Journal: The Journal of Cell Biology

Article Title: Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

doi: 10.1083/jcb.201405009

Figure Lengend Snippet: d- cer-C6 treatment alters Golgi membrane organization. (A) HeLa cells expressing mannosidase II–GFP (MannII-GFP) were treated with ethanol, 20 µM l- cer-C6, or 20 µM d- cer-C6 for 4 h. The localization of the Golgi markers mannosidase II–GFP and GRASP65 was monitored by immunofluorescence microscopy. (B) HeLa cells were treated with ethanol, 20 µM l- cer-C6, or 20 µM d- cer-C6 for 4 h, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. (C) HeLa cells were transfected with sialyltransferase-GFP (ST-GFP) and treated with ethanol, 20 µM l- cer-C6, or 20 µM d- cer-C6 for 4 h. The localization of ST-GFP and TGN46 was monitored by immunofluorescence microscopy. (D) Quantitation of the relative colocalization of the different proteins in the experiments shown in A–C, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. Bars, 5 µm. **, P < 0.01.

Article Snippet: Sheep anti–human TGN46 was obtained from AbD Serotec.

Techniques: Membrane, Expressing, Immunofluorescence, Microscopy, Transfection, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

doi: 10.1083/jcb.201405009

Figure Lengend Snippet: d- cer-C6–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l- cer-C6, or 20 µM d- cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. **, P < 0.01.

Article Snippet: Sheep anti–human TGN46 was obtained from AbD Serotec.

Techniques: Membrane, Transfection, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunofluorescence, Microscopy, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

doi: 10.1083/jcb.201405009

Figure Lengend Snippet: d- cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d- cer-C6, or 20 µM l- cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d- cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d- cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l- cer-C6, or 20 µM d- cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments ( n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.

Article Snippet: Sheep anti–human TGN46 was obtained from AbD Serotec.

Techniques: Glycoproteomics, Western Blot, Isolation, Transfection, Control, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

doi: 10.1083/jcb.201405009

Figure Lengend Snippet: d- cer-C6 treatment causes permanent segregation of a Golgi-resident enzyme from its substrate. (A) Scheme of the trapping procedure using rapamycin (Rap.)-mediated dimerization of FRB and FKBP domains. The domain structure of the chimeric constructs TGN46-FRB-GFP and ST-FKBP-RFP is shown relative to the Golgi membrane. Transmembrane (TM), FRB, and FKBP domains and the N and C termini of the proteins are indicated. (B) HeLa cells expressing TGN46-FRB-GFP and ST-FKBP-RFP were treated with 20 µM l- cer-C6 or 20 µM d- cer-C6 for 4 h, after which 100 µM cycloheximide and DMSO or 500 nM rapamycin was added to the culture media for an additional 2 h. Cells were then fixed for fluorescence microscopy. (C) Quantitation of the relative colocalization of TGN46-FRB-GFP and ST-FKBP-RFP in the experiment shown in B, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from four independent experiments. (D) HeLa cells expressing TGN46-FRB-GFP and ST-FKBP-RFP were treated with DMSO or 500 nM rapamycin for 2 h, after which 100 µM cycloheximide and 20 µM l- cer-C6 or 20 µM d- cer-C6 were added to the culture media for an additional 4 h. Cells were then fixed, and the localization of TGN46-FRB-GFP and ST-FKBP-RFP was monitored by fluorescence microscopy. (E) Quantitation of the relative colocalization of TGN46-FRB-GFP and ST-FKBP-RFP in the experiment shown in D, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from four independent experiments. **, P < 0.01. Bars, 5 µm.

Article Snippet: Sheep anti–human TGN46 was obtained from AbD Serotec.

Techniques: Construct, Membrane, Expressing, Fluorescence, Microscopy, Quantitation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of PAC1-CHO. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Activity Assay, Western Blot, Over Expression

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP - , and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP - cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP - transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP - cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Knockdown, shRNA, Western Blot, Expressing, Transfection, Control, Produced, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Cooperation between MYC and β-catenin in liver tumorigenesis requires Yap/Taz

doi: 10.1101/819631

Figure Lengend Snippet: MYC and β-cat Ex3 activation cooperate in transformation in liver and fibroblasts. (A) Kaplan Meyer disease-free survival curves for mice of the indicated genotypes (all in the presence of the Alb-CreER T2 transgene). The number of mice (n) and the median survival are indicated. p-values were calculated with the log-rank test. (B) Western blot analysis of β-catenin and MYC protein expression in wild-type (wt) liver and representative R26-lslMYC and β-cat Ex3 ;R26-lslMYC liver tumor samples. Vinculin was used as loading control. Each tumor is identified by its unique reference number. (C) Hematoxylin and Eosin staining of representative liver sections from the indicated genotypes. Bars: 400 µm (H&E 5x) or 100 µm (H&E 5x). (D) Left: representative pictures of the colony forming assay of 3T9 MycER;S33Y cells plated in 50% (v/v) methylcellulose and cultured for 7-10 days in the presence of 1 μM Doxycycline and/or 400 nM 4-OHT, as indicated. This experiment was performed four times, each with technical triplicates. Bar: 1000 µm. Right: quantification of the total number of colonies with a diameter >10 μM in each condition. Bar plot represent the average and standard deviation for the 4 biological replicates. (E) Representative pictures of CD1-nude mice 6 weeks after injection of 3×10 6 3T9 MycER;S33Y cells and fed with Tamoxifen (Tam) and/or Doxycyline (Doxy), as indicated. The fraction of tumor-bearing mice in each experimental group is indicated below each photograph.

Article Snippet: After addition of 6X Laemmli buffer (375 mM Tris-HCl, 9% SDS, 50% glycerol, 9% beta-mercatoethanol and 0.03% bromophenol blue), lysates were boiled (5 minute at 100°C) and then electrophoresed on pre-cast 4-15% gradient Polyacrilamide gels (Bio-Rad, #5678084), transferred onto methylcellulose membranes (Bio-Rad, #1704271) and protein expression detected with the indicated primary antibodies (Supplementary Table 5).

Techniques: Activation Assay, Transformation Assay, Western Blot, Expressing, Control, Staining, Cell Culture, Standard Deviation, Injection

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A and B, TLR3 colocalized with SREC-I in HEK 293-TLR3-SREC-I overexpressing cells. HEK293 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hours. Cells were then incubated without (A) or with (B) Poly I:C (10ug/ml) on ice for 30 min. Cells (A) and (B) were then fixed with 4% para formaldehyde and permeabilized with 0.1% Triton X100 (A) or not (B). Cells were then stained for FLAG with anti-FLAG M2 antibody (green) C, HEK293 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hr. Cells were then incubated Poly I:C (10ug/ml) on ice for 30 min followed by incubation with warm media at 37°C for 20 min. Cells were then fixed with 4% PFA and permeabilized with 0.1% Triton X100. Cells were stained with anti-FLAG antibody (green). D, HEK293 cells were transfected with FLAG-SREC-1 or TLR3-CFP for 18 hours. Cells were incubated with Poly I:C (10μg/ml) for 1 hour and then fixed and stained with anti-FLAG M2 ab (green). E, Differentiated THP1 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hr. Cells were then incubated with Poly I:C (10μg/ml) for 1 hr and then fixed and permeabilized as in D. Cells were stained for SREC-I (green), with anti FLAG ab, LAMP1 with anti LAMP1 (red). TLR3-CFP is in blue. Experiments were repeated twice with reproducible findings. Scale bar 2 micron.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, Staining

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: HEK293 cells stably expressing TLR3 was transfected with SREC-I-GFP for 22 hr. Cells were then treated with or without 10ug/ml Poly I:C for 30 min. Cell lysates were collected and SREC-I-GFP was immunoprecipitated using anti-GFP antibody and anti GFP ab and then the precipitated complexes were subjected to SDS-PAGE analysis followed by blotting for TLR3 using anti-TLR3 antibody. Experiments were carried out twice, reproducibly.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, B and C, HEK293-TLR3 cells were transfected with SREC-I or untransfected HEK293-TLR3 cells were then incubated with or without Poly I:C (10μg/ml) for 2 hr. Cell lysates were collected and then subjected to SDS-PAGE and western blotting with appropriate antibodies. D, Bone marrow derived macrophages (BMDM) were transfected with siRNA of SREC-I for 72 hr. Cells were then incubated with Poly I:C (or not) as in A. Cell were lysed and equal amount of protein were subjected to SDS-PAGE and western blotting using anti phospho-p65 antibody and anti p65 antibody. E, BMDM cells were treated as in D and then cell lysates were subjected to SDS-PAGE and western blotting using antibodies shown in E. F, HEK293-TLR3 cells were transfected with SREC-I or not and also NFkβ-SEAP/CMV-SEAP constructs. Cells were incubated with Poly I:C (10μg/ml)/ODN2395 (10μg/ml), a non TLR3 ligand. NFkβ activity was measured as instructed by NFkβ-SEAporter assay kit. Similar results were observed in two separate experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, SDS Page, Western Blot, Derivative Assay, Construct, Activity Assay

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1 cells were transfected with TLR3 or TLR3 and SREC-I expression plasmids for 22 hr. Cells were incubated with 10ug Poly I:C for 12 hr and then assayed for cytokine production using a human cytokine multianylate ELISA array kit according to manufacturer’s protocol. B, THP1 cells treated as in (A) and then assayed for chemokines using multianylate ELISA array kit according to Manufacturer’s protocol. Data represent the mean of two independent experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1 cells were transfected with TLR3/SREC-I or TLR3 and SREC-I expression plasmids for 22 hr. Cells were incubated with Poly I:C (10ug/ml) for 12 hr and then assayed for IL-8 release. B, THP1 cells were transfected with SREC-I and TLR3 for 22 hr and then incubated with the ERK inhibitor (PD98059), p38 inhibitor (SB203580) or the JNK inhibitor (JNK inhibitor II) (10uM) for 1 hr right before incubation with incubation with Poly I:C for 12 hr. IL-8 secretion was assayed according to manufacturer’s instructions. C, THP1 cells were treated as in A and then IL-6 release was assayed according to manufacturer’s instruction. D, THP1 cells were treated as in B and IL-6 release was assayed. E, THP1 cells were treated with PMA (5–10ng/ml) for 72 hours. Cells were then transfected with siRNA SREC-I or siRNA TLR3. Cells were incubated with Poly I:C for 12 hr and then Ifnβ release was measured according to manufacturer’s protocol. F. BMDM cells were transfected with siRNA SREC-I/TLR3 or not. Wild type or TLR3 knocked down (siRNA) cells were also treated with 1–5 ng/ml of LPS for 12hrs. Cells were then incubated with Poly I:C for 12 hr. Secreted IL-6 in media was measured according to manufacturer’s protocol. Experiment was repeated twice. Data shown are the mean ±SD of results from those two experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Expressing, Incubation

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1-TLR3 cells were transfected with or without SREC-I for 22 hr and then incubated with 10ug Poly I:C for indicated time. IL-8 release from cells was then assayed. B, THP1-TLR3 cells were transfected as in A and then incubated with or without Bafilomycin A (0.2 uM for 20 min) or PP2 (10uM for 12 hr). Cells were incubated with Poly I:C for 12 hr and then IL-8 release from THP1 cells were assayed. C, Differentiated THP1 cells were transfected with siRNA SREC-I for 72 hr and then cells were treated with Poly I:C (10μg/ml) for 2 hr. Cells were then lysed and equal amount of protein in lysate were subjected to SDS-PAGE and western blotting using appropriate antibodies. Experiments were carried out twice reproducibly.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, SDS Page, Western Blot

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: Double stranded RNA species Poly I:C interacts with macrophages leading to recruitment of SREC-I and TLR3 to the cell surface where they form a membrane complex that interacts with c-Src. The latter kinase then regulates endocytosis of the SREC-I/TLR3/Poly I:C complexes in endosomes. The complex finally resides in endosomes with low intravesicular pH, marked with lysosomal protein LAMP1. TLR3 is able to signal from such complexes and launches NFkB, MAPK and IRF3 signaling. (IRF3 is activated by the kinase TBK1). Activated NFkB, AP-1 and C/EBPβ are known to interact with the IL-8 gene while activated IRF3 leads to synthesis of Ifn-β.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Membrane