Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. Schematic diagram of the engineered F-TrCP-Shc ubiquitin ligase to target the ErbB RTKs. B. F-TrCP-Shc binds to ErbB2. 293T cells were transiently transfected with the indicated plasmids. ErbB2 was immunoprecipitated with the anti-ErbB2 antibody and immunoblotted with the ErbB2, FLAG and β-actin antibodies. C. Degradation of endogenous ErbB2 by the engineered F-TrCP-Shc. 293T cells were transiently transfected with increasing doses of F-TrCP-Shc or F-TrCP-Shc(m), and levels of endogenous ErbB2, F-TrCP fusions and β-actin were determined by Western blotting. The experiment was repeated three times. D. F-TrCP-Shc promotes ubiquitination of ErbB2. 293T cells were transfected with the indicated plasmids for in vivo ubiquitination assays. E–F. F-TrCP-Shc-induced ErbB2 degradation is proteasome-dependent. SKBR3 and BT474 cells expressing F-TrCP-Shc, F-TrCP-Shc(m) or pBMN-GFP were treated with 50 μM MG132 or 100 μM choloroquine. The levels of endogenous ErbB2, F-TrCP fusions and β-actin were determined by Western blotting.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, In Vivo, Expressing

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: SKBR3 (A) and BT474 (B) cells infected with the indicated recombinant retroviruses were lysed and 50 μg of each cell lysate was analyzed by SDS-PAGE and Western blotting with the indicated antibodies. C. SKBR3 and BT474 cells were plated in 6-well plates for 24 hours, trypsinized and washed in FACS buffer. The live cells were stained with anti-ErbB2 antibody (Santa Cruz) against the extracellular domain of ErbB2. Results are the mean for three replicate experiments. D. Dose-dependent degradation of endogenous ErbB2 upon infection of adenoviral F-TrCP-Shc was determined by Western blotting with the indicated antibodies. E. Depletion of ErbB2 on the cell membrane by adenoviral F-TrCP-Shc. ErbB2 (red) was detected by immunofluorescent staining with the anti-ErbB2 antibody (R&D Systems). Infected cells were marked by GFP expression from the adenoviral vector. The nuclei were visualized by DAPI staining (blue). White arrow, a low F-TrCP-Shc expressing SKBR3 cell.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, Recombinant, SDS Page, Western Blot, Staining, Membrane, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. BT474 cells were infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP, sorted and analyzed for inhibition of MAPK and PI3K signaling by Western blotting as in Fig. 4A. B. Retroviral expression of F-TrCP-Shc in BT474 cells inhibited cell growth. Results are the mean for three replicate experiments. * indicates P<0.05 for pBMN-F-TrCP-Shc-infected cells compared to those with pBMN-GFP or pBMN-F-TrCP-Shc(m). C–D. Apoptotic induction of BT474 cells by F-TrCP-Shc, as determined by Annexin V-FACS analysis and PARP1 cleavage by Western blotting. For FACS analysis, cells were stained with Annexin-V-APC and 7-AAD, and the percentage of early ( right bottom ) and late apoptotic ( right top ) populations are indicated.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, Inhibition, Western Blot, Retroviral, Expressing, Staining

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: The engineered F-TrCP-Shc E3 ligase sensitizes BT474 breast cancer cells to cytotoxic killing by cisplatin and decreases their transforming ability. A–B. BT474 or MCF-10A cells infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP retroviruses were treated with the indicated amounts of cisplatin for 48 hours, and the cell viability was measured by the XTT assay. Results are represented as the mean ± standard deviation (n=3), for BT474 cells. C–D. Focus formation assay. Retroviral vector infected BT474 cells were seeded in 6-well plates and cultured for 10 days to allow for colony formation. The colonies were fixed, stained and photographed. The colony numbers were counted and the colony formation ratio was calculated according to the formula: colony formation ratio ( % ) = ( number of colonies / number of cells seeded ) × 100 . Results are represented as the mean ± standard deviation (n=3). * indicates P<0.05 for pBMN-F-TrCP-Shc-infected cells compared to those with pBMN-GFP or pBMN-F-TrCP-Shc(m).

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, XTT Assay, Standard Deviation, Tube Formation Assay, Retroviral, Plasmid Preparation, Cell Culture, Staining

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. Nude mice were injected subcutaneously in the right flank with 5×10 6 BT474 cells infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP retroviruses. Tumor volumes were measured over a 4-week period and calculated by the formula: ( length × width 2 ) / 2 . B. Tumor weight (g) was recorded at the end of the experiment. Columns, mean value of tumor weight; bars, SD. C. Representative images of tumor-bearing mice. * P<0.05 or **P<0.01 indicate tumors expressing pBMN-F-TrCP-Shc compared to those infected with pBMN-GFP or pBMN-F-TrCP-Shc(m).

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Injection, Infection, Expressing

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: TRPV1-ir on human skin and cultured human HFs. A: TRPV1-ir (with diaminobenzidine as a chromogen) on inner root sheath, ORS, and matrix (MK) keratinocytes (DP). Inset, negative control of TRPV1 staining. B: Negative control of TRPV1 staining. TSA (C, D) and AP (E, F) TRPV1-ir on cultured anagen (C, E) and catagen (IFN-γ-induced; D, F) human HFs. G/1, G/2: Double-fluorescence immunolabeling of TRPV1+ (red, G/1) and NKI/beteb+ (green, G/2) cells in the human cultured HFs. Note the lack of co-localization of TRPV1-ir on HF melanocytes. Nuclei were counterstained by DAPI (blue fluorescence in B–D; also in G, but not shown for clarity). H: Q-PCR analysis of TRPV1 expression in the human HF. Data of TRPV1 expression were normalized to the expression of GAPDH of the same sample and are expressed as relative mRNA amounts as a function of startup cDNA (relative amount of 1 was defined as the detection threshold). NTC, nontemplate (negative) control; TRPV1/C6 and TRPV1/CHO, C6 and CHO cells stably expressing the recombinant rat TRPV1 (positive controls). Values are mean ± SEM of three independent determinations. Original magnifications: ×100 (A), ×200 (B–G).

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Cell Culture, Negative Control, Staining, Fluorescence, Immunolabeling, Expressing, Stable Transfection, Recombinant

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: The activation of TRPV1 inhibits HF elongation. A: Cultured HFs (18 per group) were treated with either the vehicle (DMSO, control) or with various concentrations of capsaicin (Caps) for the time indicated. B: Cultures were treated with the vehicle (control), 10 μmol/L capsaicin (Caps), or 10 μmol/L capsaicin and 100 nmol/L iodo-RTX (Caps + I-RTX). Length measurements were made on individual HFs using a light microscope with an eyepiece measuring graticule. All data were compared with those of the control group, and analyzed by two-tailed unpaired t-test. Results are expressed as mean ± SD. *, Significant (P < 0.05) differences. Two to three additional experiments yielded similar results.

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Activation Assay, Cell Culture, Control, Light Microscopy, Two Tailed Test

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: The activation of TRPV1 inhibits proliferation and induces apoptosis in cultured HFs. Co-immunolabeling of proliferating (Ki-67+, red fluorescence) and apoptotic (TUNEL+, green fluorescence) cells on control (A) and capsaicin-treated (10 μmol/L for 5 days, B) cultured HFs. Nuclei were counterstained by DAPI (blue fluorescence). MK, matrix keratinocytes. C: Statistical analysis of number of Ki-67 and TUNEL+ cells as compared to the number of DAPI+ cells on several HFs per group. Data are expressed as mean ± SD. *, Significant (P < 0.05) differences. Original magnifications, ×400.

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Activation Assay, Cell Culture, Immunolabeling, Fluorescence, TUNEL Assay, Control

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: The activation of TRPV1 induces catagen transformation in cultured HFs. Cultured HFs were treated with either the vehicle (A) or with 10 μmol/L capsaicin for 5 days (B), then processed for H&E staining, and the percentage of HFs in anagen or catagen state, according to previously well-defined criteria (narrower hair bulb and depigmentation in catagen HFs), was determined (C). MK, matrix keratinocytes. Results are expressed as mean ± SEM. *, Significant (P < 0.05) differences. Two to three additional experiments yielded similar results. Original magnifications, ×100.

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Activation Assay, Transformation Assay, Cell Culture, Staining

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: The activation of TRPV1 does not affect terminal differentiation but up-regulates TGF-β2 expression in cultured HFs. Fluorescence immunolabeling of differentiation marker filaggrin on control (A) and capsaicin-treated (10 μmol/L for 3 days, B) HFs (the isthmus region exhibiting the most intense staining is shown). Note the lack of effect of capsaicin on filaggrin expression (see text for statistics). Fluorescence immunolabeling of TGF-β2 on control (C) and capsaicin-treated (10 μmol/L for 2 days, D) HFs. MK, matrix keratinocytes. Note the up-regulation of TGF-β2 by capsaicin (see text for statistics). Nuclei were counterstained by DAPI (blue fluorescence). E: RT-PCR analysis of TGF-β2 (and β-actin, used as an internal control) mRNA expression in cultured HFs. Lane 1, reaction without template (negative control); lane 2, control at day 1; lane 3, 10 μmol/L capsaicin treated at day 1; lane 4, control at day 2; lane 5, 10 μmol/L capsaicin treated at day 2. bp, predicted product sizes. F: mRNA of transcripts were quantified by densitometry and the values of TGF-β2 were normalized to those of β-actin. Data of the capsaicin-treated groups, obtained in three independent experiments, are expressed as mean ± SEM values as a percentage of the daily matched control samples regarded as 100%. *, Significant (P < 0.05) differences. Original magnifications: ×100 (A, B); ×400 (C, D).

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Activation Assay, Expressing, Cell Culture, Fluorescence, Immunolabeling, Marker, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal:

Article Title: A Hot New Twist to Hair Biology

doi:

Figure Lengend Snippet: Identification of TRPV1 in cultured ORS and HaCaT keratinocytes, the activation of which elevates [Ca2+]i and inhibits proliferation. A: TRPV1-ir (green fluorescence) in cultured ORS keratinocytes. Nuclei were counterstained by DAPI (blue fluorescence). Inset, negative control of TRPV1 staining. B: Western blot analysis of TRPV1 expression on cell lysates of ORS and HaCaT keratinocytes, and of HDFs. Arrow indicates predicted molecule size (95 kd). C: Q-PCR analysis of TRPV1 expression in cultured cells and in the human HFs (for the human HFs, data are identical to those shown in Figure 1H). Data of TRPV1 expression were normalized to the expression of GAPDH of the same sample and are expressed as relative mRNA amounts as a function of startup cDNA (relative amount of 1 was defined as the detection threshold). NHEK, normal human epidermal keratinocytes. Values are mean ± SEM of three independent determinations. D: Fura-2 AM-loaded cells were challenged with various concentrations of capsaicin or with 10 μmol/L capsaicin and 100 nmol/L iodo-RTX (I-RTX) together in Tyrode’s solution containing 1.8 mmol/L calcium, and [Ca2+]i was determined as described in Materials and Methods. Data are expressed as mean ± SEM obtained on 15 to 20 cells per group. *, Significant (P < 0.05) differences compared to the resting [Ca2+]i. **, Significant (P < 0.05) effect of I-RTX to prevent the action of 10 μmol/L capsaicin to increase [Ca2+]i. E: A BrdU proliferation assay. Cells were plated in 96-well multititer plates in quadruplicates and were treated with various concentrations of capsaicin (Caps) or with 30 μmol/L capsaicin and 100 nmol/L iodo-RTX (I-RTX) together for 72 hours. For determining the extracellular calcium dependence, experiments were performed in low-Ca2+ SFM (0.4 mmol/L) and high-Ca2+ SFM (2 mmol/L). Data are expressed as mean ± SEM as a percentage of the matched control values regarded as 100%. *, Significant (P < 0.05) differences compared to control (vehicle-treated) proliferation. **, Significant (P < 0.05) effect of I-RTX to prevent the action of 30 μmol/L capsaicin to inhibit proliferation. Original magnification, ×630 (A).

Article Snippet: PCR amplification was performed by using the TaqMan primers and probes (assay ID, Hs00218912_m1 for human TRPV1; assay ID, Rn00583117_m1 for rat TRPV1) using the TaqMan universal PCR master mix protocol (Applied Biosystems).

Techniques: Cell Culture, Activation Assay, Fluorescence, Negative Control, Staining, Western Blot, Expressing, Proliferation Assay, Control

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Different cell types composing intestinal wall were examined for the presence of Fc gamma receptors. Villin and α-SMA were used to confirm cell type-specific phenotypes (A). Immunofluorescence analysis of CD64-expressing CCD-18Co fibroblasts. Cells expressing CD64 at the plasma membrane are indicated by arrows. Size bar: 50 µm (B). Intestinal fibroblasts CCD-18Co (C), intestinal epithelial cells Caco2 BBE (D) and THP-1 cells (E) were pre-incubated with anti-TNF therapeutics, and subsequently treated with human recombinant TNF to induce pro-inflammatory response. Values on Y axis represent mRNA expression levels relative to ß-actin. Columns represent mean values of four independent experiments measured in triplicate. Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Immunofluorescence, Expressing, Clinical Proteomics, Membrane, Incubation, Recombinant

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Peripheral blood of healthy donors (n = 4) and IBD patients (n = 3) was treated with different anti-TNFs and subsequently with soluble TNF to induce pro-inflammatory responses in PBMCs and plasma. The mRNA expression levels of CD64 (A), CD32 (B) and CD16 (C) Fc gamma receptors in healthy individuals and IBD patients were assessed by RT-PCR as compared to THP-1 cells (set to 1). The extent of inhibition by each drug was determined by measuring mRNA levels of ICAM-1 (D), TNF (E), and IL-8 (F) after 2 h of TNF stimulation, as well as by the release of IL-8 after 24 h of combined TNF and anti-TNF stimulation (G). The inhibitory efficacies were determined after eliminating intra-individual differences by setting the TNF-induced responses to 100% for each donor and target (H–K). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Clinical Proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Both ADA and IFX immune complexes were added to THP-1 cells to activate CD64 receptor in the presence (+FCS) or absence (−FCS) of serum. ADA/TNF complexes were more potent in inducing phospho-tyrosine signaling (A), but IFX either alone or in complex with TNF induced phosphorylation of distinct tyrosine residues (enlarged insert, right panel). Activation of CD64 downstream signaling leads to elevated transcription of GM-CSF (B, C), MCP-1 (D, E) and IL-8 (F, G) genes. Human serum (HS) was used as a positive control for activation of Fc gamma receptor(s). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Phospho-proteomics, Activation Assay, Positive Control

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: CCD-18Co fibroblasts (A), Ko77 fibroblasts (C) and THP-1 cells (E) were pre-incubated with human IgG1 isotype (10 µg/ml) prior to subsequent incubation with infliximab (1 µg/ml) and TNF (50 ng/ml). Columns show mean values from a single, representative experiment measured in triplicates. Error bars indicate SD of three measurements. The recovery of anti-TNF activity was also evaluated as percentage of inhibition of TNF-induced responses for each cell line (B, D, F).

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Incubation, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Blocking of Fc fragments of adalimumab and infliximab changes their inhibitory efficacy in Ko77 fibroblasts (A). Graph shows mean values from four independent experiments measured in triplicates. Error bars represent SD. Fab fragments of anti-TNFs have different inhibitory efficacies as compared to the whole IgG1 molecules in THP-1 cells (B). Columns represent mean values of three independent experiments measured in triplicates and set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Treatment with IFN-γ elevates the mRNA expression levels of CD64 (A), which result in the loss of efficacy of IFX and ADA (B). Decreased expression levels of CD64 (C) correlate with increased inhibitory efficacy of IFX (D). PMA treatment decreases the expression levels of CD64 (E), which results in complete recovery of efficacy of IFX (F). Graphs show mean values of at least three independent experiments measured in triplicates and columns are set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: IFN-γ induces its receptor downstream signaling involving JAK and STAT proteins (A). STAT dimers elevate the transcription of the CD64 (FCGR1) gene (B). Translated CD64 protein is targeted into plasma membrane, where it resides in an inactive state until binding immune complexes IFX-TNF (C). Propagation of downstream signaling (D) occurs through immunoreceptor tyrosine-based activation motif (ITAM) leading to the transcription of relevant target genes (E) via parallel activation of p38, JNK and ERK kinases . Production and release of IL-8 and other pro-inflammatory factors contribute to the limited outcome of IFX treatment.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

doi: 10.1371/journal.pone.0036301

Figure Lengend Snippet: Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in . pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.

Article Snippet: The human probes used were: MYOC (Hs00165345_m1), CSTA (Hs00193257_m1), CXCL2 (Hs00235956_m1), IGF1 (Hs00153126_m1), MMP1 (Hs00233958_m1), MMP3 (Hs00968305_m1), MMP12 (Hs00899662_m1), SFRP1 (Hs00610060_m1), STC1 (Hs00174970_m1), SNCA (Hs01103383_m1), RAB39B (Hs00293395_m1) and THBD (Hs000264920_s1).

Techniques: Recombinant, Expressing, Generated, Transfection, SDS Page, Western Blot, Electroporation

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in the IRF3−/− MEFs. (A) MEFs derived from wild-type (WT) and IRF3−/−, IRF7−/−, or MyD88−/− mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and IRF3−/− mice were inoculated with rBV-GFP at an MOI of 100, incubated at 4°C for 30 min, and washed three times with PBS. The total cellular DNA was extracted, and the amounts of baculovirus genome were quantified by real-time PCR. Data represent means ± SD from 2 independent experiments. (C) MEFs derived from WT and IRF3−/− mice were inoculated with 2 doses of rBV-GFP (MOI of 100 or 20). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. (D) MEFs derived from WT and IRF3-deficient mice were inoculated with 2 doses of rBV-GFP (MOI of 100 and 20). At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, IRF3, or β-actin, respectively.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, SDS Page

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: IRF3-dependent enhancement of gene transduction by recombinant baculovirus. (A) Cell extracts of MEFs derived from WT or IRF3−/− mice and FLAG-mIRF3-transduced IRF3−/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IRF3, or β-actin, respectively. (B) MEFs derived from WT or IRF3−/− mice and FLAG-mIRF3-transduced IRF3-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent the means ± SD from 3 independent experiments. (C) MEFs were inoculated with rBV-luc at an MOI of 100. At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. IRF3 (green) was stained with the appropriate antibodies, followed by staining with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained by DAPI.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, SDS Page, Luciferase, Activity Assay, Staining

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus vectors through the STING/TBK1/IRF3 axis in MEFs. (A) MEFs derived from wild-type (WT) and STING- or ZBP1-deficient mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, the GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B and C) MEFs derived from WT and STING- or ZBP1-deficient mice were inoculated with rBV-luc (MOI of 100) or rBV-GFP (MOI of 100). At 24 h after inoculation, the luciferase activity of cell lysates was determined (B), and the production of IFN-β in the culture supernatant was determined by sandwich ELISA (C). (D) The MEFs were inoculated with wild-type baculovirus (AcNPV) (MOI of 100). At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. STING (green) and ER-calnexin (red) were stained with the appropriate antibodies, followed by staining with Alexa Fluor 488- or Alexa Fluor 555-conjugated second antibodies, respectively. Nuclei were stained by DAPI.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Luciferase, Activity Assay, Sandwich ELISA, Staining

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: RLR signaling pathways participate in the suppression of gene transduction in MEFs upon infection with recombinant baculovirus. (A) MEFs derived from wild-type (WT) and IPS-1-, TBK1-, ZBP1-, or IRF3-deficient mice were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments. (B) MEFs derived from WT and IPS-1−/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, GFP expression was detected by microscopic observation after fixation in 4% paraformaldehyde. (C) MEFs derived from WT or IPS-1-deficient mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Cell extracts of MEFs derived from WT or IPS-1−/− mice and FLAG-mIPS-1-transduced IPS-1−/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IPS-1, or β-actin, respectively. (E) MEFs derived from WT or IPS-1−/− mice and FLAG-mIPS-1-transduced IPS-1-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Protein-Protein interactions, Transduction, Infection, Recombinant, Derivative Assay, Luciferase, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, SDS Page

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in HCV replicon-harboring cells. (A) Huh7OK1 cells (cured) and the HCV replicon-harboring cells derived from genotype 1a (RMT strain), 1b (con1 strain), and 2a (JFH1 strain) were inoculated with rBV-luc (MOI of 100) or VSV-luc at an MOI of 5 and (B) with rBV-luc at MOI of 5, 10, 25, and 50. At 24 h after inoculation, the luciferase activity of cell lysates was determined. (C) Huh7 cells were inoculated with rBV-GFP (MOI of 100) or VSV-GFP (NCP mutant) at an MOI of 0.05. At 24 h after inoculation, total RNA was extracted, and the expression of IP-10, ISG15, and IL-8 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Huh7 cells infected with HCVcc at an MOI of 1 and incubated for 72 h were inoculated with rBV-GFP (MOI of 100) in the presence or absence of human recombinant IFN-α (rIFN-α) (100 U/ml). At 24 h after inoculation, the cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, NS5A, or β-actin, respectively. (E) Relative luciferase activity in Huh7 cells with IRF3, IPS-1, or STING knocked down. Cells were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity of cell lysates was determined (right panel). Luciferase activity is normalized to control shNC cells, and data represent the means ± SD from 2 independent experiments. Total RNA was extracted, and the expression of IRF3, IPS-1, or STING mRNA was determined by real-time PCR (left panel). Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Infection, Incubation, SDS Page, Control