Journal: International Journal of Molecular Sciences

Article Title: Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease

doi: 10.3390/ijms21103539

Figure Lengend Snippet: Expression of secreted frizzled-related proteins (sFRPs) and Wnt signaling in vascular smooth muscle cells (VSMCs) exposed to vascular calcification (VC) induction medium (high-phosphate, angiotensin II, and vitamin (D) were measured by Western blotting. Six replicates per condition were performed. The expression levels of β-catenin ( A ) and Wnt3a ( B ) were significantly increased and Wnt5a ( C ) expression was decreased in VC induction medium compared with the control. The expression levels of sFRP1−3 ( D – F ) were not affected by the chronic kidney disease environment. The expression of sFRP4 ( G ) was increased and that of sFRP5 ( H ) was decreased in VC induction medium compared with the control. Data are expressed as means ± standard errors of the mean from six independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Serum sFRP5 concentrations were assayed and samples were stored at −70 °C shortly after measurement of VC using a human sFRP5 kit (EK1472; Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease

doi: 10.3390/ijms21103539

Figure Lengend Snippet: Secreted frizzled-related protein 5 (sFRP5) inhibited osteoblastic trans-differentiation of vascular smooth muscle cells (VSMCs) cultured in vascular calcification (VC) induction media (high-phosphate, angiotensin II, and vitamin (D). The protein level of RUNX2 was determined using Western blotting, and calcification was confirmed visually by von Kossa staining. Six replicates per condition were performed. ( A ) Treatment with sFRP5 of VSMCs in VC induction medium decreased the expression of RUNX2, and neutralization with anti-sFRP5 restored the expression of RUNX2 to control immunoglobulin G levels; ( B ) VSMCs cultured in VC induction medium with different additional interventions and stained with von Kossa stain are shown. Six replicates per condition were performed. VSMCs incubated in VC induction medium showed significantly increased staining compared with the control. Treatment with sFRP5 led to the attenuation of staining, and the addition of anti-sFRP5 resulted in increased staining. Scale bar, 100 µm. Data are expressed as means ± standard errors of the means from six independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Serum sFRP5 concentrations were assayed and samples were stored at −70 °C shortly after measurement of VC using a human sFRP5 kit (EK1472; Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Cell Culture, Western Blot, Staining, Expressing, Neutralization, Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease

doi: 10.3390/ijms21103539

Figure Lengend Snippet: The protective effect of secreted frizzled-related protein 5 (sFRP5) against vascular smooth muscle cell (VSMC) differentiation was mediated by the inhibition of noncanonical (β-catenin–independent) Wnt signaling. VSMCs were cultured with vascular calcification (VC) induction medium in the presence or absence of sFRP5. Six replicates per condition were performed. The protein expressions of ROCK-2 and total and phosphorylated JNK were determined by Western blotting. ( A ) Culture in VC induction medium induced an increase in ROCK-2 expression, and treatment with sFRP5 led to a decrease in ROCK-2 expression. The addition of anti-sFRP5 restored the expression of ROCK-2; ( B ) Culture in VC induction medium increased JNK phosphorylation. Treatment with sFRP5 led to decreased JNK phosphorylation, and neutralization with anti-sFRP5 reversed this effect. Data are expressed as means ± standard errors of the means from six independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Serum sFRP5 concentrations were assayed and samples were stored at −70 °C shortly after measurement of VC using a human sFRP5 kit (EK1472; Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Cell Culture, Western Blot, Expressing, Phospho-proteomics, Neutralization

Journal: International Journal of Molecular Sciences

Article Title: Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease

doi: 10.3390/ijms21103539

Figure Lengend Snippet: Expression of secreted frizzled-related proteins (sFRPs) and Wnt signaling in an animal model of adenine-induced chronic kidney disease (CKD) were measured by Western blotting, and serum concentrations of sFRP5 were measured in human subjects. For each model, six rats were included; three representative results from each group are presented. The expression of sFRP4 was increased ( A ) and that of sFRP5 was decreased ( B ) in the CKD with vascular calcification (VC) group compared with the control group. The expression levels of ROCK-2 ( C ) and phosphorylation of JNK ( D ) were significantly increased in the CKD group compared with the control group. The expression of β-catenin ( E ) and Wnt3a ( F ) was significantly increased and that of Wnt5a ( G ) was decreased in the CKD with VC group compared with the control group. ( H ) The serum concentration of sFPR5 was significantly lower in patients on hemodialysis (HD) than in subjects with normal renal function. Among patients on HD, subjects with VC had significantly lower levels of serum sFRP5 than did subjects without VC. Three representative samples per group are shown ( A – G ). Data are expressed as means ± standard errors of the means from six samples per group. * p < 0.05, ** p < 0.01.

Article Snippet: Serum sFRP5 concentrations were assayed and samples were stored at −70 °C shortly after measurement of VC using a human sFRP5 kit (EK1472; Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Animal Model, Western Blot, Control, Phospho-proteomics, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease

doi: 10.3390/ijms21103539

Figure Lengend Snippet: Schematic diagram showing the pathways involved in the pathogenesis of vascular calcification (VC) in chronic kidney disease (CKD). As CKD progresses, secreted frizzled-related protein 5 (sFRP5) and Wnt5a levels decrease, while Wnt3a levels increase. Wnt3a can activate the canonical or noncanonical Wnt signaling pathway. With regard to the pathogenesis of VC, Wnt3a activates noncanonical (ROCK/JNK) Wnt signaling, which leads to the trans-differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. sFRP5 inhibits Wnt3a-mediated activation of the noncanonical Wnt signaling pathway, leading to the attenuation of the osteoblastic trans-differentiation of VSMCs. Wnt5a inhibits canonical Wnt signaling, which leads to the suppression of osteoblast activation following bone loss in CKD. The solid arrows indicate activation, the dashed lines with blocked ends indicate inhibition, and the dashed arrows indicate reductions in the CKD environment.

Article Snippet: Serum sFRP5 concentrations were assayed and samples were stored at −70 °C shortly after measurement of VC using a human sFRP5 kit (EK1472; Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Inhibition

Journal: Journal of neuropathology and experimental neurology

Article Title: Apoptosis and proliferation markers in diffusely infiltrating astrocytomas: profiling of 17 molecules.

doi: 10.1097/01.jnen.0000235857.79502.c3

Figure Lengend Snippet: FIGURE 3. (A) Western blot anal- ysis of caspases and inhibitor of apoptosis proteins in normal brain tissue (NBT, lanes 1Y7), diffuse astrocytoma (lanes 9Y15), anaplas- tic astrocytoma (lanes 17Y21), and glioblastoma multiforme (lanes 24Y31). Controls included glioma cell line U251 (lanes 8 and 22), melanoma cell line A875 (lane 16), and a carcinoma tissue sample (lane 23), which were known to express BIRC5 and caspase 3. GAPDH was used as internal con- trol. (B) Western blot analysis of BIRC5 in one original blot. Simul- taneous probing of BIRC5 and GAPDH illustrate the specificity of the antibodies. Absence of BIRC5 (first 5 lanes from left) in 5 astro- cytoma samples and presence of BIRC5 in glioma cell U251 (lane 6) and a carcinoma sample (lane 7). A prestained broad-range (11Y170 kDa) protein ladder was used for SDS-PAGE and aligned to the blot on the right.

Article Snippet: The following primary antibodies at indicated dilutions were used for IHC: CASP3 (rabbit polyclonal, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA), CASP6 (goat polyclonal, 1:100; Santa Cruz), CASP7 (goat polyclonal, 1:100; Santa Cruz), CASP8 (goat polyclonal, 1:100; Santa Cruz), CASP9 (rabbit polyclonal, 1:100; Santa Cruz), CASP10 (goat polyclonal, 1:100; Santa Cruz), CASP14 (goat polyclonal, 1:300; Santa Cruz), CIAP1 (rabbit polyclonal, 1:100; Santa Cruz), CIAP2 (rabbit polyclonal, 1:100; Santa Cruz), Xlinked inhibitor of apoptosis protein (XIAP; rabbit polyclonal, 1:300; ProteinTech Group, Inc., Chicago, IL), BIRC5 (Survivin) (rabbit polyclonal, 1:500; R and D Systems, Inc., Minneapolis, MN), LIVIN (goat polyclonal, 1:500; R and D), APAF1 (rabbit polyclonal, 1:100; Santa Cruz), SMAC (goat polyclonal, 1:300; Santa Cruz), BCL2 (mouse monoclonal, 1:200; Zymed Laboratories Inc., San Francisco, CA), PHH3 (rabbit polyclonal, 1:500; Upstate Group LLC, Charlottesville, VA), and Ki67 (MIB1) (mouse monoclonal, 1:100; DakoCytomation, Glostrup, Denmark).

Techniques: Western Blot, SDS Page

Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: HSP90 pharmacological inhibition or HSP90α knockout increases the pro‐apoptotic and DNA damage effect of CDDP in Pt‐res TOV‐112D cells. (A) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D Pt‐res cl. 7 and (B) cl. 2 cells, untreated or treated for 48 h (upper panels) or 72 h (lower panels), with CDDP and/or ganetespib at IC 50 72 h doses of parental cells. (C) Western blot analysis of γH2AX, PARP1 and caspase 3 cleavage, BAX and BCL2 expression, in TOV‐112D Pt‐res pool 2 cells untreated or treated with CDDP and/or ganetespib at the doses indicated above. β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software. (D) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D, TOV‐112D Pt‐res cl. 7 and in HSP90α knockout TOV‐112D Pt‐res KO#2 cells, untreated or treated for 48 or 72 h with CDDP at IC 50 72 h doses of parental cells.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: Inhibition, Knock-Out, Flow Cytometry, Staining, Western Blot, Expressing, Software

Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: Potentiation of CDDP antitumor effect induced by ganetespib in vivo CDDP‐resistant TOV‐112D xenograft model. TOV‐112D Pt‐res pool 2 cells (5 × 10 6 ) were s.c. injected into NSG mice as described in . When tumors were established, mice (five/group) were treated once a week for two weeks, with CDDP (2.5 mg·kg −1 i.p.), ganetespib (GANE; 75 mg·kg −1 i.p.), both drugs in combination, or their respective vehicles (UNT). (A) Relative TV measured at prespecified time points (Means ± SEM). (B) TVs at cutoff when mice were sacrificed. Data are shown as means ± SEM. (C) Mice body weight as surrogate indicator of toxicity for in vivo experiment reported in A. Body weight was measured three times/week. (D) TGD, indicating the mean rate of tumor growth in the treatment groups relative to control untreated mice (see ). Statistically significant results calculated with one‐way ANOVA test are reported (* P < 0.05, ** P < 0.01, and *** P < 0.001). (E) Western blot analysis of γH2AX, cleaved PARP1, cleaved caspase 3, HSP90α, and total HSP90, in lysates from three representative xenograft tumor samples from each treatment group (see ). β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software using the mean value of the three samples for each experimental group.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: In Vivo, Injection, Western Blot, Expressing, Software

Journal: Science Advances

Article Title: Engineering bacterial outer membrane vesicles as transdermal nanoplatforms for photo-TRAIL–programmed therapy against melanoma

doi: 10.1126/sciadv.aba2735

Figure Lengend Snippet: Part I: Preparation of I-P-OMVs. (i) Transformation of E. coli with pDNA-TRAIL (T– E. coli ). (ii) Isolation of OMVs from T– E. coli . (iii) Detoxification of OMVs with lysozyme. (iv) Modification of OMVs with RGP forming P-OMVs. (v) Conjugation of ICG to P-OMVs forming I-P-OMVs. Part II: Topical application of I-P-OMVs induces photo-TRAIL treatment in skin melanoma. (i) I-P-OMVs penetrate through skin and target to melanoma. (ii) NIR irritation triggers ICG to induce hyperthermia effect and secret singlet oxygen that clears primary melanoma spheroids promptly. (iii) Photothermal effect induces the deformation of OMVs that release TRAIL, followed by their binding to death receptors in melanoma cells surface, activating the apoptosis in residual melanoma cells. (iv) I-P-OMVs+NIR treatments prevents the metastatic potential of melanoma through interfering the relevant genes and proteins.

Article Snippet: The TRAIL protein in OMVs was determined by ELISA (Boster, China).

Techniques: Transformation Assay, Isolation, Modification, Conjugation Assay, Binding Assay

Journal: Science Advances

Article Title: Engineering bacterial outer membrane vesicles as transdermal nanoplatforms for photo-TRAIL–programmed therapy against melanoma

doi: 10.1126/sciadv.aba2735

Figure Lengend Snippet: ( A ) TEM images of T– E. coli with their derived OMVs. Scale bars, 2 μm and 200 nm. ( B ) Confocal laser scanning microscopy (CLSM) image of Dil-OMVs. Scale bar, 2 μm. ( C ) Size distribution of OMVs measured by DLS. ( D ) WB analysis of S3 protein (a classical protein contained within E. coli ) and TRAIL protein in bacterial cells and their derived OMVs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( E ) Size distribution and ( F ) zeta potential (ZP) of OMVs, R-OMVs, and P-OMVs. ( G ) TEM images of OMVs, R-OMVs, and P-OMVs. Scale bars, 200 nm.

Article Snippet: The TRAIL protein in OMVs was determined by ELISA (Boster, China).

Techniques: Derivative Assay, Confocal Laser Scanning Microscopy, Zeta Potential Analyzer

Journal: Science Advances

Article Title: Engineering bacterial outer membrane vesicles as transdermal nanoplatforms for photo-TRAIL–programmed therapy against melanoma

doi: 10.1126/sciadv.aba2735

Figure Lengend Snippet: ( A ) Photothermal response of I-P-OMVs (0 to 5 μg/ml) to NIR irritation (2 W/cm 2 for 3 min) ( n = 3). ( B ) Changes in the DPBF absorbance spectra in the presence of I-P-OMVs under NIR irradiation (0, 0.6, and 2 W/cm 2 ). ( C ) DPBF absorbance in the presence of I-P-OMVs at 412 nm under NIR irradiation (0, 0.6, and 2 W/cm 2 ) ( n = 3). ( D ) TRAIL release profiles at 37°C without or with NIR irradiation (2 W/cm 2 ) ( n = 3). ( E ) CLSM images of 3D tumor spheroids incubated with Dil-OMVs, Dil-R-OMVs, and Dil-P-OMVs. ( F and G ) CM-Dil fluorescence intensity in different depths of 3D tumor spheroids incubated with Dil-OMVs (F), Dil-R-OMVs (F), and Dil-P-OMVs (G). All data are represented as means ± SD. *** P < 0.001 and ** P < 0.01.

Article Snippet: The TRAIL protein in OMVs was determined by ELISA (Boster, China).

Techniques: Irradiation, Incubation, Fluorescence

Journal: Science Advances

Article Title: Engineering bacterial outer membrane vesicles as transdermal nanoplatforms for photo-TRAIL–programmed therapy against melanoma

doi: 10.1126/sciadv.aba2735

Figure Lengend Snippet: ( A ) The distribution of OMVs-GFP, R-OMVs-GFP, and P-OMVs-GFP in skin slice. Scale bars, 100 nm. ( B ) The accumulative transdermal amounts of TRAIL protein ( n = 3). ( C ) Scanning electron microscopy (SEM) (c 1 ) and TEM (c 2 , c 3 , and c 4 ) images of skin tissues after topical application of DiI-P-OMVs. Scale bars, 1 μm. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)–P-OMVs were indicated by red arrows. ( D ) Schematic illustration of the distribution of OMVs in skin and their transdermal routes. ( E ) In vivo fluorescence imaging of tumor-bearing mice after topically applied with Dil-P-OMVs. ( F ) Fluorescent images of tumors and major organs ( G ) Therapeutic regimen of I-P-OMVs+NIR in mice with B16F10 melanoma. ( H to J ) The tumors size, relapse rates, and body weights in mice during tested periods. ( K ) The TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling), cleaved caspase 3, Fontana-Masson (12 hours, day 6, and day 12), S100-β (day 12), and CD63 (day 12) staining of tumors. ( L to R ) Quantification of (K)-positive cells in the tumors ( n = 3). Scale bars, 100 nm. I to V represent the ICG+NIR, I-TRAIL+NIR, I-OMVs+NIR, I-R-OMVs+NIR, and I-P-OMVs+NIR groups, respectively. All data are represented as means ± SD. *** P < 0.001, ** P < 0.01, and * P < 0.05. n.s., not significant.

Article Snippet: The TRAIL protein in OMVs was determined by ELISA (Boster, China).

Techniques: Electron Microscopy, In Vivo, Fluorescence, Imaging, TUNEL Assay, End Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TNFα and IFNβ do not trigger cell apoptosis. A, poly(I-C) up-regulated the expression of TNFα in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. The supernatant was harvested to detect the TNFα secretion by ELISA. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the medium control. B, TNFα (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. C, TNFα (50 ng/ml) induced NF-κB signaling in immortalized HUVECs. D, TNFα neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs. E, IFNβ (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. F, IFNβ neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs.

Article Snippet: Human TRAIL ELISA kit was purchased from Boster Biochemicals (Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TRAIL-DR4/5 and Noxa trigger the extrinsic and intrinsic pathways, respectively. A and B, RT-PCR results show that poly(I-C) (37 °C for 24 h) up-regulated the gene expression of TRAIL, DR4, and DR5 in immortalized (A) and 1 μg/ml poly(I-C) pretreated primary (B) HUVECs. C, poly(I-C) up-regulated the protein expression of TRAIL in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C), were re-treated with the indicated concentrations of poly(I-C) for 24 h. TRAIL in the cell lysates was assayed by ELISA. *, p < 0.05 compared with the control. D, TRAIL neutralization repressed the cell apoptosis induced by poly(I-C) in immortalized HUVECs. *, p < 0.05 compared with the poly(I-C) treatment group. E and F, RT-PCR results show the effect of poly(I-C) (37 °C for 24 h) on the gene expression of Bcl-2 and Noxa in immortalized (E) and primary (F) HUVECs. G and H, Western blot results show the effect of poly(I-C) on the protein expression of Bcl-2 and Noxa in immortalized (G) and primary (H) HUVECs. I, inhibition of TLR3 repressed the poly(I-C)-induced down-regulation of Bcl-2 and up-regulation of Noxa in immortalized HUVECs. Cells were transiently transfected with human TLR3 shRNA plasmid and then treated with 2 μg/ml poly(I-C) for 24 h. The protein expression of TLR3, Bcl-2 and Noxa was detected by Western blot.

Article Snippet: Human TRAIL ELISA kit was purchased from Boster Biochemicals (Wuhan, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization, Western Blot, Inhibition, Transfection, shRNA, Plasmid Preparation