Journal: Journal of Biological Chemistry

Article Title: TAK1-ECSIT-TRAF6 Complex Plays a Key Role in the TLR4 Signal to Activate NF-κB

doi: 10.1074/jbc.m114.597187

Figure Lengend Snippet: FIGURE 4. TAK1-ECSIT-TRAF6 complex is functionally important for the TLR4-mediated signaling. A, NF-B reporter assay in HEK293T cells transfected with the indicated vectors, mock, wild type (wt) ECSIT, ECSIT 1–300 truncated mutant, or ECSIT 257–431 truncated mutant, in the presence or absence of LPS. Error bars, S.D. from triplicate samples. B, THP-1 cells were infected with lentiviral particles containing shRNA targeting human ECSIT or control lentiviral particles according to the manufacturer’s protocol. Control THP-1 (ctrl) and ECSITKD THP-1 cells were cultured in puromycin-containing medium (4 g/ml) for 2 weeks to select stable clones, and IB with anti-ECSIT or anti-GAPDH antibody was performed to evaluate the knockdown efficacy. C, Western blot assay in control (ctrl) THP-1 and ECSITKD THP-1 cells with anti-ECSIT, anti-phospho-MEKK1, anti-MEKK1, anti-phospho-TAK1, anti-TAK1, anti-phospho-IKK, anti-IKK, or anti-IKK antibody or anti-GAPDH as a loading control. Control THP-1 and ECSITKD THP-1 cells were treated with or without the addition of 100 ng/ml LPS for the times indicated. D, control or ECSITKD THP-1 cells were treated with or without LPS, as indicated, and then the endogenous expressions of TAK1 were analyzed by a Western blotting assay. A TAK1 kinase assay was performed, according to the manufacturer’s protocol. As a positive control, active c-TAK1 (2.5 nM) was used in this study. Error bars, S.D. triplicate samples. *, p 0.01; **, p 0.05.

Article Snippet: Knockdown by Lentiviral Particles—THP-1 cells were infected with lentiviral particles containing shRNA targeting human ECSIT (Santa Cruz Biotechnology, Inc., sc-77224-V), shRNA targeting human TAK1 (sc-36606-V), shRNA targeting human TRAF6 (sc-36717-V), or control lentiviral particles (sc-108080) according to the manufacturer’s protocol.

Techniques: Reporter Assay, Transfection, Mutagenesis, Infection, shRNA, Control, Cell Culture, Clone Assay, Knockdown, Western Blot, Kinase Assay, Positive 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: 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: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 3. Involvement of EGFR/AKT/CREB pathway in REG4-mediated macrophage polarization to M2. (A) Representative western blots of phosphorylated EGFR, AKT and CREB in macrophage cultures treated with REG4 (10 nM) for different time intervals indicated. (B) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively. Data are mean ± SEM from four independent experiments. *p<0.05, **P<0.01 compared to untreated cells. (C) Representative western blotting images showing the phosphorylation of EGFR, AKT and CREB in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. (D) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively, in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. Data are mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with REG4 treatment alone. (E) Representative western blots of CD163, CD68, and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. (F) Quantitative analysis of the immunoblots of CD163, CD68 and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. Data are mean ± SEM from three independent donors. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Phospho-proteomics, Infection, Control, shRNA

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 4. The conditioned medium of Panc1 cells induces macrophage polarization to M2. (A) Western blots of REG4 in the culture medium or cell lysate of Panc1, AsPC1 and BxPC3 cells. Shown are representatives of three independent experiments with similar results. (B) Representative western blots of CD206 and CD163 in macrophage cultures treated with or without the conditioned medium of Panc1, AsPC1 and BxPC3 cell cultures, respectively. (C) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with or without the conditioned medium of Panc1 (CMPanc1), AsPC1 (CMAsPC1) and BxPC3 (CMBxPC3) cell cultures, respectively. Data are shown by mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with CMPanc1 treatment. (D) Western blots of REG4 in the culture medium and cell lysate of Panc1 cells infected with or without lentiviral REG4 shRNA. Shown are representatives of three independent experiments with similar results. (E) Representative western blots of CD206 and CD163 in macrophage cultures treated with the conditioned medium Panc1 cells infected with lentiviral control shRNA (CMPanc1/shCon) or REG4 shRNA (CMPanc1/shREG4). (F) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with CMPanc1/shCon or CMPanc1/shREG4. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Infection, shRNA, Control

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

Article Title: Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear receptors PPARγ and TR4 for survival.

doi: 10.4049/jimmunol.1103038

Figure Lengend Snippet: FIGURE 1. PPARg, TR4, and LXRa loss of function modulates intracellular H37Ra load. (A) Loss of function (RNAi: 40 nM; knockdown efficiency ∼95%) of PPARg and TR4 reduces and that of LXRa augments intracellular H37Ra load as monitored by CFUs and by monitoring the percentage of dead bacteria in stable knockdown cell lines (shRNA lentiviral par- ticles stably inserted) by flow cytometry (B). CFU counts are plotted as mean 6 SD, and flow cytometry results are plotted as median. (C) Evaluation of M. tuberculosis survival by im- aging experiments. The extent of colocalization of GFP-H37Ra with acidified lysosomes (stained with LysoTracker; Invitrogen) was de- termined in control and stable knockdown cell lines of PPARg, TR4, and LXRa and the double knockdown of PPARg and TR4. Images shown for each group are those obtained for myco- bacteria (GFP-H37Ra), acidified lysosomes (LysoTracker), or a merge of the two (Merge). Original magnification 360. (D) The overlap coefficient (at a scale of 1) of GFP-H37Ra with the LysoTracker for 20 consecutive infected macrophages. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. Error bars show SD. *p , 0.05, significant differences with H37Ra control, two-tailed Student t test.

Article Snippet: For stable cell lines, cells were infected with short hairpin RNA (shRNA) lentiviral particles containing three to five expression constructs encoding target-specific nucleotide shRNA as per the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Knockdown, Bacteria, shRNA, Stable Transfection, Cytometry, Staining, Control, Infection, Two Tailed Test