCterm-xSox11-FLAG that removes 265 amino acids and contains only the N-terminus and HMG domain. C-G Immunoprecipitation (IP) of ΔCterm-xSox11-FLAG, ΔN46-xSox11-FLAG or ΔHMG-xSox11-FLAG with xPou3f2-HA or xNgn2-MYC. Proteins were immunoprecipitated using either FLAG (red), HA (green), or MYC (blue) antibodies. Samples were analyzed by WB with anti-FLAG-HRP, anti-HA-HRP, or anti-MYC-HRP. Inputs were ran as control.
    Figure Legend Snippet: Sox11 N-terminus is essential for protein-protein interactions A Schematic of xSox11domains including HMG domain (yellow), transactivation domain (orange) and FLAG tag (red) along with xSox11 deletion constructs. ΔN46-xSox11-FLAG that removes 46 amino acids upstream of the HMG domain, ΔHMG-xSox11-FLAG that removes the 72 amino acid HMG domain, and the ΔCterm-xSox11-FLAG that removes 265 amino acids and contains only the N-terminus and HMG domain. C-G Immunoprecipitation (IP) of ΔCterm-xSox11-FLAG, ΔN46-xSox11-FLAG or ΔHMG-xSox11-FLAG with xPou3f2-HA or xNgn2-MYC. Proteins were immunoprecipitated using either FLAG (red), HA (green), or MYC (blue) antibodies. Samples were analyzed by WB with anti-FLAG-HRP, anti-HA-HRP, or anti-MYC-HRP. Inputs were ran as control.

    Techniques Used: FLAG-tag, Construct, Immunoprecipitation, Western Blot

    Xenopus Sox11 interacts with xPou3f2 and xNgn2, but not xNgn1 A-D Immunoprecipitation (IP) of xSox11-FLAG and xPou3f2-HA (A), xNgn2-MYC (B) or xNgn1-HA (C) from in vitro translated proteins. Proteins were immunoprecipitated using either FLAG (red), HA (green) or MYC (blue) antibodies. Samples were analyzed by western blot (WB) indicated on the right with FLAG-HRP, MYC-HRP or HA-HRP. Inputs were run as control.
    Figure Legend Snippet: Xenopus Sox11 interacts with xPou3f2 and xNgn2, but not xNgn1 A-D Immunoprecipitation (IP) of xSox11-FLAG and xPou3f2-HA (A), xNgn2-MYC (B) or xNgn1-HA (C) from in vitro translated proteins. Proteins were immunoprecipitated using either FLAG (red), HA (green) or MYC (blue) antibodies. Samples were analyzed by western blot (WB) indicated on the right with FLAG-HRP, MYC-HRP or HA-HRP. Inputs were run as control.

    Techniques Used: Immunoprecipitation, In Vitro, Western Blot

    32) Product Images from "Neuronal hemoglobin affects dopaminergic cells' response to stress"

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.458

    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated

    Techniques Used: Western Blot, Fractionation, Immunofluorescence

    AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated
    Figure Legend Snippet: AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Infection

    Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)
    Figure Legend Snippet: Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)

    Techniques Used: Western Blot, Expressing, FACS

    Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated
    Figure Legend Snippet: Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated

    Techniques Used: Western Blot, Cell Fractionation, Immunofluorescence, Expressing

    33) Product Images from "The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification"

    Article Title: The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008583

    TMEM98 prevents MYRF self-cleavage and binds to the C-terminal part of MYRF. (A) Co-immunoprecipitation experiment where HEK293T cells were transiently transfected with the indicated epitope-tagged expression constructs and immunoprecipitated with anti-V5.The two MYRF constructs were either full-length (MYRF) or lacked exon 19 (MYRF Δ19). Western blot analysis of the inputs (left) and immunoprecipitated fractions (right) probed with anti-MYC (Cell Signaling Technology, 2276), anti-FLAG (Cell Signaling Technology, 2368) and anti-V5 antibodies are shown. Anti-tubulin antibody was used to probe the input Western as a loading control. The Western of the input samples shows that MYRF cleaves when transfected alone but remains largely intact when co-transfected with TMEM98-V5. The Western of the immunoprecipitated fractions shows that intact MYC-MYRF-FLAG and the C-terminal part tagged with FLAG are co-immunoprecipitated with TMEM98-V5 indicating that TMEM98 interacts with the C-terminal part of MYRF. Uncropped Western blot images are shown in S9 Fig . (B-D) ARPE-19 cells were transiently transfected with TMEM98-V5 and/or MYC-MYRF-FLAG and immunostained with anti-V5 (magenta), anti-MYC (Cell Signaling Technology, 2278) (red) and anti-FLAG (Biolegend, 637302) (green) antibodies as indicated. DAPI staining is in blue. (C) When transfected alone MYC-MYRF-FLAG cleaves and the N-terminal part tagged with MYC translocates to the nucleus whilst the C-terminal part tagged with FLAG is membrane-bound. (D) When MYC-MYRF-FLAG is co-transfected with TMEM98-V5 it remains intact and colocalises with TMEM98-V5 in the membrane. Scale bars represent 20 μm.
    Figure Legend Snippet: TMEM98 prevents MYRF self-cleavage and binds to the C-terminal part of MYRF. (A) Co-immunoprecipitation experiment where HEK293T cells were transiently transfected with the indicated epitope-tagged expression constructs and immunoprecipitated with anti-V5.The two MYRF constructs were either full-length (MYRF) or lacked exon 19 (MYRF Δ19). Western blot analysis of the inputs (left) and immunoprecipitated fractions (right) probed with anti-MYC (Cell Signaling Technology, 2276), anti-FLAG (Cell Signaling Technology, 2368) and anti-V5 antibodies are shown. Anti-tubulin antibody was used to probe the input Western as a loading control. The Western of the input samples shows that MYRF cleaves when transfected alone but remains largely intact when co-transfected with TMEM98-V5. The Western of the immunoprecipitated fractions shows that intact MYC-MYRF-FLAG and the C-terminal part tagged with FLAG are co-immunoprecipitated with TMEM98-V5 indicating that TMEM98 interacts with the C-terminal part of MYRF. Uncropped Western blot images are shown in S9 Fig . (B-D) ARPE-19 cells were transiently transfected with TMEM98-V5 and/or MYC-MYRF-FLAG and immunostained with anti-V5 (magenta), anti-MYC (Cell Signaling Technology, 2278) (red) and anti-FLAG (Biolegend, 637302) (green) antibodies as indicated. DAPI staining is in blue. (C) When transfected alone MYC-MYRF-FLAG cleaves and the N-terminal part tagged with MYC translocates to the nucleus whilst the C-terminal part tagged with FLAG is membrane-bound. (D) When MYC-MYRF-FLAG is co-transfected with TMEM98-V5 it remains intact and colocalises with TMEM98-V5 in the membrane. Scale bars represent 20 μm.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Western Blot, Staining

    34) Product Images from "Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer"

    Article Title: Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer

    Journal: Science Advances

    doi: 10.1126/sciadv.abc2578

    c-Myc induces neuronal repressor REST to mediate lineage conversion. ( A ) Expression of REST in NCI-H1963 cells with LacZ overexpression and NCI-H1963 with c-Myc overexpression ( n = 3). ( B ) Protein expression of c-Myc and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells. ( C ) Protein expression of Rest and ASCL1, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells to overexpress Rest. ( D ) Protein expression of c-Myc, Rest, and ASCL1, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells genetically deleted for REST and overexpressed with either LacZ as control or c-Myc. ( E ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in geneticall y engineered NCI-H1963 cells treated with vehicle [dimethyl sulfoxide (DMSO)] or 10 μM Rest inhibitor (X5050) for 48 hours. ( F ) Rest ChIP-qPCR in NCI-H1963 c-Myc–overexpressed cells and LacZ-overexpressed cells (negative control) at the ASCL1 and MYCL loci with 7q31 and HOPX as negative control loci. One representative of three biological replicates is shown. ( G ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells treated with vehicle (DMSO) or 0.5 μM Notch inhibitor (DBZ) for 48 hours. ( H ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells treated with vehicle (DMSO) or 10 μM Notch inhibitor (DAPT) for 48 hours. ( I ) c-Myc ChIP-qPCR in NCI-H1963 c-Myc–overexpressed cells and LacZ-overexpressed cells (negative control) at the ASCL1 and REST loci with 7q31 and HOPX as negative control loci. One representative of three biological replicates is shown. ( J ) Graphical schematic representing role of c-Myc and L-Myc as lineage-determining factors in SCLC.
    Figure Legend Snippet: c-Myc induces neuronal repressor REST to mediate lineage conversion. ( A ) Expression of REST in NCI-H1963 cells with LacZ overexpression and NCI-H1963 with c-Myc overexpression ( n = 3). ( B ) Protein expression of c-Myc and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells. ( C ) Protein expression of Rest and ASCL1, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells to overexpress Rest. ( D ) Protein expression of c-Myc, Rest, and ASCL1, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells genetically deleted for REST and overexpressed with either LacZ as control or c-Myc. ( E ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in geneticall y engineered NCI-H1963 cells treated with vehicle [dimethyl sulfoxide (DMSO)] or 10 μM Rest inhibitor (X5050) for 48 hours. ( F ) Rest ChIP-qPCR in NCI-H1963 c-Myc–overexpressed cells and LacZ-overexpressed cells (negative control) at the ASCL1 and MYCL loci with 7q31 and HOPX as negative control loci. One representative of three biological replicates is shown. ( G ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells treated with vehicle (DMSO) or 0.5 μM Notch inhibitor (DBZ) for 48 hours. ( H ) Protein expression of c-Myc, L-Myc, ASCL1, and Rest, as well as vinculin, as a loading control in genetically engineered NCI-H1963 cells treated with vehicle (DMSO) or 10 μM Notch inhibitor (DAPT) for 48 hours. ( I ) c-Myc ChIP-qPCR in NCI-H1963 c-Myc–overexpressed cells and LacZ-overexpressed cells (negative control) at the ASCL1 and REST loci with 7q31 and HOPX as negative control loci. One representative of three biological replicates is shown. ( J ) Graphical schematic representing role of c-Myc and L-Myc as lineage-determining factors in SCLC.

    Techniques Used: Expressing, Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    35) Product Images from "RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling"

    Article Title: RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1166

    Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.
    Figure Legend Snippet: Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.

    Techniques Used: Mutagenesis, Expressing, Transfection, Construct, Western Blot, Fractionation, Marker

    36) Product Images from "From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria"

    Article Title: From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria

    Journal: Human mutation

    doi: 10.1002/humu.23983

    Overview of relative mRNA expression levels per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to quantitative analysis of mRNA expression applying qRT-PCR. Data are expressed as fold-change (mean +/− SD) normalized to the relative expression of the respective ASL wildtype plasmids ( A-E ; n=3 for each experiment). Red columns illustrate FLAG-tagged expression vectors, blue columns represent MYC-tagged plasmids. Variants associated with a premature stop codon are indicated by an asterisk (*).
    Figure Legend Snippet: Overview of relative mRNA expression levels per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to quantitative analysis of mRNA expression applying qRT-PCR. Data are expressed as fold-change (mean +/− SD) normalized to the relative expression of the respective ASL wildtype plasmids ( A-E ; n=3 for each experiment). Red columns illustrate FLAG-tagged expression vectors, blue columns represent MYC-tagged plasmids. Variants associated with a premature stop codon are indicated by an asterisk (*).

    Techniques Used: Expressing, Variant Assay, Transfection, Plasmid Preparation, Cell Culture, Quantitative RT-PCR

    Overview of protein expression levels per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to protein expression analysis using standard Western blot technique. Briefly, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on nitrocellulose membranes using the Trans-Blot® Turbo Transfer System (BioRad). Expression of FLAG-or MYC-tagged ASL variants was visualized using anti-FLAG-or anti-MYC antibodies on two identical gels, which were carried in parallel ( A-E ). Equal protein loading in cell lysates was confirmed by immunoblotting using an anti-β-actin antibody. Activity of β-galactosidase per variant combination was measured applying the β-galactosidase enzyme assay system (Promega). Data are expressed as mean in mU/mg protein ( A-E . Variants associated with a premature stop codon are indicated by an asterisk (*). Red letters illustrate FLAG-tagged expression vectors, blue letters represent MYC-tagged plasmids.
    Figure Legend Snippet: Overview of protein expression levels per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to protein expression analysis using standard Western blot technique. Briefly, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on nitrocellulose membranes using the Trans-Blot® Turbo Transfer System (BioRad). Expression of FLAG-or MYC-tagged ASL variants was visualized using anti-FLAG-or anti-MYC antibodies on two identical gels, which were carried in parallel ( A-E ). Equal protein loading in cell lysates was confirmed by immunoblotting using an anti-β-actin antibody. Activity of β-galactosidase per variant combination was measured applying the β-galactosidase enzyme assay system (Promega). Data are expressed as mean in mU/mg protein ( A-E . Variants associated with a premature stop codon are indicated by an asterisk (*). Red letters illustrate FLAG-tagged expression vectors, blue letters represent MYC-tagged plasmids.

    Techniques Used: Expressing, Variant Assay, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page, Activity Assay, Enzymatic Assay

    Overview of enzymatic ASL activities per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and enzymatic ASL activities determined applying a spectrophotometric assay as described under Material and methods . Data are expressed as mean +/− SD in % of ASL wildtype activity ( A-E , n=3 for each experiment). Variants associated with a premature stop codon are indicated by an asterisk (*).
    Figure Legend Snippet: Overview of enzymatic ASL activities per variant combination. COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and enzymatic ASL activities determined applying a spectrophotometric assay as described under Material and methods . Data are expressed as mean +/− SD in % of ASL wildtype activity ( A-E , n=3 for each experiment). Variants associated with a premature stop codon are indicated by an asterisk (*).

    Techniques Used: Variant Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Spectrophotometric Assay, Activity Assay

    37) Product Images from "Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes"

    Article Title: Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12876

    Cooperative effects of OTX015 with a panel of drugs in ALCL in vitro models A. The combination of OTX015-CEP28122 effectively down-regulated MYC protein levels by Western blot in TS-Supm2 cells (β-actin was used as a loading control), resulting in a pronounced G1 cell cycle arrest with limited effects on cell viability according to FACScan. The effect was maximal after 48 h. B. OTX015 exposure (500 nM) for 30 minutes to 24 h in Karpas299 cells resulted in robust down-regulation of GLI1 mRNA, a known target of GANT61, following an initial rapid upregulation according to qRT-PCR. Exposure of in Karpas299 cells to increasing OTX015 concentrations for 24 h showed that the mRNA down-regulation was dose-dependent. C. The effects on cell viability of the GLI inhibitor GANT61 and OTX015 were measured as single agents and in combination according to the qRT-PCR. Concomitant exposure of OTX015 (500 nM) and GANT61 (2.5 or 5 μM) increased the percentage of dead cells after 24 and 48 h exposure. D. Cells treated with combined ibrutinib (10 μM) and OTX015 (500 nM) had reduced levels of P-STAT3 and P-ITK protein compared to treatment with either single agent, as per Western blot. β-actin was used as a loading control. E. Metabolic readout of ALCL cell lines treated with OTX015 (500 nM) in combination with ibrutinib (1.2 or 5 μM) for 48 h, determined using an ATPlite assay. F. Cell viability was determined after 48 h in multiple cell lines exposed to 500 nM OTX015 in combination with ibrutinib (2, 5, 10 μM), according to the ATPlite assay.
    Figure Legend Snippet: Cooperative effects of OTX015 with a panel of drugs in ALCL in vitro models A. The combination of OTX015-CEP28122 effectively down-regulated MYC protein levels by Western blot in TS-Supm2 cells (β-actin was used as a loading control), resulting in a pronounced G1 cell cycle arrest with limited effects on cell viability according to FACScan. The effect was maximal after 48 h. B. OTX015 exposure (500 nM) for 30 minutes to 24 h in Karpas299 cells resulted in robust down-regulation of GLI1 mRNA, a known target of GANT61, following an initial rapid upregulation according to qRT-PCR. Exposure of in Karpas299 cells to increasing OTX015 concentrations for 24 h showed that the mRNA down-regulation was dose-dependent. C. The effects on cell viability of the GLI inhibitor GANT61 and OTX015 were measured as single agents and in combination according to the qRT-PCR. Concomitant exposure of OTX015 (500 nM) and GANT61 (2.5 or 5 μM) increased the percentage of dead cells after 24 and 48 h exposure. D. Cells treated with combined ibrutinib (10 μM) and OTX015 (500 nM) had reduced levels of P-STAT3 and P-ITK protein compared to treatment with either single agent, as per Western blot. β-actin was used as a loading control. E. Metabolic readout of ALCL cell lines treated with OTX015 (500 nM) in combination with ibrutinib (1.2 or 5 μM) for 48 h, determined using an ATPlite assay. F. Cell viability was determined after 48 h in multiple cell lines exposed to 500 nM OTX015 in combination with ibrutinib (2, 5, 10 μM), according to the ATPlite assay.

    Techniques Used: In Vitro, Western Blot, Quantitative RT-PCR

    OTX015 modulates MYC and BRD expression A. MYC mRNA and protein levels were reproducibly down-regulated after 72-h OTX015 exposure (100 - 1000 nM) in ALK+ cell lines by qRT-PCR and Western blot respectively. CT, DMSO-treated controls. B. 24 h exposure to OTX015 (250, 500 nM) led to dose-dependent down-regulation of MYC protein levels and G1 cell cycle arrest. β-actin was used as a protein loading control. C. 24 h exposure to OTX015 resulted in down-regulation of BRD2, BRD3 and BRD4 RNA and protein levels by qRT-PCR and Western blot. β-actin was used as a protein loading control.
    Figure Legend Snippet: OTX015 modulates MYC and BRD expression A. MYC mRNA and protein levels were reproducibly down-regulated after 72-h OTX015 exposure (100 - 1000 nM) in ALK+ cell lines by qRT-PCR and Western blot respectively. CT, DMSO-treated controls. B. 24 h exposure to OTX015 (250, 500 nM) led to dose-dependent down-regulation of MYC protein levels and G1 cell cycle arrest. β-actin was used as a protein loading control. C. 24 h exposure to OTX015 resulted in down-regulation of BRD2, BRD3 and BRD4 RNA and protein levels by qRT-PCR and Western blot. β-actin was used as a protein loading control.

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

    38) Product Images from "Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment"

    Article Title: Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment

    Journal: Nature Communications

    doi: 10.1038/ncomms13616

    Molecular effects of TUG1 inhibition in mouse xenograft model. ( a ) RNA-FISH analysis of TUG1 (red) and Notch1 (green) in tumour cells of CTRL -DDS (upper panels) and TUG1 -DDS-treated mice (bottom panels). ( b ) Immunostaining of CD15 (red) and Nestin -EGFP (green) in CTRL -DDS (upper panels) and TUG1 -DDS-treated tumour (bottom panels). Asterisk indicates blood vessel in CTRL -DDS treated mice. Tumour areas are surrounded with the dashed line in TUG1 -DDS-treated mice. Scale bars, 100 μm. ( c ) Expression levels of miR-145, SOX2 , MYC and TUG1 target genes ( BDNF , NGF and NTF3 ) were examined by qPCR in tumour cells derived from the mouse xenograft. Relative expression levels compared with that in the CTRL -DDS-treated tumour are indicated on the y -axis ( n =4). Error bars indicate s.e.m. * P
    Figure Legend Snippet: Molecular effects of TUG1 inhibition in mouse xenograft model. ( a ) RNA-FISH analysis of TUG1 (red) and Notch1 (green) in tumour cells of CTRL -DDS (upper panels) and TUG1 -DDS-treated mice (bottom panels). ( b ) Immunostaining of CD15 (red) and Nestin -EGFP (green) in CTRL -DDS (upper panels) and TUG1 -DDS-treated tumour (bottom panels). Asterisk indicates blood vessel in CTRL -DDS treated mice. Tumour areas are surrounded with the dashed line in TUG1 -DDS-treated mice. Scale bars, 100 μm. ( c ) Expression levels of miR-145, SOX2 , MYC and TUG1 target genes ( BDNF , NGF and NTF3 ) were examined by qPCR in tumour cells derived from the mouse xenograft. Relative expression levels compared with that in the CTRL -DDS-treated tumour are indicated on the y -axis ( n =4). Error bars indicate s.e.m. * P

    Techniques Used: Inhibition, Fluorescence In Situ Hybridization, Mouse Assay, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay

    Analysis of TUG1 transcripts for maintenance of stemness features of GSCs. ( a – c ) Effects of TUG1 overexpression on cell viability ( a ), apoptosis ( b ) and expression of the stemness-associated genes ( SOX2 , MYC , Nestin and CD15 ) ( c ) in GSCs treated with γ-secretase inhibitor (RO4929097). Plasmid vectors expressing each TUG1 exon (1–2,132, 2,133–2,910 and 2,911–7,115 nucleotides corresponding to exon 1, exon 2 and exon 3, respectively) were transfected. Viable cells were assessed by trypan blue staining ( a ). The number of apoptotic cells were counted by FACS analysis with 7-AAD and PE Annexin V staining ( b ). Expression levels of stemness-associated genes were analysed by qRT-PCR. y -axis indicates relative expression level compared to that seen in DMSO-treated cells ( c ). Values are indicated relative to abundance in DMSO-treated cells. * P
    Figure Legend Snippet: Analysis of TUG1 transcripts for maintenance of stemness features of GSCs. ( a – c ) Effects of TUG1 overexpression on cell viability ( a ), apoptosis ( b ) and expression of the stemness-associated genes ( SOX2 , MYC , Nestin and CD15 ) ( c ) in GSCs treated with γ-secretase inhibitor (RO4929097). Plasmid vectors expressing each TUG1 exon (1–2,132, 2,133–2,910 and 2,911–7,115 nucleotides corresponding to exon 1, exon 2 and exon 3, respectively) were transfected. Viable cells were assessed by trypan blue staining ( a ). The number of apoptotic cells were counted by FACS analysis with 7-AAD and PE Annexin V staining ( b ). Expression levels of stemness-associated genes were analysed by qRT-PCR. y -axis indicates relative expression level compared to that seen in DMSO-treated cells ( c ). Values are indicated relative to abundance in DMSO-treated cells. * P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Transfection, Staining, FACS, Quantitative RT-PCR

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    Incubation:

    Article Title: Identification of MYC as an antinecroptotic protein that stifles RIPK1–RIPK3 complex formation
    Article Snippet: Immunofluorescence Analysis.HT-29 cells were plated on confocal dishes (100350, SPL). .. Cells were incubated with immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) containing anti-MYC (1:100; 5605, Cell Signaling) or anti-RIPK3 antibodies (1:50; 13526. .. Samples were washed with PBS three times and then incubated with Alexa Fluor 488 anti-rabbit (1:200; A-11008, Thermo Fisher Scientific) supplemented in immunofluorescence blocking buffer for 1 h. HeLa cells were cultured on confocal dishes (100350, SPL).

    Article Title: Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration
    Article Snippet: Proteins were then transferred to a PVDF membrane (cat. no. 1620177; Bio-Rad) for 90 min at 100 mV which was then blocked using 5% BSA (cat. no. A2153-100G; Sigma) diluted in 1X TBS with 1% Tween-20 (cat. no. 9005-64-5). .. The blots were incubated with rabbit monoclonal L1-CAM (cat. no. ab20148), HMGA1 (cat. no. ab129153), FABP5 (cat. no. ab84028), survivin (cat. no. ab469) (all from Abcam, Cambridge MA, USA) and MYCN antibodies (cat. no. 9405; Cell Signaling Technology, Danvers, MA, USA) and with mouse monoclonal GAPDH antibody (cat. no. ab9484) (Abcam) overnight (all diluted 1:1,000 in TBS-T +5% BSA blocking solution). .. The blots were washed 5 times for 5 min with TBST and then incubated with secondary antibodies [goat anti-rabbit (cat. no. 170-5046) or goat anti-mouse (cat. no. 170-5047) HRP-conjugated secondary antibodies from Bio-Rad] for 1 h. After washing, the blots were incubated with Clarity Western ECL substrate (cat. no. 1705060; Bio-Rad) for 3 min and imaged using the Bio-Rad© ChemiDoc system and analyzed using ImageLab® software.

    Immunofluorescence:

    Article Title: Identification of MYC as an antinecroptotic protein that stifles RIPK1–RIPK3 complex formation
    Article Snippet: Immunofluorescence Analysis.HT-29 cells were plated on confocal dishes (100350, SPL). .. Cells were incubated with immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) containing anti-MYC (1:100; 5605, Cell Signaling) or anti-RIPK3 antibodies (1:50; 13526. .. Samples were washed with PBS three times and then incubated with Alexa Fluor 488 anti-rabbit (1:200; A-11008, Thermo Fisher Scientific) supplemented in immunofluorescence blocking buffer for 1 h. HeLa cells were cultured on confocal dishes (100350, SPL).

    Blocking Assay:

    Article Title: Identification of MYC as an antinecroptotic protein that stifles RIPK1–RIPK3 complex formation
    Article Snippet: Immunofluorescence Analysis.HT-29 cells were plated on confocal dishes (100350, SPL). .. Cells were incubated with immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) containing anti-MYC (1:100; 5605, Cell Signaling) or anti-RIPK3 antibodies (1:50; 13526. .. Samples were washed with PBS three times and then incubated with Alexa Fluor 488 anti-rabbit (1:200; A-11008, Thermo Fisher Scientific) supplemented in immunofluorescence blocking buffer for 1 h. HeLa cells were cultured on confocal dishes (100350, SPL).

    Article Title: Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration
    Article Snippet: Proteins were then transferred to a PVDF membrane (cat. no. 1620177; Bio-Rad) for 90 min at 100 mV which was then blocked using 5% BSA (cat. no. A2153-100G; Sigma) diluted in 1X TBS with 1% Tween-20 (cat. no. 9005-64-5). .. The blots were incubated with rabbit monoclonal L1-CAM (cat. no. ab20148), HMGA1 (cat. no. ab129153), FABP5 (cat. no. ab84028), survivin (cat. no. ab469) (all from Abcam, Cambridge MA, USA) and MYCN antibodies (cat. no. 9405; Cell Signaling Technology, Danvers, MA, USA) and with mouse monoclonal GAPDH antibody (cat. no. ab9484) (Abcam) overnight (all diluted 1:1,000 in TBS-T +5% BSA blocking solution). .. The blots were washed 5 times for 5 min with TBST and then incubated with secondary antibodies [goat anti-rabbit (cat. no. 170-5046) or goat anti-mouse (cat. no. 170-5047) HRP-conjugated secondary antibodies from Bio-Rad] for 1 h. After washing, the blots were incubated with Clarity Western ECL substrate (cat. no. 1705060; Bio-Rad) for 3 min and imaged using the Bio-Rad© ChemiDoc system and analyzed using ImageLab® software.

    Immunoprecipitation:

    Article Title: PinX1t, a Novel PinX1 Transcript Variant, Positively Regulates Cardiogenesis of Embryonic Stem Cells
    Article Snippet: The details of RNA immunoprecipitation can be found in Data . .. Briefly, anti‐myc antibody (9B11; Cell Signaling Technology, Inc, Danvers, MA) at a dilution ratio of 1:250 and isotype antibody IgG2a (Abcam, Cambridge, UK) were used for the immunoprecipitation. ..

    SDS Page:

    Article Title: Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation
    Article Snippet: Western blot analysis Cells were lysed in buffer (50mM TRIS, 0.5% sodium deoxycholate, 1.0% NP-40, 0.1% SDS, 150mM NaCl, 2mM EDTA) supplemented with protease inhibitors (Roche). .. Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R & D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals). .. Membranes were then incubated with a peroxidase-conjugated antibody.

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    Cell Signaling Technology Inc anti myc
    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with <t>anti-FLAG</t> ( α -globin) and <t>anti-MYC</t> ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated
    Anti Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal anti myc
    Actinonin is unlikely to inhibit the peptide deformylase of P. falciparum . ( A ) Western blot of the parental line (NF54attB-pCRISPR) and the <t>PDF-myc</t> parasites grown with our without aTC for 24 hr. Induction of the second copy of PfPDF (PDF-myc) with 4 uM aTC results in two bands, the top lighter band representing unprocessed PDF-myc, and the bottom darker band representing processed PDF-myc. PfAldolase is used as a loading control. Induction with 0.125–4 uM aTC results in similar amount of PDF-myc induction (data not shown). ( B ) Western blot for PDF-myc of parasites with or without their apicoplast. An accumulation of unprocessed PDF-myc is observed when the apicoplast is missing, due to loss of the transit peptide cleavage that usually occurs upon import to the apicoplast. This has been shown previously for apicoplast-resident proteins and is consistent apicoplast localization ( Yeh and DeRisi, 2011 ). ( C ) Dose dependent parasites growth inhibition by actinonin in the presence of 4 uM aTC does not change the actinonin EC 50 . This experiment was also performed under IPP rescue conditions, to confirm apicoplast specificity of actinonin and with a range of aTC concentrations (0.125–4 uM) to insure max expression of PDF-myc (data not shown). Error bars represent the SEM of 3 biological replicates. ( D ) Parasite growth after one or two replication cycles after treatment with actinonin, chloramphenicol, or both actinonin and chloramphenicol normalized to growth of an untreated control. Treatment with actinonin alone inhibited growth after the first replication cycle, whereas treatment with chloramphenicol alone inhibited growth after the second replication cycle. Co-treatment with chloramphenicol, which targets apicoplast translation, did not suppress effects of actinonin treatment, which was inconsistent with actinonin targeting the peptide deformylase (PDF) of the apicoplast. This experiment was tried using a range of concentrations of actinonin and chloramphenicol to insure the data was not the result of partial inhibition. All concentrations that lead to apicoplast-specific death gave this phenotype (data not shown).
    Rabbit Monoclonal Anti Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated

    Journal: Cell Death & Disease

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    doi: 10.1038/cddis.2016.458

    Figure Lengend Snippet: Neurochemical intoxication accumulates insoluble Hb in the nucleolus. Differentiated Hb cells were treated with MPP + ( a , d , h ) or rotenone ( b , f , i ) at the indicated concentrations for 16 h. ( a and b ) Western blotting analysis of soluble and insoluble fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. α -Tubulin was used to visualize specifically soluble fraction. β -Actin was used as a loading control. (FLAG n =3, MYC n =2; FLAG n =4, MYC n =2) ( c ) Densitometric analysis of insoluble FLAG ( α -globin) level. Insoluble α -globin level was normalized to β -actin. Untreated cells was used as reference and set to 100%. ( n =3, n =3) ( d and f ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =3) ( e and g ) Quantification of globin chain aggregates in the cells. An average of 120 randomly chosen cells were counted for quantification of each condition. Values are expresses as a percentage relative to the total. ( h and i ) Solubilized double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-NPM antibodies. Nuclei were marked by DAPI. Scale bar 5 μ m. ( n =2, n =2) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated

    Article Snippet: The following primary antibodies were used: anti-TH 1:1000 (Sigma-Aldrich or Millipore), anti-FLAG 1:100 (Sigma-Aldrich), anti-MYC 1:100 (Cell Signaling) and anti-Hemoglobin 1:1000 (MP Biomedicals).

    Techniques: Western Blot, Fractionation, Immunofluorescence

    AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated

    Journal: Cell Death & Disease

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    doi: 10.1038/cddis.2016.458

    Figure Lengend Snippet: AAV9-mediated delivery of Hb in SNpc inhibits improvement of motor performance and triggers Hb aggregates in DA cells. ( a ) Scheme of experimental settings. ( b ) Latency in rotarod test in AAV9-control ( n =18) and AAV9-Hb ( n =19) mice at 5, 10, 15 and 20 r.p.m. ( c ) Immunohistochemistry of coronal sections of AAV9-control and AAV9-Hb mouse brain. TH + fibres in the striatum were stained with anti-TH antibody. Scale bar 800 μ m. ( d ) Immunohistochemistry of coronal sections of AAV9-Hb and AAV9-control mouse brain. SN was stained with anti-TH antibody. Scale bar 200 μ m. ( e ) Densitometric analysis of TH + fibres in AAV9-Hb ( n =18) and AAV9-control ( n =18) mice. Values are expresses as a percentage relative to the contralateral side of AAV9-control mice, arbitrary set to 100%. ( f ) Quantitative analysis of TH + cells number in AAV9-Hb ( n =16) and AAV9-control ( n =15) mice. Values are expressed as the number of total TH + cells relative to the contralateral side of AAV9-control mice. ( g ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) antibody. Arrow indicates α -globin aggregates. Nuclei were visualized with DAPI (4,6-diamidino-2-phenylindole). Scale bar 50 μ m. ( h ) Immunohistochemistry of coronal sections of AAV9-Hb mouse brain. SN was stained with anti-TH antibody. Infected cells were stained with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Arrow indicates globin aggregates. Scale bar 10 μ m. All data are represented as mean±S.E.M. Data were evaluated statistically by one-way analysis of variance. Resulting P -values are indicated

    Article Snippet: The following primary antibodies were used: anti-TH 1:1000 (Sigma-Aldrich or Millipore), anti-FLAG 1:100 (Sigma-Aldrich), anti-MYC 1:100 (Cell Signaling) and anti-Hemoglobin 1:1000 (MP Biomedicals).

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Infection

    Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)

    Journal: Cell Death & Disease

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    doi: 10.1038/cddis.2016.458

    Figure Lengend Snippet: Hb increases susceptibility to cell death in cellular model of PD. Differentiated Hb cells (Hb) and control cells (control) were treated with MPP + ( a–c ) or rotenone ( d–f ) at the indicated concentrations for 16 h. ( a and d ) Western blotting analysis of cleaved Caspase-3 (cleaved Casp3) expression. α - and β -Globins were detected with anti-FLAG and anti-MYC antibodies, respectively. For normalization, the levels of pro-Caspase-3 (pro-Casp3) were detected. β -Actin was used as a loading control. ( n =3, n =3) ( b and e ) FACS analysis. Percentage of subG1 cells is expressed as fold increased relative to untreated cells, arbitrary set to 1. ( n =3, n =4) ( c and f ) WST-1 analysis. In graph are represented the percentage of viable cells relative to untreated cells, arbitrary set to 100%. ( n =4, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. Resulting P -values are indicated (NS=not significant)

    Article Snippet: The following primary antibodies were used: anti-TH 1:1000 (Sigma-Aldrich or Millipore), anti-FLAG 1:100 (Sigma-Aldrich), anti-MYC 1:100 (Cell Signaling) and anti-Hemoglobin 1:1000 (MP Biomedicals).

    Techniques: Western Blot, Expressing, FACS

    Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated

    Journal: Cell Death & Disease

    Article Title: Neuronal hemoglobin affects dopaminergic cells' response to stress

    doi: 10.1038/cddis.2016.458

    Figure Lengend Snippet: Neurochemical intoxication increases Hb in the nucleus. Differentiated Hb cells were treated with MPP + ( a and b ) or rotenone ( c–f ) at the indicated concentrations for 16 h. ( a and c ) Western blotting analysis of cellular fractionation was carried out with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Anti-TH and anti-UBF antibodies were used to visualize specifically cytoplasm and nucleus compartment, respectively. β -Actin was used as a loading control. ( n =5, n =4) ( b and d ) Double immunofluorescence was performed with anti-FLAG ( α -globin) and anti-MYC ( β -globin) antibodies. Nuclei were marked by DAPI (4,6-diamidino-2-phenylindole). Scale bar 5 μ m. ( n =3, n =4) ( e ) Western blotting analysis of H3K4me3 and H3K9me2 expression. For normalization, the total levels of H3 and β -actin were detected. Levels of pro-Caspase-3 (pro-Casp3) and cleaved Caspase-3 (cleaved Casp3) were also monitored. ( n =3) ( f ) Densitometric analysis of H3K4me3 and H3K9me2 expression. H3K4me3 and H3K9me2 levels were normalized to H3 and β -actin. Untreated control cells was used as reference and set to 100%. ( n =3, n =3) Values are mean±S.D. Data were evaluated statistically by Student's t -test. The P -values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate. Resulting P -values are indicated

    Article Snippet: The following primary antibodies were used: anti-TH 1:1000 (Sigma-Aldrich or Millipore), anti-FLAG 1:100 (Sigma-Aldrich), anti-MYC 1:100 (Cell Signaling) and anti-Hemoglobin 1:1000 (MP Biomedicals).

    Techniques: Western Blot, Cell Fractionation, Immunofluorescence, Expressing

    Verification of proteomics data. (A) Representative western blot images of L1-cell adhesion molecule (L1-CAM), MYCN proto-oncogene, bHLH transcription factor (MYCN), high mobility group protein A1 (HMGA1), survivin and fatty-acid binding protein 5 (FABP5) protein expression in the IMR-32 compared to the SK-N-SH cells. (B) Densitometric analysis verified the proteomics data and revealed the significant overexpression of L1-CAM, MYCN, HMGA1, survivin and FABP5 in the IMR-32 compared to the SK-N-SH cells. Experiments were run in triplicate and repeated > 3 times. Results represent the means ± SEM; * P

    Journal: International Journal of Oncology

    Article Title: Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration

    doi: 10.3892/ijo.2018.4236

    Figure Lengend Snippet: Verification of proteomics data. (A) Representative western blot images of L1-cell adhesion molecule (L1-CAM), MYCN proto-oncogene, bHLH transcription factor (MYCN), high mobility group protein A1 (HMGA1), survivin and fatty-acid binding protein 5 (FABP5) protein expression in the IMR-32 compared to the SK-N-SH cells. (B) Densitometric analysis verified the proteomics data and revealed the significant overexpression of L1-CAM, MYCN, HMGA1, survivin and FABP5 in the IMR-32 compared to the SK-N-SH cells. Experiments were run in triplicate and repeated > 3 times. Results represent the means ± SEM; * P

    Article Snippet: The blots were incubated with rabbit monoclonal L1-CAM (cat. no. ab20148), HMGA1 (cat. no. ab129153), FABP5 (cat. no. ab84028), survivin (cat. no. ab469) (all from Abcam, Cambridge MA, USA) and MYCN antibodies (cat. no. 9405; Cell Signaling Technology, Danvers, MA, USA) and with mouse monoclonal GAPDH antibody (cat. no. ab9484) (Abcam) overnight (all diluted 1:1,000 in TBS-T +5% BSA blocking solution).

    Techniques: Western Blot, Chick Chorioallantoic Membrane Assay, Binding Assay, Expressing, Over Expression

    Transcriptional knockdown experiments reveal an interplay between tumorigenic proteins. (A) Western blot analysis revealed a concomitant significant downregulation of MYCN proto-oncogene, bHLH transcription factor (MYCN) protein expression following the knockdown of L1-cell adhesion molecule (L1-CAM), high mobility group protein A1 (HMGA1) and fatty-acid binding protein 5 (FABP5) by siRNA in the IMR-32 cells compared to the controls. (B) Conversely, MYCN knockdown by siRNA led to the concomitant significant downregulation of L1-CAM, HMGA1 and FABP5 protein expression, whereas (C) the combined knockdown of L1-CAM and FABP5 by siRNA led to the concomitant significant downregulation of HMGA1 protein expression. (D) MYCN, HMGA1 and FABP5 knockdown and the combined dual-target knockdown led to the concomitant downregulation of survivin protein expression. Non-adjacent bands of western blot experiments were re-aligned side by side to increase the clarity of the presented data and designated by a straight vertical line. Experiments were run in triplicate and repeated > 3 times. Results represent the means ± SEM; * P

    Journal: International Journal of Oncology

    Article Title: Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration

    doi: 10.3892/ijo.2018.4236

    Figure Lengend Snippet: Transcriptional knockdown experiments reveal an interplay between tumorigenic proteins. (A) Western blot analysis revealed a concomitant significant downregulation of MYCN proto-oncogene, bHLH transcription factor (MYCN) protein expression following the knockdown of L1-cell adhesion molecule (L1-CAM), high mobility group protein A1 (HMGA1) and fatty-acid binding protein 5 (FABP5) by siRNA in the IMR-32 cells compared to the controls. (B) Conversely, MYCN knockdown by siRNA led to the concomitant significant downregulation of L1-CAM, HMGA1 and FABP5 protein expression, whereas (C) the combined knockdown of L1-CAM and FABP5 by siRNA led to the concomitant significant downregulation of HMGA1 protein expression. (D) MYCN, HMGA1 and FABP5 knockdown and the combined dual-target knockdown led to the concomitant downregulation of survivin protein expression. Non-adjacent bands of western blot experiments were re-aligned side by side to increase the clarity of the presented data and designated by a straight vertical line. Experiments were run in triplicate and repeated > 3 times. Results represent the means ± SEM; * P

    Article Snippet: The blots were incubated with rabbit monoclonal L1-CAM (cat. no. ab20148), HMGA1 (cat. no. ab129153), FABP5 (cat. no. ab84028), survivin (cat. no. ab469) (all from Abcam, Cambridge MA, USA) and MYCN antibodies (cat. no. 9405; Cell Signaling Technology, Danvers, MA, USA) and with mouse monoclonal GAPDH antibody (cat. no. ab9484) (Abcam) overnight (all diluted 1:1,000 in TBS-T +5% BSA blocking solution).

    Techniques: Western Blot, Expressing, Chick Chorioallantoic Membrane Assay, Binding Assay

    MYC, MYCL and MYCN inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway

    Journal: Oncotarget

    Article Title: Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation

    doi: 10.18632/oncotarget.8826

    Figure Lengend Snippet: MYC, MYCL and MYCN inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway

    Article Snippet: Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R & D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals).

    Techniques: Inhibition, Activation Assay

    Omomyc induces growth suppression in SCLC cells A. Status of the MYC family genes, TP53 , and RB1 in SCLC cell lines used in this study. mut: mutated. Predominant type of the cell cycle arrest, occurrence of apoptosis and levels of p21, p27 and p16 after MYC inhibition by Omomyc are shown. B. Immunoblot analysis for the expression of MYC, MYCL or MYCN in SCLC cells. Media were changed 24 hr before collection of the cells. C. Growth curve of SCLC cells in the presence or absence of doxycycline (DX). Cumulative population doubling level (PDL) was calculated by adding the PDLs of the previous passages. Data are shown as the mean ± SD of four counts from a single representative experiment. P-values were calculated by unpaired two-tailed t-test. *p

    Journal: Oncotarget

    Article Title: Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation

    doi: 10.18632/oncotarget.8826

    Figure Lengend Snippet: Omomyc induces growth suppression in SCLC cells A. Status of the MYC family genes, TP53 , and RB1 in SCLC cell lines used in this study. mut: mutated. Predominant type of the cell cycle arrest, occurrence of apoptosis and levels of p21, p27 and p16 after MYC inhibition by Omomyc are shown. B. Immunoblot analysis for the expression of MYC, MYCL or MYCN in SCLC cells. Media were changed 24 hr before collection of the cells. C. Growth curve of SCLC cells in the presence or absence of doxycycline (DX). Cumulative population doubling level (PDL) was calculated by adding the PDLs of the previous passages. Data are shown as the mean ± SD of four counts from a single representative experiment. P-values were calculated by unpaired two-tailed t-test. *p

    Article Snippet: Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R & D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals).

    Techniques: Inhibition, Expressing, Two Tailed Test

    Actinonin is unlikely to inhibit the peptide deformylase of P. falciparum . ( A ) Western blot of the parental line (NF54attB-pCRISPR) and the PDF-myc parasites grown with our without aTC for 24 hr. Induction of the second copy of PfPDF (PDF-myc) with 4 uM aTC results in two bands, the top lighter band representing unprocessed PDF-myc, and the bottom darker band representing processed PDF-myc. PfAldolase is used as a loading control. Induction with 0.125–4 uM aTC results in similar amount of PDF-myc induction (data not shown). ( B ) Western blot for PDF-myc of parasites with or without their apicoplast. An accumulation of unprocessed PDF-myc is observed when the apicoplast is missing, due to loss of the transit peptide cleavage that usually occurs upon import to the apicoplast. This has been shown previously for apicoplast-resident proteins and is consistent apicoplast localization ( Yeh and DeRisi, 2011 ). ( C ) Dose dependent parasites growth inhibition by actinonin in the presence of 4 uM aTC does not change the actinonin EC 50 . This experiment was also performed under IPP rescue conditions, to confirm apicoplast specificity of actinonin and with a range of aTC concentrations (0.125–4 uM) to insure max expression of PDF-myc (data not shown). Error bars represent the SEM of 3 biological replicates. ( D ) Parasite growth after one or two replication cycles after treatment with actinonin, chloramphenicol, or both actinonin and chloramphenicol normalized to growth of an untreated control. Treatment with actinonin alone inhibited growth after the first replication cycle, whereas treatment with chloramphenicol alone inhibited growth after the second replication cycle. Co-treatment with chloramphenicol, which targets apicoplast translation, did not suppress effects of actinonin treatment, which was inconsistent with actinonin targeting the peptide deformylase (PDF) of the apicoplast. This experiment was tried using a range of concentrations of actinonin and chloramphenicol to insure the data was not the result of partial inhibition. All concentrations that lead to apicoplast-specific death gave this phenotype (data not shown).

    Journal: eLife

    Article Title: Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

    doi: 10.7554/eLife.29865

    Figure Lengend Snippet: Actinonin is unlikely to inhibit the peptide deformylase of P. falciparum . ( A ) Western blot of the parental line (NF54attB-pCRISPR) and the PDF-myc parasites grown with our without aTC for 24 hr. Induction of the second copy of PfPDF (PDF-myc) with 4 uM aTC results in two bands, the top lighter band representing unprocessed PDF-myc, and the bottom darker band representing processed PDF-myc. PfAldolase is used as a loading control. Induction with 0.125–4 uM aTC results in similar amount of PDF-myc induction (data not shown). ( B ) Western blot for PDF-myc of parasites with or without their apicoplast. An accumulation of unprocessed PDF-myc is observed when the apicoplast is missing, due to loss of the transit peptide cleavage that usually occurs upon import to the apicoplast. This has been shown previously for apicoplast-resident proteins and is consistent apicoplast localization ( Yeh and DeRisi, 2011 ). ( C ) Dose dependent parasites growth inhibition by actinonin in the presence of 4 uM aTC does not change the actinonin EC 50 . This experiment was also performed under IPP rescue conditions, to confirm apicoplast specificity of actinonin and with a range of aTC concentrations (0.125–4 uM) to insure max expression of PDF-myc (data not shown). Error bars represent the SEM of 3 biological replicates. ( D ) Parasite growth after one or two replication cycles after treatment with actinonin, chloramphenicol, or both actinonin and chloramphenicol normalized to growth of an untreated control. Treatment with actinonin alone inhibited growth after the first replication cycle, whereas treatment with chloramphenicol alone inhibited growth after the second replication cycle. Co-treatment with chloramphenicol, which targets apicoplast translation, did not suppress effects of actinonin treatment, which was inconsistent with actinonin targeting the peptide deformylase (PDF) of the apicoplast. This experiment was tried using a range of concentrations of actinonin and chloramphenicol to insure the data was not the result of partial inhibition. All concentrations that lead to apicoplast-specific death gave this phenotype (data not shown).

    Article Snippet: For anti-PDF immunoblots, membranes were probed with 1:2000 rabbit monoclonal anti-MYC (Cell Signaling Technology 2278S, Danvers, MA), followed by 1:20,000 rabbit polyclonal anti-PfAldolase (Abcam ab207494, UK) and 1:10,000 donkey anti-rabbit 800 (LiCor Biosciences).

    Techniques: Western Blot, Inhibition, Expressing