Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: ITGA5 Controls IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs were transduced with shITGA5, shITGB1 or a non relevant shRNA (shNR), total RNA was used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were transduced with a lentiviral vector encoding ITGA5, or treated with the agonist peptide CRRETAWAC (100 μg/ml) (CRRETAWAC) or with a non relevant control peptide (GRGESP; 100 μg/ml), or with a conformation-dependent anti-α5 monoclonal antibody (SNAKA51; 10 μg/ml), and IGF2 and IGFBP2 mRNA expression was determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR, Plasmid Preparation, Control

Journal: BMC Cell Biology

Article Title: Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

doi: 10.1186/1471-2121-11-44

Figure Lengend Snippet: Inhibition of ITGA5-Induced Signalling Abrogates IGF2/IGFBP2 Expression in hMSCs . Adult hMSCs transduced with a lentiviral vector encoding ITGA5 or treated with the agonist peptide (CRRETAWAC; 100 μg/ml), and control cells were transiently transfected with a specific shRNA targeting FAK. Total RNA was collected and used for quantitative RT-PCR analysis of IGF2 ( A ) and IGFBP2 ( B ). Adult hMSCs were treated with the MEK inhibitor U0126 (10 μM), or the PI3K inhibitor wortmannin (50 nM) for 24 hours and IGF2 and IGFBP2 mRNA levels were determined by quantitative RT-PCR analysis ( C-D ). Results are expressed as mean ± SD of treated over control ratio after normalization to 18 S expression. *: significant difference with untreated cells ( P <0.05).

Article Snippet: Non relevant shRNA (scrambled sequence that does not lead to specific degradation of any known cellular mRNA), ITGB1 shRNA and human FAK shRNA lentiviral particles (mixtures of viral particles containing 3 target-specific constructs that encode shRNA designed to knock down gene expression) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Inhibition, Expressing, Transduction, Plasmid Preparation, Control, Transfection, shRNA, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

doi: 10.1093/nar/gkr198

Figure Lengend Snippet: Nef and HSF1 proteins are co-recruited on HSE of HSP40 promoter in HIV-1-infected cells. ( A ) HSF1 is required for Nef-mediated activation of HSP40 promoter. HSF1 and Sp1 depleted 293T cells were co-transfected with HSP40-luc and Nef expression vectors and analyzed for luciferase activity. Gene silencing efficiency of HSF1 and Sp1 siRNAs are shown as inset. ( B ) Both HSF1 and Nef activate HSP40 promoter-driven gene expression in 293T cells as analyzed by luciferase assay. ( C ) Schematic representation of HSP40 promoter showing the position of primers used in ChIP analysis. ( D ) Nef is recruited on HSP40 promoter during HIV-1 infection. ChIP analysis was performed with HIV-1-infected CEM-GFP cells using Nef antibody followed by PCR amplification using F2 and R1 primers. ( E ) Nef and HSF1 are co-recruited on HSP40 promoter as analyzed by sequential ChIP. Primary immunoprecipitation was performed with Nef and secondary immunoprecipitaion with HSF1 or Sp1 antibody followed by PCR analysis using F2 and R1 primers. ( F ) Nef and HSF1 is recruited specifically at HSE elements on HSP40 promoter. Sequential ChIP analysis was performed as in E above using either F1 and R2 or F2 and R1 primer sets. The error bars represent the mean ± SEM of three independent experiments. Statistical analysis was performed using Student's t- test, with the levels of significance defined as * P < 0.05.

Article Snippet: Control and Sp1 siRNA pools were from Santa Cruz Biotechnology, USA and HSF1 siRNA pool was obtained from Dharmacon, USA.

Techniques: Infection, Activation Assay, Transfection, Expressing, Luciferase, Activity Assay, Gene Expression, Amplification, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

doi: 10.1093/nar/gkr198

Figure Lengend Snippet: HIV-1 Nef physically interacts and co-localize with HSF1 both in vitro and in vivo. ( A ) HSF1 and Nef interact in 293T cells overexpressing HA-Nef and Flag-HSF1 as analyzed by co-immunoprecipitation. The input indicates lysate prepared from 293T cells co-transfected with HA-Nef and Flag-HSF1 expression vectors. ( B ) Nef co-immunoprecipitates with HSF1 in HIV-1-infected CEM-GFP cells. The input indicates lysate prepared from CEM-GFP cells infected with HIV-1. ( C ) Nef and Sp1 do not interact in HIV-1-infected CEM-GFP cells. The input indicates lysate prepared from CEM-GFP cells infected with HIV-1. ( D ) Proline-rich motif of Nef is important for interaction with HSF1. Lysates of HEK-293T cells expressing different HA-tagged Nef and its mutants were used for His-pull down with purified His-tagged HSF1. The input indicates the lysates prepared from wild-type and mutant Nef transfected 293T cells. ( E ) Nef and HSF1 co-localize in Nef and HSF1 expressing cells. Immunofluorescence studies were performed with 293T cells transfected with Nef and HSF1 vectors (left column), pNL4-3 transfected 293T cells (middle column) and HIV-1-infected Jurkat cells using Nef and HSF1 antibody (right column). The bottom panel in each column is a magnified image of one cell from the merged image panel. Arrows indicate co-localization of Nef and HSF1.

Article Snippet: Control and Sp1 siRNA pools were from Santa Cruz Biotechnology, USA and HSF1 siRNA pool was obtained from Dharmacon, USA.

Techniques: In Vitro, In Vivo, Immunoprecipitation, Transfection, Expressing, Infection, Purification, Mutagenesis, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

doi: 10.1093/nar/gkr198

Figure Lengend Snippet: HSF1 overexpression increases HIV-1 replication whereas it's silencing leads to inhibition of HIV-1 replication. ( A ) HSF1 overexpression increases virus production. Culture supernatants of 293T cells transfected with pNL4-3 and HSF1 vectors were analyzed for virus production using p24 antigen capture ELISA. HSF1 overexpression is shown in the inset ( B ) HSF1 overexpression leads to increased virus production in HIV-1 NL4-3-infected Jurkat cells. ( C ) HSF1 downregulation reduces HIV-1 virus production. Culture supernatants from 293T cells co-transfected with pNL4-3 and increasing concentrations of HSF1 siRNA were analyzed for virus production using p24 ELISA. Efficiency of gene silencing was checked by RT–PCR (inset). ( D ) HSF1 silencing leads to inhibition of virus production in HIV-1-infected Jurkat cells. Jurkat cells were first transfected with HSF1 siRNA followed by infection with NL4-3 virus. Cells were analyzed for HSF1 silencing by RT–PCR as shown in (inset) and culture supernatant was used for p24 ELISA. The error bars represent the mean ± SEM of two independent experiments. Statistical analysis was performed using Student's t- test, with the levels of significance defined as * P < 0.05.

Article Snippet: Control and Sp1 siRNA pools were from Santa Cruz Biotechnology, USA and HSF1 siRNA pool was obtained from Dharmacon, USA.

Techniques: Over Expression, Inhibition, Virus, Transfection, Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

doi: 10.2147/ijn.s63717

Figure Lengend Snippet: Figure 4 Confocal laser scanning microscopy images of different cell lines and different transfection time in HUVECs. Notes: (A) Different cell lines after incubation with the RPM/siRNA complexes for 6 hours. (B) The cellular uptake and localization of RPM/siRNA complexes in HUVECs at 1 or 6 hours after transfection. Cell nuclei were counterstained with DAPI (blue) and siRNA was labeled with Cy5 (red). Scale bar is 20 μm. Abbreviations: Cy5, indodicarbocyanine-5; DAPI, 4′,6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; RPM, cyclo(Arg-Gly-Asp-d-Phe-Lys)-8- amino-3,6-dioxaoctanoic acid-β-maleimidopropionic acid; siRNA, small interfering RNA.

Article Snippet: Samples were collected at different time points, and electrophoresed in a 2% agarose gel. cell culture Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution.

Techniques: Confocal Laser Scanning Microscopy, Transfection, Incubation, Labeling, Small Interfering RNA

Journal: International Journal of Nanomedicine

Article Title: Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

doi: 10.2147/ijn.s63717

Figure Lengend Snippet: Figure 3 Confocal laser scanning microscopy images of HUVECs that were transfected with different RPM/siRNA complexes. Notes: (A) Confocal laser scanning microscopy images of HUVECs after 6 hours’ incubation of RPM/siRNA complexes which contain different volume ratio between RPM and siRNA. (B) High-definition DIC images of HUVECs when the volume ratio between RPM and siRNA is 1.5:1. Cell nuclei were counterstained with DAPI (blue) and siRNA was labeled with Cy5 (red). Scale bar is 20 μm. Abbreviations: Cy5, indodicarbocyanine-5; DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast; HUVECs, human umbilical vein endothelial cells; RPM, cyclo(Arg-Gly-Asp-d-Phe-Lys)-8-amino-3,6-dioxaoctanoic acid-β-maleimidopropionic acid; siRNA, small interfering RNA.

Article Snippet: Samples were collected at different time points, and electrophoresed in a 2% agarose gel. cell culture Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution.

Techniques: Confocal Laser Scanning Microscopy, Transfection, Incubation, Labeling, Small Interfering RNA

Journal: International Journal of Nanomedicine

Article Title: Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

doi: 10.2147/ijn.s63717

Figure Lengend Snippet: Figure 6 Cytotoxicity and gene silencing in vitro. Notes: (A) Cell viability by CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, People’s Republic of China). (B) RT-qPCR analysis of VEGFR2 mRNA levels expressed in HUVECs after a 48-hour transfection (100 nM siRNA:siVEGFR2). All qPCR experiments were done in triplicate and VEGFR2 mRNA expression was normalized to the expression of GAPDH. (C) Western blot analysis of VEGFR2 protein and GAPDH protein expressed in HUVECs after a 72-hour transfection (100 nM siRNA:VEGFR2). Three independent experiments were performed. (D) Quantitative analysis of VEGFR2 protein expression levels. The expression of targeted protein was relative to the expression of GAPDH protein. *P0.05, **P0.01, compared with untreated; #P0.05, ##P0.01, compared with RPM; &&P0.01, compared with RPM/control siRNA;

Article Snippet: Samples were collected at different time points, and electrophoresed in a 2% agarose gel. cell culture Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution.

Techniques: In Vitro, CCK-8 Assay, Quantitative RT-PCR, Transfection, Expressing, Western Blot, Control

Journal: International Journal of Nanomedicine

Article Title: Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

doi: 10.2147/ijn.s63717

Figure Lengend Snippet: Figure 5 Confocal laser scanning microscopy images of HUVECs that were treated with different ways. Notes: The HUVECs were incubated with anti-integrin αvβ3 antibody for 30 minutes prior to transfection with RPM/siRNA for 6 hours (A) or incubated with the RAPM/ siRNA complexes for 6 hours (B). Cell nuclei were counterstained with DAPI (blue) and siRNA was labeled with Cy5 (red). Scale bar is 20 μm. Abbreviations: c(RADfk), cyclo(Arg-Ala-Asp-d-Phe-Lys); c(RGDfk), cyclo(Arg-Gly-Asp-d-Phe-Lys); Cy5, indodicarbocyanine-5; DAPI, 4′,6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; MAL, β-maleimidopropionic acid; PEG, 8-amino-3,6-dioxaoctanoic acid; RAPM, c(RADfk)-PEG-MAL; RPM, c(RGDfk)-PEG- MAL; siRNA, small interfering RNA.

Article Snippet: Samples were collected at different time points, and electrophoresed in a 2% agarose gel. cell culture Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution.

Techniques: Confocal Laser Scanning Microscopy, Incubation, Transfection, Labeling, Small Interfering RNA

Journal: Nature neuroscience

Article Title: A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

doi: 10.1038/nn.4584

Figure Lengend Snippet: Figure 1 EGFR inhibition triggers an adaptive response in glioma cells. (a) Western blot showing EGFR levels in established GBM cell lines and patient- derived primary GBM neurospheres. U87 vector indicates U87MG cells transfected with an empty vector. U251 is an established GBM cell line. β-actin was used as a loading control. (b) Patient-derived primary GBM neurospheres (GBM9) were exposed to erlotinib (1 µM) for the indicated times followed by western blot with the indicated antibodies; pMet, pAxl, pERK, pEGFR, pSTAT3 and pAkt specifically detect phosphorylated isoforms. (c,d) A similar experiment in GBM neurospheres derived from two different patients (GBM39 and SK987). (e) U87EGFR cells were treated with erlotinib (1 µM) for the indicated times followed by western blot with the indicated antibodies. (f) A similar experiment was conducted in U87EGFRvIII cells. (g–j) Axl was inhibited using the specific inhibitor R428 (1 µM). Cells were exposed to erlotinib followed by western blot. Erlotinib-induced ERK activation is inhibited when the Axl inhibitor is used in both established GBM cell lines and patient-derived neurospheres. (k–n) siRNA knockdown of Axl results in an inhibition of erlotinib-induced ERK activation in both established cell lines and patient-derived neurospheres. Control siRNA or Axl siRNA was transfected into cells (for 48 h), followed by addition of erlotinib for 48 h and western blot with indicated antibodies. Western blots shown in a–n are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 11.

Article Snippet: For transient silencing we used a pool of siRNA sequences directed against human TNFR1, Axl, JNK1, JNK2 or control (scrambled), obtained from Santa Cruz. siRNAs were introduced into cells using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Derivative Assay, Plasmid Preparation, Transfection, Control, Activation Assay, Knockdown

Journal: Nature neuroscience

Article Title: A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

doi: 10.1038/nn.4584

Figure Lengend Snippet: Figure 2 EGFR inhibition–induced Axl and ERK activation is mediated by JNK. (a,b) Patient-derived primary GBM neurospheres were exposed to erlotinib for 48 h in the presence or absence of the JNK inhibitor SP600125 (1 µM) or p38 inhibitor SB203580 (10 µM), followed by western blot with the indicated antibodies. DMSO indicates vehicle alone. (c,d) U87EGFRwt or U87EGFRvIII cells were exposed to erlotinib for 48 h in the presence or absence of SP600125 or SB203580, followed by western blot with the indicated antibodies. (e,f) siRNA knockdown for JNK1 and JNK2 (siJNK) was conducted in GBM9 and GBM39 neurospheres, followed by exposure to erlotinib for 48 h and western blot with the indicated antibodies. siCtrl indicates scrambled control siRNA. (g) A similar experiment was done in U87EGFRwt cells. (h–k) JNK is activated in response to erlotinib in patient- derived primary neurospheres, as well as in established GBM cell lines, as determined by the phosphorylation of JNK. Western blots shown in a–k are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 12. (l) A luciferase reporter assay shows that EGFR inhibition with erlotinib results in an increase in AP-1 transcriptional activity in GBM9 and U87EGFRwt cells. Erlotinib was used for 24 h (1 µM). DMSO was used as a control (Ctrl). GBM9: Ctrl versus erlotinib: P = 0.0056, t = 5.43, d.f. = 4, **P < 0.01; U87EGFRwt: Ctrl versus erlotinib: P = 0.0061, t = 5.31, d.f. = 4, **P < 0.01. Data are presented as mean ± s.e.m. Significant difference analyzed by an unpaired Student’s t-test (n = 3 biologically independent experimental replicates).

Article Snippet: For transient silencing we used a pool of siRNA sequences directed against human TNFR1, Axl, JNK1, JNK2 or control (scrambled), obtained from Santa Cruz. siRNAs were introduced into cells using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Inhibition, Activation Assay, Derivative Assay, Western Blot, Knockdown, Control, Phospho-proteomics, Luciferase, Reporter Assay, Activity Assay

Journal: Nature neuroscience

Article Title: A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

doi: 10.1038/nn.4584

Figure Lengend Snippet: Figure 4 EGFR inhibition leads to increased TNF signaling that triggers an adaptive signaling pathway. (a,b) EGFR inhibition leads to an increase in TNF mRNA in patient-derived GBM9 and GBM39 neurospheres. Cells were exposed to erlotinib (100 nM) for the times indicated followed by real-time quantitative PCR for TNF mRNA. (a) 0 versus 24 h: P = 0.0019, t = 7.22, d.f. = 4. (b) 0 versus 4 h: P = 0.0102, t = 4.58, d.f. = 4; 0 versus 24 h: P = 0.0021, t = 7.10, d.f. = 4. (c,d) A similar experiment was conducted in U87EGFRwt and U87EGFRvIII cells using an erlotinib concentration of 1 µM. (c) 0 versus 4 h: P = 0.0018, t = 7.41, d.f. = 4; 0 versus 24 h: P = 0.0012, t = 8.20, d.f. = 4. (d) 0 versus 4 h: P = 0.0030, t = 6.46, d.f. = 4; 0 versus 24 h: P = 0.0054, t = 5.47, d.f. = 4. (e) A TNF ELISA was performed on supernatants from erlotinib treated U87EGFRwt and U87EGFRvIII cells (1 µM) and GBM9 and GBM39 neurospheres (100 nM). U87EGFRwt: 0 versus 24 h: P = 0.0056, t = 5.42, d.f. = 4; 0 versus 48 h: P = 0.0006, t = 10.4, d.f. = 4; U87EGFRVIII: 0 versus 24 h: P = 0.0022, t = 6.98, d.f. = 4; 0 versus 48 h: P = 0.0083, t = 4.86, d.f. = 4; GBM9: 0 versus 24 h: P = 0.01, t = 4.6, d.f. = 4; 0 versus 48 h: P = 0.0043, t = 5.84, d.f. = 4; GBM39: 0 versus 24 h: P = 0.0189, t = 3.82, d.f. = 4; 0 versus 48 h: P = 0.0024, t = 6.81, d.f. = 4. Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-test (n = 3 biologically independent experimental replicates). (f) Time course of TNF upregulation in mouse tumors exposed to erlotinib 50 mg/kg for the indicated time points after formation of subcutaneous tumors (n = 3). Tumors were removed after erlotinib exposure, followed by TNF ELISA on protein extracts. 0 versus 1 d: P = 0.0045, t = 5.77, d.f. = 4; 0 versus 2 d: P = 0.0002, t = 13.92, d.f. = 4; 0 versus 7 d: P = 0.0245, t = 3.52, d.f. = 4. Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001 from a two-tailed unpaired Student’s t-test. (g) Signal transduction in tumors exposed to erlotinib (50 mg/kg) for the indicated time points. (h) A neutralizing antibody to TNF (TNF Ab) (2 µg/ml) blocked erlotinib-induced activation of Axl, ERK and JNK in GBM9 and GBM39 neurospheres and U87EGFRwt and U87EGFRvIII cell lines, while control antibody (Ctrl Ab) had no effect. The control antibody was normal mouse IgG. (i) siRNA knockdown of TNFR1 (siTNFR1) blocked erlotinib-induced activation of Axl, ERK and JNK in GBM9 and GBM39 neurospheres and in U87EGFRwt and U87EGFRvIII cell lines, while control (scrambled) siRNA (siCtrl) had no effect. Western blots shown in g–i are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 13.

Article Snippet: For transient silencing we used a pool of siRNA sequences directed against human TNFR1, Axl, JNK1, JNK2 or control (scrambled), obtained from Santa Cruz. siRNAs were introduced into cells using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Inhibition, Derivative Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transduction, Activation Assay, Control, Knockdown, Western Blot

Journal: Nature neuroscience

Article Title: A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

doi: 10.1038/nn.4584

Figure Lengend Snippet: Figure 6 TNF inhibition sensitizes glioma cells to EGFR inhibition. (a,b) AlamarBlue cell viability assay in GBM9 or GBM39 neurospheres. Etanercept (100 nM) sensitizes cells to EGFR inhibition with erlotinib. Etanercept and erlotinib were added to GBM9 or GBM39 neurospheres concurrently and AlamarBlue assay was done after 72 h. DMSO was used as a control. (a) Erlotinib versus erlotinib + etanercept: P = 0.0027, t = 6.59, d.f. = 4. (b) Erlotinib versus erlotinib + etanercept: P = 0.0044, t = 6.59, d.f. = 4. (c) A similar experiment was performed in U87EGFRwt cells. Erlotinib versus erlotinib + etanercept: P = 0.0056, t = 5.41, d.f. = 4. (d,e) TNFR1 was silenced using siRNA (siTNFR1) in GBM9 and GBM39 cells and cells were exposed to erlotinib for 72 h in stem cell medium without EGF for 72 h, followed by AlamarBlue assay. (d) Erlotinib + scrambled control siRNA (siCtrl) versus erlotinib + siTNFR1: P = 0.0014, t = 7.95, d.f. = 4. (e) Erlotinib + siCtrl versus erlotinib + siTNFR1: P = 0.0041, t = 5.90, d.f. = 4. (f) A similar experiment was done in U87EGFRwt cells. Erlotinib + siCtrl versus erlotinib + siTNFR1: P = 0.0021, t = 7.11, d.f. = 4. (g–i) Thalidomide sensitizes GBM9 and GBM39 cells to EGFR inhibition with erlotinib. Thalidomide (1 µM) and erlotinib were added to GBM9 and GBM39 neurospheres (100 nM) or U87EGFRwt cells (1 µM) concurrently and AlamarBlue assay was done after 72 h. (g) Erlotinib versus erlotinib + thalidomide: P = 0.0030, t = 6.42, d.f. = 4. (h) Erlotinib versus erlotinib + thalidomide: P = 0.0027, t = 6.59, d.f. = 4. (i) Erlotinib versus erlotinib + thalidomide: P = 0.0013, t = 8.11, d.f. = 4. (j,k) Etanercept or thalidomide blocks erlotinib-induced activation of JNK, Axl and ERK in GBM9 and GBM39 neurospheres, as shown by western blot. Control antibody (Ctrl Ab) is normal mouse IgG. (l) A similar experiment was conducted in U87EGFRwt cells. Western blots shown in j–l are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 14. (m,n) Exogenous TNF protects GBM9 and GBM39 neurospheres from erlotinib-induced cell death. TNF (1 ng/ml) and erlotinib (1 µM) were added to cells concurrently and AlamarBlue cell viability assay was done after 72 h. (m) Erlotinib versus erlotinib + TNF: P = 0.0018, t = 7.41, d.f. = 4. (n) Erlotinib versus erlotinib + TNF: P = 0.0087, t = 4.79, d.f. = 4. Data are presented as mean ± s.e.m.; **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-test (n = 3 biologically independent experimental replicates).

Article Snippet: For transient silencing we used a pool of siRNA sequences directed against human TNFR1, Axl, JNK1, JNK2 or control (scrambled), obtained from Santa Cruz. siRNAs were introduced into cells using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Inhibition, Viability Assay, Alamar Blue Assay, Control, Activation Assay, Western Blot, Two Tailed Test

Journal: Annals of Translational Medicine

Article Title: B3GNT3 as a prognostic biomarker and correlation with immune cell infiltration in lung adenocarcinoma

doi: 10.21037/atm-22-493

Figure Lengend Snippet: Knockdown of B3GNT3 inhibited the proliferation, cloning and invasion of LUAD cells. (A) qPCR shows the relative levels of B3GNT3 after transfection of siRNA. (B) 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) analysis of the proliferation of A549 and PC9 cells after 24, 48 and 72 h. (C) Colony formation assay of the cloning ability of A549 and PC9 cells (crystal violet staining, 10×). (D) Transwell cell assays demonstrate cell invasion with crystal violet in siB3GNT3-transfected A549 and PC9 cells compared with non-specific scramble (scr) siRNA as a control group, 10×. siRNA, small interfering RNA. *, P<0.05; **, P<0.01; ***, P<0.001. LUAD, lung adenocarcinoma; qPCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: Cell culture LUAD cell lines (A549 and PC9) and human bronchial epithelial cell line BEAS-2B were purchased from the American Type Culture Collection.

Techniques: Knockdown, Cloning, Transfection, Colony Assay, Staining, Control, Small Interfering RNA, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: De-ubiquitinating enzyme, USP11, promotes transforming growth factor β -1 signaling through stabilization of transforming growth factor β receptor II

doi: 10.1038/cddis.2016.371

Figure Lengend Snippet: USP11 regulates TGF β -1 signaling pathway. ( a ) MRC5 cells were infected with cont shRNA (−) or USP11 shRNA lentivirus for 3 days as described in the Materials and Methods section, and then cells were treated with TGF β -1 (2 ng/ml) for 30 min. USP11 and phosphorylated and total SMAD2/3 levels were analyzed by western blotting. ( b ) MRC5 cells were transfected with cont siRNA (−) or USP11 siRNA for 3 days, and then cells were treated with TGF β -1 (2 ng/ml) for 30 min. USP11, total, and phosphorylated SMAD2/3 levels were analyzed by western blotting. ( c ) MRC5 cells were infected with cont shRNA (−) or USP11 shRNA lentivirus for 3 days, and then cells were treated with TGF β -1 (2 ng/ml) for 20 h. USP11, FN, SMA, and β -actin protein levels were analyzed by western blotting. ( d ) MRC5 cells were transfected with USP11-HA plasmids (0–4 μ g) for 2 days, and then cells were treated with TGF β -1 (2 ng/ml) for 30 min. USP11-HA, phosphorylated and total SMAD2/3, and β -actin levels were analyzed by western blotting. ( e ) MRC5 cells were transfected with USP11-HA plasmids (2 μ g) for 2 days, and then cells were treated with TGF β -1 (2 ng/ml) for 20 h. USP11-HA, FN, SMA, and β -actin protein levels were analyzed by western blotting. Western blotting images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. Representative of experiments performed at least three independent times

Article Snippet: Immobilized protein A/G beads, FN, α-SMA, T β RI, HA tag, USP11, V5 tag, T β RII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, shRNA, Western Blot, Transfection, Derivative Assay

Journal: Cell Death & Disease

Article Title: De-ubiquitinating enzyme, USP11, promotes transforming growth factor β -1 signaling through stabilization of transforming growth factor β receptor II

doi: 10.1038/cddis.2016.371

Figure Lengend Snippet: USP11 regulates T β RII stability in human lung fibroblast cells. ( a ) MRC5 cells were infected with cont shRNA (−) or USP11 shRNA lentivirus for 3 days, and then USP11, T β RII, and β -actin levels were analyzed by western blotting. ( b ) MRC5 cells were infected with cont shRNA (−) or USP11 shRNA lentivirus for 3 days, and then total RNA was extracted. Tgfbr2 and Usp11 gene expression were examined by RT-real time PCR. ( c ) MRC5 cells were co-transfected with T β RII-V5 and empty vector or USP11-HA plasmids for 48 h, and then cells were treated with CHX (20 μ g/ml) for 0–4 h. T β RII-V5, USP11-HA, and β -actin levels were analyzed by western blotting. Intensities of T β RII-V5 were analyzed by the ImageJ software and then compared between the two groups. ( d ) MRC5 cell lysates were subjected to immunoprecipitation with IgG or a T β RII antibody, followed by USP11 and T β RI immunoblotting. Input lysates were analyzed by USP11, T β RI, and T β RII immunoblotting. ( e ) MRC5 cells grown on glass-bottom dishes were co-transfected with USP11-HA and T β RII-V5 plasmids for 48 h. Localization of USP11-HA (green) and T β RII-V5 (red) in MRC5 cells were examined by immunostaining. Nuclei were stained by DAPI (4,6-diamidino-2-phenylindole; blue). USP11-HA and T β RII-V5 co-localization on the plasma membrane are indicated by white arrows; two protein co-localization in the cytoplasm are indicated by black arrows. Bars, 50 μ m. Representative images are shown. ( f ) MRC5 cells were transfected with plasmids encoding T β RII-V5 full-length (FL), C571, C586 deletion mutants, or K205R mutants for 48 h. Cell lysates were subjected to immunoprecipitation with a V5 antibody, followed by USP11 immunoblotting. Input lysates were analyzed by USP11 and V5 immunoblotting. Western blotting images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. Representative of experiments performed at least three independent times

Article Snippet: Immobilized protein A/G beads, FN, α-SMA, T β RI, HA tag, USP11, V5 tag, T β RII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, shRNA, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Software, Immunoprecipitation, Immunostaining, Staining, Clinical Proteomics, Membrane, Derivative Assay

Journal: Cell Death & Disease

Article Title: De-ubiquitinating enzyme, USP11, promotes transforming growth factor β -1 signaling through stabilization of transforming growth factor β receptor II

doi: 10.1038/cddis.2016.371

Figure Lengend Snippet: USP11 de-ubiquitinates T β RII. ( a ) MRC5 cells were infected with cont shRNA or USP11 shRNA lentivirus for 3 days. Cell lysates were subjected to immunoprecipitation with a T β RII antibody, followed by ubiquitin immunoblotting. Input lysates were analyzed by T β RII, USP11, and β -actin immunoblotting. ( b ) MRC5 cells were co-transfected with T β RII-V5 and empty vector or USP11-HA plasmids for 48 h, and then cell lysates were subjected to immunoprecipitation with a V5 antibody, followed by ubiquitin immunoblotting. Input lysates were analyzed by HA, V5, and β -actin immunoblotting. Western blotting images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. Representative of experiments performed at least three independent times

Article Snippet: Immobilized protein A/G beads, FN, α-SMA, T β RI, HA tag, USP11, V5 tag, T β RII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, shRNA, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Derivative Assay

Journal: Cell Death & Disease

Article Title: De-ubiquitinating enzyme, USP11, promotes transforming growth factor β -1 signaling through stabilization of transforming growth factor β receptor II

doi: 10.1038/cddis.2016.371

Figure Lengend Snippet: T β RII and USP11 are increased in the lungs from bleomycin-induced fibrosis model and IPF patients ( a ) C57BL/6 mice were challenged with intranasal injection of bleomycin for 3 weeks. P-SMAD2, T β RII, USP11, and β -actin levels were analyzed by western blotting. ( b ) Intensities of T β RII and USP11 were analyzed by the ImageJ software. ( c ) Human normal and IPF lung tissues were fixed and immunostained with T β RII and USP11 antibodies. Scale bars in × 10 image, 400 μ m; scale bars in × 60 images: 50 μ m. Representative images (from three per each group) are shown

Article Snippet: Immobilized protein A/G beads, FN, α-SMA, T β RI, HA tag, USP11, V5 tag, T β RII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Injection, Western Blot, Software

Journal: Cell Death & Disease

Article Title: De-ubiquitinating enzyme, USP11, promotes transforming growth factor β -1 signaling through stabilization of transforming growth factor β receptor II

doi: 10.1038/cddis.2016.371

Figure Lengend Snippet: USP11 de-ubiquitinates and stabilizes T β RII. T β RII stability is regulated by its poly-ubiquitination. Destabilization of T β RII attenuates TGF β -1 signaling. USP11 stabilizes T β RII through de-ubiquitination of T β RII. MTX, an inhibitor of USP11, promotes ubiquitination and degradation of T β RII, thus mitigating TGF β -1 signaling

Article Snippet: Immobilized protein A/G beads, FN, α-SMA, T β RI, HA tag, USP11, V5 tag, T β RII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Ubiquitin Proteomics

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: List of PCR primers used in the study

Article Snippet: Control siRNA and mouse LRP1 siRNA pool (CGCUGACCCUAUUUGAAGAtt, UCUUCAAAUAGGGUCAGCGtt; CCUUCAGCAUCGAUGUGUUtt,AACACAUCGAUGCUGAAGGtt; CUACCUACAAGAUGUAUGAtt, UCAUACAUCUUGUAGGUAGtt)were purchased from Santa Cruz Biotechnology. siRNA transfection of BV-2 microglial cells was performed (Lipofectamine TM 2000; Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) promoted microglial migration through low-density lipoprotein receptor-related protein (LRP)1. (A, B) BV-2 microglial cells were transiently transfected with control small interfering (si)RNA or LRP1-specific siRNA. After 48 hours, a scratch wound was made. Cells were treated with or without mouse PAI-1 protein (100 ng/ml), followed by (A) the wound-healing assay and (B) the Boyden chamber assay, as described in Figure . Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, NS = not significant (lower panel). (Upper panels) Representative images of each assay. (C, D) Knockdown of LRP1 gene expression by siRNA was confirmed by using (C) reverse transcriptase PCR, (D) dot blotting (upper panel), and western blotting (lower panel). β-actin and the α-tubulin were used as internal controls. (E) BV-2 microglial cells were treated with mouse PAI-1 protein (100 ng/ml) and RAP protein (5 μg/ml) as indicated. The fold increase in migration distance was measured using the wound-healing assay. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control (lower panel). (Upper panel) Representative images also shown.

Article Snippet: Control siRNA and mouse LRP1 siRNA pool (CGCUGACCCUAUUUGAAGAtt, UCUUCAAAUAGGGUCAGCGtt; CCUUCAGCAUCGAUGUGUUtt,AACACAUCGAUGCUGAAGGtt; CUACCUACAAGAUGUAUGAtt, UCAUACAUCUUGUAGGUAGtt)were purchased from Santa Cruz Biotechnology. siRNA transfection of BV-2 microglial cells was performed (Lipofectamine TM 2000; Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Migration, Transfection, Control, Wound Healing Assay, Boyden Chamber Assay, Knockdown, Gene Expression, Reverse Transcription, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-1 was involved in the plasminogen activator inhibitor type 1 (PAI-1)-enhanced microglial motility. (A) BV-2 microglial cells were treated with mouse PAI-1 protein (100 ng/ml) or interferon (IFN)-γ (50 U/ml), and cell lysates were collected at 30 minutes after the treatment. The levels of phosphorylated STAT1 (pSTAT1 at Tyr701) or total STAT1 protein were then evaluated by western blotting analysis. Ponceau S staining was performed to confirm the equal loading of the samples. (B) BV-2 microglial cells were transfected with control small interfering (si)RNA or low-density lipoprotein receptor-related protein (LRP)1 siRNA. The cells were harvested 48 hours after transfection and used for the experiments. Cells were treated with mouse PAI-1 protein (100 ng/ml) for 30 minutes. Phosphorylated STAT1 or total STAT1 was measured by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. Values indicate the results of densitometric quantification normalized to α-tubulin. (C) BV-2 microglial cells were transfected with control siRNA or LRP1 siRNA. The cells were harvested at 48 hours after transfection and then treated with mouse IFN-γ (50 U/ml) for 30 minutes. Phosphorylated STAT1 was detected by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. (D) BV-2 microglial cells were pretreated with AG490 (JAK-specific inhibitor; 20 μmol/l) for 30 minutes before the treatment with mouse PAI-1 protein (100 ng/ml), and then cell migration was evaluated by the wound-healing assay. Results are mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control (lower panel). Representative images are shown (upper panel; original magnification × 150).

Article Snippet: Control siRNA and mouse LRP1 siRNA pool (CGCUGACCCUAUUUGAAGAtt, UCUUCAAAUAGGGUCAGCGtt; CCUUCAGCAUCGAUGUGUUtt,AACACAUCGAUGCUGAAGGtt; CUACCUACAAGAUGUAUGAtt, UCAUACAUCUUGUAGGUAGtt)were purchased from Santa Cruz Biotechnology. siRNA transfection of BV-2 microglial cells was performed (Lipofectamine TM 2000; Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Western Blot, Staining, Transfection, Control, Migration, Wound Healing Assay

Journal: Oncology Letters

Article Title: MKP-1 overexpression is associated with chemoresistance in bladder cancer via the MAPK pathway

doi: 10.3892/ol.2020.11741

Figure Lengend Snippet: MKP-1 and MAPK expression in RT112 cells transfected with NC and MKP-1 siRNA. (A) Relative MKP-1 expression in the siNC and siMKP-1 groups was examined using reverse transcription-quantitative PCR. **P<0.01. (B) Representative microscopic images of siNC and siMKP-1 treated cells captured in both 2D and 3D environments under a phase contrast microscope. (C) MKP-1 expression of the siNC and siMKP-1 groups, as determined via western blotting. GAPDH was used as the internal control. (D) Phosphorylated and total ERK1/2 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (E) Phosphorylated and total p38 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (F) Phosphorylated and total JNK protein expression levels of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. MKP-1, mitogen activated protein kinase phosphatase-1; NC, negative control; siMKP-1, MKP-1 small interfering RNA; siNC, small interfering negative control.

Article Snippet: 3D Cell Culture Gel (cat. no. P720M-10) was purchased from Col-Tgel Med ( http://www.101bio.com/P720_3D_cell_culture_gel.php ).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Microscopy, Western Blot, Negative Control, Small Interfering RNA

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: Nucleolar stress induces PICT1 degradation in various cell lines. HeLa, MCF-7, H1299, U2OS, HL-60, HuH-7, PC-3, and HEK293 cells were treated with 1 mm FUrd for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures” (A, C, and E). The calculated PICT1/ACTB value of each control (0 h) was set to 100%, and normalized values are presented in B, D, and F. A and B, HeLa, MCF-7, H1299, and U2OS; C and D, HL-60 and HuH-7; E and F, PC-3 and HEK293.

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: Proteasome inhibitors, but not lysosome inhibitors, block nucleolar stress-induced PICT1 degradation in H1299 cells. H1299 cells were treated with proteasome (A) or lysosome (B) inhibitors for 5 min, followed by the addition of the indicated stress inducers. After incubation for 6 h, cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures.” The calculated PICT1/ACTB value of each control experiment was set to 100%, and normalized values are presented as average ± range of duplicate experiments (A). MG132, 10 μm MG132; Epoxo, 1 μm epoxomicin; Lacta, 1 μm lactacystin; CA-074, 20 μm CA-074Me; PepA, 25 μm pepstatin A; E64d, 30 μm E64d; ActD, 5 nm actinomycin D; Doxo, 1 μm doxorubicin; FUrd, 1 mm FUrd.

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Blocking Assay, Incubation, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: Nucleolar stress does not alter PICT1 ubiquitination. A and B, HeLa cells grown on 6-well plates were transfected with 1.5 μg of pMT107 (His-Ub, +) or pCMV5 (His-Ub, −) in combination with 3.5 μg of Myc-PICT1/pCAGGS (Myc-PICT1, +) or pCAGGS (Myc-PICT1, −). After incubation for 2 day, the cells were treated for 7 h with 10 μm MG132 or vehicle (DMSO). Ubiquitinated proteins were pulled down with Ni2+-NTA beads and subjected to immunoblot analysis. C, H1299 cells grown on 6-cm dishes were transfected with 4 μg each of pMT107 and pEF1 (His-Ub) or 4 μg each of pMT107 and Myc-PICT1/pEF1 (His-Ub+Myc-PICT1). After a 4-h incubation, the cells were trypsinized and plated onto four wells of 6-well plate, and incubated for 1 day. The cells were treated for 6 h with 1 mm FUrd and/or 1 μm epoxomicin (Epoxo). Ubiquitinated proteins were pulled down with Ni2+-NTA beads and subjected to immunoblot analysis as described under “Experimental Procedures.” D, HeLa cells grown on 6-cm dishes were transfected with 5 μg of pMT107 (His-Ub, +) or pcDNA3 (His-Ub, −) in combination with 5 μg of FLAG-PTEN(T382A/T383A)/pCMV5 (PTEN(TA/TA), +) or pCMV5 (PTEN(TA/TA), −). After incubation for 2 days, the cells were treated for 8 h with 10 μm MG132 or vehicle (DMSO). Ubiquitinated proteins were pulled down with Ni2+-NTA beads and subjected to immunoblot (IB) analysis.

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: Pharmacological and genetic inhibition of the E1 enzyme does not affect nucleolar stress-induced PICT1 degradation in H1299 cells. A, H1299 cells were treated with 1 μm epoxomicin (Epoxo), 10 μg/ml Pyr41, or vehicle (DMSO) for 5 min, followed by the addition of 1 mm FUrd, 5 nm ActD, DMSO, or no addition (−). After incubation for 6 h, cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures.” B–D, HeLa cells were transfected with indicated siRNAs using Lipofectamine RNAi Max according to the manufacturer's protocol. B, synthesized siRNAs with target sequence. C, after a 72-h incubation, RNAs were extracted, and the expression of UBE1, UBA6, and ACTB mRNAs was analyzed by RT-PCR. Primers used are represented. D, cells were also treated for the indicated times with 5 ng/ml ActD. Cell lysates were then subjected to immunoblot analysis as described under “Experimental Procedures.”

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Inhibition, Incubation, Western Blot, Transfection, Synthesized, Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: PICT1 deletion mutant (d342–449) is resistant to nucleolar stress-induced degradation and shows nuclear localization. A, H1299 cells were transfected with Myc-PICT1/pEF1 (WT) or Myc-PICT1(d342–449)/pEF1 (d342–449). After a 2-day incubation, intracellular localization of ectopically expressed Myc-tagged PICT1 protein was analyzed by immunofluorescence assay as described under “Experimental Procedures.” One hundred cells were analyzed for each sample, and localization patterns of Myc-tagged PICT1 are presented in the right panel. No, nucleolar; No/N, nucleolar and nuclear; N, nuclear; N/C, nuclear and cytosolic; C, cytosolic. B, H1299/Myc-PICT1(d342–449) cells were treated with 10 ng/ml doxycycline for 24 h to induce expression of PICT1(d342–449) mutant protein in cells. The cells were treated with 1 mm FUrd for the indicated times, and cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures.” The calculated PICT1/ACTB value of each control (0 h) was set to 100%, and normalized values are presented in the right panel. PICT1(d342–449), blue; endogenous PICT1, red. C, comparable amount of immunopurified Myc-PICT1 proteins (WT and d342–449) were mixed and subjected to 20 S proteasome-mediated in vitro degradation assay. After incubation for the indicated times, Myc-tagged proteins and mouse IgG heavy chain (IgG-H) was detected by immunoblot analysis as described under “Experimental Procedures.”

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Mutagenesis, Transfection, Incubation, Immunofluorescence, Expressing, Western Blot, Control, In Vitro, Degradation Assay

Journal: The Journal of Biological Chemistry

Article Title: Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein *

doi: 10.1074/jbc.M114.571893

Figure Lengend Snippet: Nucleolar stress does not alter intracellular localization of PICT1. H1299 cells were treated for 6 h with 1 μm epoxomicin (Epoxo), 5 nm ActD, 1 mm FUrd, alone or together as indicated. Intracellular localization of endogenous PICT1 and nucleolin was analyzed by immunofluorescence assay as described under “Experimental Procedures.”

Article Snippet: Cell Culture H1299 (human non-small cell lung carcinoma, ATCC CRL-5803), HeLa (human cervical carcinoma, ATCC CCL-2), U2OS (human osteosarcoma, ATCC HTB-96), MCF-7 (human breast adenocarcinoma, ATCC HTB-22) cells, HuH-7 cells (human hepatoma, JCRB 0403), and HEK293 (human embryonic kidney, ATCC CRL-1573) were cultured at 37 °C in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Immunofluorescence

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 1 NUAK1 co-immunoprecipitates with p53. Equal amount of cell extracts from A549 cells were immunoprecipitated with anti-NUAK1 antibody or normal rabbit IgG as negative control and were western blotted with anti-p53 antibody. Fifty percent of protein before immunoprecipitation was kept for input and was subjected to western blotting with anti-NUAK1 and anti- LKB1 antibodies. Vec, LKB1 and KDM indicate that A549 cells were stably transfected with vector control, wild-type (WT) LKB1 and kinase-deficient LKB1, respectively; L þ s and L þ c indicate that cells that stably expressed WT LKB1 were transiently transfected with NUAK1 siRNA pool or control siRNA as a control. Glucose: cells were incubated in medium without glucose for 2 h; glucose þ : no glucose starvation treatment.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 2 NUAK1 directly phosphorylates p53. (a) In vitro phosphorylation of p53 by E. coli-produced and LKB1-activated NUAK1. In kinase buffer that contained (g-32P) ATP, His-p53 was incubated with His-NUAK1 (N), or His-NUAK1 incubated with active- LKB1 and then isolated (NL) or His-NUAK1 incubated with heat-inactivated active-LKB1 and then isolated (NiL). After separation by SDS–PAGE, proteins were transferred to PVDF membrane and detected by autoradiography. His-p53 and His-NUAK1 were also detected by anti–p53 and anti-NUAK1 antibodies. (b) In vitro phosphorylation of p53 by E. coli-produced mutants of NUAK1. His-p53 was incubated with His-NUAK1 (N), His-NUAK1 (T211E) (TE) or His-NUAK1 (T211D) (TD), and then detected by autoradiography. His-p53, His-NUAK1 and mutants were also examined by western blotting. (c) In vitro phosphorylation of p53 by NUAK1 and mutants produced in HEK293T cells. His-p53 was incubated with His-NUAK1 (N), T211A mutant (TA) or kinase-dead mutant K84A (KA) and then detected by autoradiography. Western blotting was the same as in (b). (d) Phosphorylation of p53 in Hep3B cells. Hep3B cells were stably transfected with vector control (Vec) or WT p53 (p53) or p53 and transiently expressed NUAK1 (p þ N) or p53 and NUAK1 with ATM siRNA pool (pNs) or p53 and NUAK1 with control siRNA (pNc). After incubated in glucose þ or glucose medium for 2 h, cells were lysed and subjected to western blotting with phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53, anti-NUAK1, anti-ATM and b-actin antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vitro, Phospho-proteomics, Produced, Incubation, Isolation, SDS Page, Membrane, Autoradiography, Western Blot, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Control

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 3 LKB1 activation of NUAK1 stimulates phosphorylation of p53. (a) LKB1-dependent p53 phosphorylation requires NUAK1. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) or WT LKB1 and transiently transfected with control siRNA (L þ c). Cells were incubated in glucose þ or glucose medium for 2 h. Western blotting was done using phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53 antibody, anti-NUAK1 antibody, anti-LKB1 antibody and b-actin antibody. (b) Requirement of NUAK1 kinase activity in LKB1-dependent p53 phosphorylation. A549 cells stably transfected with LKB1 ( þ ) or vector control () were transiently transfected with NUAK1 ( þ ), T211A (TA), kinase-dead mutant K84A (KA) or vector control (Vec), and treated under glucose starvation for 2 h. Cells were lysed and western blotting was performed as in (a). (c) In vivo phosphorylation assay of NUAK1 by LKB1. A549 cells that stably expressed WT LKB1 , vector control (Vec) or kinase-deficient LKB1 (KDM) were transiently transfected with WT NUAK1 or NUAK1 T211A mutation. Transfection was the same in (d), (e) and (f). The cells were subjected to glucose starvation for 2 h and incubated for 3 h with [32P] Pi (300 cpm/pmol; Furi). Cells were then lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The immunoprecipitates were separated by SDS–PAGE and subjected to autoradiography. (d) Cells were treated with 200 mM AMP. (e) In vitro kinase assay of NUAK1. After being subjected to glucose starvation for 2 h, cells were lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The in vitro kinase activity of immunoprecipitates was assayed by measuring the 32P labeling of SAMS peptide. One unit of activity was defined as 1 nmol SAMS peptide phosphorylated per minute. (f) Cells were treated with 200 mM AMP.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Activity Assay, Mutagenesis, In Vivo, Immunoprecipitation, SDS Page, Autoradiography, In Vitro, Kinase Assay, Labeling

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 4 Cell cycle arrest induced by LKB1/NUAK1 requires p53. (a) A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 ( þ ). Cells stably expressed WT LKB1 were also transiently transfected with ( þ ) or without () WT NUAK1, NUAK1 siRNA pool (siRNA) or control siRNA (Ctl-si). After synchronization, cells were treated with glucose medium. Cells were then harvested, stained with propidium iodide and analyzed by flow cytometry. Each analysis was carried out in triplicate and also in (b). (b) A549 cells were stably transfected with vector control (Vec) or WT LKB1 ( þ ), and transiently transfected with p53 ( þ ), vector control (Vec), p53 S15A mutant (S15A), p53 S392A mutant (S392A), p53 siRNA pool (siRNA) or control siRNA (Ctl-si). Cells were treated as in (a) and subjected to flow cytometry analysis.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Staining, Cytometry, Mutagenesis

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 5 p21/WAF1 transcriptional activity induced by LKB1/ NUAK1. (a) Quantitative RT–PCR analysis of p21/WAF1 transcription. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM), WT LKB1 (LKB1) and transiently transfected with NUAK1 siRNA pool (L þ s), or WT LKB1 and transiently transfected with control siRNA (L þ c). RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA and displayed as fold change relative to the value obtained from cells transfected with vector control, which was set at 1. (b) Western blotting of endogenous p21 protein. Transfection was the same as in (a).

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 6 NUAK1 interacts with p53 in the nucleus and binds to p21/WAF1 promoter. (a) Endogenous NUAK1 was present in the p53RE region of p21/WAF1 promoter. A549 cells were stably transfected with vector control (Vec), wild-type LKB1 (LKB1), kinase- deficient LKB1 (KDM), or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s). Cells were subjected to glucose starvation for 2 h. ChIP was done using anti-NUAK1 antibody and normal rabbit IgG was used as a negative control for the specificity of the immunoprecipitation. Cells transiently transfected with NUAK1 siRNA pool were included as a negative control. Quantitative PCR was done with the specific primers for p53RE of p21/WAF1 promoter and the bars represent normalized quantitative PCR values expressed as percentage of input. (b) ChIP assay of NUAK1 at p21/WAF1 TATA-50UTR region. (c) ChIP assay of p53 at p21/WAF1 promoter p53RE region. (d) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region. (e) The binding of NUAK1 to p53RE required wild-type p53. p53-null Hep3B cells were stably transfected with wild-type p53 (p53), p53 S15A mutant (S15A), p53 S392A mutant (S392A) or vector control (Vec). ChIP assay was done as described in A and p53-expressing cells transiently transfected with NUAK1 siRNA pool were included as a negative control (p þ s). (f) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region in Hep3B cells. p53-expressing cells transiently transfected with LKB1 siRNA pool were included as a negative control (p þ s). (g) Co-immunoprecipitation analysis of endogenous NUAK1 and p53 from A549 cells that stably expressed vector control (Vec), wild-type LKB1 (LKB1), kinase-deficient LKB1 (KDM) or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) as control. Cells were treated with glucose– medium and then fractionated (N: nuclear fraction; C: cytoplasmic fraction). Equal amounts of protein from each sample were immunoprecipitated using anti-NUAK1 antibody or using normal rabbit IgG as a negative control. Fifty percent of protein before immunoprecipitation was kept for input, and was subjected to western blotting with anti-NUAK1 and anti-p53 antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Expressing, Western Blot