Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) Western blotting analysis of CYLD protein levels in PC12 cells transfected with small interfering RNA (siRNA) targeting CYLD (CYLD.siRNA) in the absence or presence of BAY 11-7085 administration. (B) Western blotting analysis of CYLD protein levels in PC12 cells transfected with vector expressing Flag-tagged wild-type CYLD (Flag-CYLD) in the absence or presence of IκBα.siRNA transfection. (C) MTT assay measuring cell viability of CYLD.siRNA-expressed PC12 cells exposed to CoCl 2 (left panel) or H 2 O 2 (right panel)at the indicated concentrations with or without BAY 11-7085 administration. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus Ctrl.siRNA; # p < 0.05 versus CYLD.siRNA, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) MTT assay measuring cell viability of Flag-CYLD-expressed PC12 cells exposed to CoCl 2 (left panel) or H 2 O 2 (right panel) at the indicated concentrations with or without IκBα.siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05 versus vector; # p < 0.05 versus Flag-CYLD, one-way ANOVA, post hoc comparisons, Tukey’s test. (E and F) Representative histograms and quantification of flow cytometry with Annexin-V/PI staining in PC12 cells exposed to 0.6 mmol/L CoCl 2 (E) or 0.4 mmol/L H 2 O 2 (F) with CYLD.siRNA transfection in the absence or presence of BAY 11-7085 administration and with Flag-CYLD transfection in the absence or presence of IκBα.siRNA transfection, respectively. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, one-way ANOVA, post hoc comparisons, Tukey’s test.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Western Blot, Transfection, Small Interfering RNA, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) Western-blotting analyses comparing the levels of IKKβ phosphorylation and total IκBα expression in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. (B) Coimmunoprecipitation assays examining the interaction between RelA and IκBα in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. IP, immunoprecipitation; WB, western-blotting. (C) Cellular ubiquitination assays comparing the poly-Ub levels of IκBα in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. PD, pull-down. (D) Western-blotting analyses detecting the levels of nuclear RelA accumulation in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. (E) ChIP analysis for RelA binding to VEGFA gene promoter in PC12 cells exposed to 0.6 mmol/L CoCl 2 and 0.4 mmol/L H 2 O 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. * p < 0.05; ** p < 0.01, one-way ANOVA, post hoc comparisons, Tukey’s test. (F) ELISA assay for VEGF release from PC12 cell cultures exposed to 0.6 mmol/L CoCl 2 and 0.4 mmol/L H 2 O 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. * p < 0.05 versus PBS; *** p < 0.001 versus CoCl 2 or H 2 O 2 plus miR-19a, one-way ANOVA, post hoc comparisons, Tukey’s test.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Western Blot, Phospho-proteomics, Expressing, Transfection, Cotransfection, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) ChIP analysis for RelA binding to miR-19a promoter in PC12 cells transfected with control.siRNA (Ctrl.siRNA) and IκBα.siRNA, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. ** p < 0.01. Two-sided Student’s t test was used to calculate the P value. (B) Western-blotting examining abundance of IκBα protein expression in PC12 cells transfected with control.siRNA (Ctrl.siRNA) and IκBα.siRNA, respectively. (C) RT-qPCR comparing levels of miR-19a mRNA expression in PC12 cells treated with CoCl 2 (left panel) or H 2 O 2 (right panel)for the indicated times in the presence of control.siRNA (Ctrl.siRNA) or IκBα.siRNA transfection. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus IκBα.siRNA, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) Luciferase assays of miR-19a promoter activity in PC12 cells treated with CoCl 2 (left panel) or H 2 O 2 (right panel)in the presence of control.siRNA (Ctrl.siRNA) or IκBα.siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus Ctrl.siRNA; # p < 0.05 versus Ctrl.siRNA plus CoCl 2 or H 2 O 2 , one-way ANOVA, post hoc comparisons, Tukey’s test. (E) RT-qPCR evaluating levels of miR-19a mRNA expression in CoCl 2 -treated PC12 cells with N-acetylcysteine (NAC) treatment in the presence or absence of BAY 11-7085 administration. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d. (F) RT-qPCR comparing levels of miR-19a mRNA expression in CoCl 2 -treated PC12 cells with IκBα.siRNA transfection in the presence or absence of N-acetylcysteine (NAC) treatment. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Binding Assay, Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) PC12 cells with miR-19a mimics transfection were treated with 0.6 mmol/L CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for the indicated times in the presence or absence of 100 ng/mL VEGF pretreatment and the cell viabilities were measured by MTT assay. Experiments were performed three times and data are expressed as mean ± s.d. ** p < 0.01 versus control; # p < 0.05 versus miR-19a, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) Caspase-3 activity assays of miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for 24h in the presence or absence of 100 ng/mL VEGF pretreatment. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, one-way ANOVA, post hoc comparisons, Tukey’s test. (C) Representative pictures (top panel) and quantification (bottom panel) from Hoechst and PI double-staining assay of miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for 24h in the presence or absence of 100 ng/mL VEGF pretreatment. Data are expressed as mean ± s.d. * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) Cellular ubiquitination assays comparing the poly-Ub levels of IκBα in miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) in the presence or absence of 100 ng/mL VEGF pretreatment. (E) Proposed schematic illustrating a pivotal role for miR-19a in promoting cell survival under OS by CYLD repression-mediated and NF-κB transactivation-dependent regulatory feedback loop.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Transfection, MTT Assay, Control, Activity Assay, Double Staining, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: Diagram of the 26 S proteasome showing multiple whole genome shRNA screen hits with the following color code: top hit (red), strong hit (>1 shRNA sequence per gene in both cell lines, dark orange), minor hit (1 shRNA sequence per gene in both cell lines, light orange), chymotrypsin-like proteolytic catalytic site (not a hit but highlighted for illustrative purposes, green). Each hit is labeled using the last two alphanumeric characters of the gene’s HUGO nomenclature; e.g., A1 = PSMA1, B5 = PSMB5, M1 = SHFM1.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: shRNA, Sequencing, Labeling

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: (A) Western blot showing protein levels of PSMA1 and PSMB5 in A549 and NCI-H460 NSCLC cells after PSMA1 shRNA knockdown compared to non-silencing shRNA control. (B) Chymotrypsin-like (CTL) proteasome activity assay in A549 (left) and NCI-H460 (right) NSCLC cells after treatment with bortezomib, or PSMA1 siRNA knockdown. All results are mean ± SEM and normalized to DMSO vehicle control. (C) Clonogenic survival assay of A549 (left) and NCI-H460 (right) following IR and bortezomib. Marked bars show the percent kill of bortezomib-treated samples compared to DMSO vehicle control at each IR dose. All results are mean ± SEM and normalized to DMSO vehicle control. (D) Apoptosis detection assay of NCI-H460 following 2 and 4 Gy IR and 50 nM bortezomib. Bars show percentage of cells in early apoptosis (left) or late apoptosis (right) via Annexin V and propidum iodide staining, respectively. All results are mean ± SD and P values were calculated using a two-tailed Student’s t test.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Western Blot, shRNA, Knockdown, Control, Activity Assay, Clonogenic Cell Survival Assay, Detection Assay, Staining, Two Tailed Test

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: (A) Visualization of a neutral comet assay showing IR induced DNA DSB in NCI-H460 cells 1, 4 and 8 hours after 40 Gy IR and pre-treated with 50 nM bortezomib compared to DMSO vehicle control. (B) Quantification of the neutral comet assay via olive moment (left) and % DNA in tail (right) at 1, 4 and 8 hours post 40 Gy IR with and without 50 nM bortezomib in NCI-H460 cells. Each data point represents 3 independent replicate experiments of at least 50 cells. All results are mean ± SD and P values were calculated using a two-tailed Student’s t test. (B) GFP reporter assay for homologous recombination after proteasome inhibition for 24 hours via bortezomib or PSMA1 siRNA knockdown in A549 (left) and NCI-H460 (right). All results are mean ± SD and normalized to DMSO vehicle control (Veh) or nonsilencing siRNA control. P values were calculated using a two-tailed Student’s t test.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Neutral Comet Assay, Control, Two Tailed Test, Reporter Assay, Homologous Recombination, Inhibition, Knockdown

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: (A) Diagram of NF-kB promoters on FANCD2 and BRCA1 genes. (B) Expression by qPCR of FANCD2 following proteasome inhibition ±30 nM (A549) or 50 nM (NCI-H460) bortezomib or ± inducible PSMA1 shRNA, and ±10 Gy IR. All values are normalized to ACTB and all results are mean ± SEM. (C) As in (B) but with BRCA1. (D) NSCLC cell survival following bortezomib and IR is partially rescued by IkBα siRNA knockdown when performed in advance. Cell viability was assayed using an ATP luminescence-based assay measuring relative luminescent units (RLU). All results are mean ± SEM.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Expressing, Inhibition, shRNA, Knockdown, Luminescence Assay

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: Proteasome inhibition by bortezomib or PSMA1 knockdown results in an increase in IκBα, which in turn decreases NF-κB binding to the promoters of FA/HR genes including FANCD2 and BRCA1 . This reduces the availability of these DNA repair proteins for recruitment to DNA damage sites, resulting in decreased RAD51 focus formation and HR following induction of DNA double strand breaks by ionizing radiation.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Inhibition, Knockdown, Binding Assay

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: 10 6 NCI-H460 cells transfected with doxycycline-inducible PSMA1 shRNA were injected into the flanks of 6–8 week-old NCr nude mice. Once tumors reached 3 mm diameter (day 0), PSMA1 knockdown was initiated with doxycycline drinking water. One week later, RT was initiated to give a total of five 4 Gy fractions every other day using a small animal radiation research platform (SARR). (A) Orthogonal images from a cone beam computed tomography (CT) scan, obtained using the SARRP, of a mouse bearing a subcutaneous xenograft. (B) Correlation of volumetric tumor measurements using the SARRP cone beam CT compared to traditional calipers. (C) Treatment schema. (D) Mice were subsequently followed until tumors reached 2 cm diameter, animals became moribund or for 100 days. (E) Kaplan-Meier analyses were performed with pairwise log-rank tests to assess differences in survival. Numbers surviving out of 10 mice per group on day 100 are indicated in brackets. For RT vs. RT+ PSMA1 knockdown, log rank P = 0.0003.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Transfection, shRNA, Injection, Knockdown, Computed Tomography

Journal: PLoS ONE

Article Title: Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0073710

Figure Lengend Snippet: (A) FANCD2 immunofluorescence in 10 Gy irradiated vs. unirradiated NCI-H460 xenografts recovered from mice with or without doxycycline-induced PSMA1 shRNA expression in tumor cells. Bar = 10 µm. (B) γ-H2AX immunohistochemistry in NCI-H460 xenograft tumors with or without doxycycline-induced PSMA1 shRNA knockdown, recovered from mice 1, 6, and 24 hours after 10 Gy irradiation. Bar = 10 µm. (C) Quantification of immunohistochemistry for γ-H2AX in (B). Cells with ≥5 foci were scored as positive (n>400 cells). All results are mean ± SEM. P values were calculated using a two-tailed Student’s t test.

Article Snippet: A549 or NCI-H460 cells were seeded into 6-well tissue culture dishes and then transfected with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (Qiagen).

Techniques: Immunofluorescence, Irradiation, shRNA, Expressing, Immunohistochemistry, Knockdown, Two Tailed Test

Journal: Cell Death Discovery

Article Title: NF-κB inhibition by dimethylaminoparthenolide radiosensitizes non-small-cell lung carcinoma by blocking DNA double-strand break repair

doi: 10.1038/s41420-017-0008-3

Figure Lengend Snippet: a Luciferase NF-κB activity assay in A549 cells treated with a range of DMAPT doses with or without 10 Gy ionizing radiation (IR). Cells were transfected with a non-phosphorylatable IκBα construct (NF-κB super-repressor (SR)) to inhibit NF-κB signaling as a positive control. Bars show mean ± SD ( n = 3). * indicates p < 0.05 by Tukey multiple comparison test. b Western blot showing IκBα Ser32 phosphorylation levels following DMAPT treatment in A549, NCI-H460, and NCI-H1299 cells. Cells were treated with DMAPT for 24 h and bortezomib for 1 h prior to harvest; bortezomib was added to permit visualization of the otherwise rapidly degraded protein. c Cell cycle analysis of A549 cells following DMAPT treatment and/or 10 Gy IR. Cells were harvested 6 h following IR or incubation without IR and analyzed by propidium iodide staining. d Quantification of c . Bars show mean ± SD ( n = 3). e Western immunoblotting for cleaved PARP in A549 cells treated with 2 Gy IR and/or 15 µM DMAPT to assess induction of apoptosis

Article Snippet: For the pSer32 specificity assay, A549 cells were seeded into six-well tissue culture dishes and then transfected using Lipofectamine RNAiMAX Reagent (56532, Qiagen) with 25 nM IκBα siRNA (#1 = si00126826, #2 = si03114630, Qiagen) or AllStars Negative Control siRNA (102781, Qiagen).

Techniques: Luciferase, Activity Assay, Transfection, Construct, Positive Control, Comparison, Western Blot, Phospho-proteomics, Cell Cycle Assay, Incubation, Staining

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Transfection efficiency of IκBα siRNA. To monitor siRNA transfection efficiency, the HCM cells were transfected with Cy3-labled IκBα siRNA using Lipofectamine 2000 reagent, which were viewed at 24 h, 48 h, and 72 h after transfection. Cy3-labled IκBα siRNA (red) was observed in the cytoplasm in HCM cells, and the transfection efficiency was 92% ( A ), 90% ( B ), and 91% ( C ) 24 h, 48 h, and 72 h after IκBα siRNA transfection, respectively.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Transfection

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Examination of IκBα mRNA and protein levels in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : IκBα mRNA expression was quantified by real-time RT–PCR. Expression levels were normalized with GAPDH . Error bars represent standard deviations (SD) calculated from three parallel experiments. B : Total cell lysates from HCM cells treated with IκBα siRNA, nonsense control siRNA (NC), and control were analyzed by western blot with IκBα antibody and GAPDH antibody. The arrows indicate IκBα (39 kDa) and GAPDH (36 kDa) bands. The bands were analyzed densitometrically, and the values were normalized with GAPDH, which are represented in the bar graph. The mRNA and protein values of IκBα siRNA, control, and nonsense control siRNA (NC) groups were determined by one-way ANOVA. The double asterisk denotes p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of ablation of IκBα on MMP-2 expression and activity 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : MMP-2 mRNA expression in in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : After IκBα siRNA transfection, the conditioned media were collected at the indicated time, concentrated, and analyzed by western blot with MMP-2 antibody. The arrow indicates MMP-2 bands that are analyzed by densitometry and the values were represented in the bar graph. C : The activity of MMP-2 is analyzed by gelatin zymography analysis. The arrows indicate pro-MMP-2 (72-kDa) and active MMP-2 (66-kDa) specific bands. The bands were analyzed by densitometry and are represented in the bar graph. The mRNA, protein, and activity values of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control were determined by one-way ANOVA. An asterisk denotes p<0.05, and a double asterisk indicates p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Expressing, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Zymography

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of knockdown IκBα on TIMP-2 and MT1-MMP expression in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. TIMP-2 ( A ) and MT1-MMP ( C ) mRNA expression in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : After IκBα siRNA transfection, the conditioned media were collected at the indicated time, concentrated, and analyzed by western blot with TIMP-2 antibody. The arrow indicates TIMP-2 (21 kDa). The bands were analyzed by densitometry and represented in the bar graph. D : Total cell lysates from HCM cells transfected with IκBα siRNA , nonsense control siRNA (NC) as well as from the control, respectively, were analyzed by western blot with MT1-MMP antibody. The arrows show MT1-MMP (66 kDa) and internal control, GAPDH (36 kDa). The bands were analyzed by densitometry and represented in the bar graph. The mRNA, protein, and activity values of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control were determined by one-way ANOVA. An asterisk denotes p<0.05, and a double asterisk indicates p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of knockdown IκBα on the expression and cellular localization of NF-κBp65 in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : NF-κBp65 mRNA expression in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : Nuclear proteins extracted from IκBα siRNA, nonsense control siRNA (NC), as well as control cells, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrow indicates the NF-κBp65 (75 kDa) band and nucleus internal control, Lamin B (67 kDa). The bands were analyzed densitometrically, and the values were normalized with Lamin B, represented in bar graph. C : Total cell lysates from HCM cells transfected with IκBα siRNA, and nonsense control siRNA (NC) as well as from the control, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrows show the NF-κBp65 (75 kDa) band and the internal control, GAPDH (36 kDa). The mRNA and protein values compared to the control and nonsense control siRNA (NC) were determined by one-way ANOVA. The double asterisk denotes p<0.01. D : The HCM cells were immunostained with NF-κBp65 antibody and analyzed by fluorescence microscopy. A weak nuclear signal of NF-κBp65 (green) was observed in control cells at 24 h, 48 h, and 72 h. After IκBα siRNA transfection, NF-κBp65 translocated from the cytoplasm into the nucleus, and a strong signal of NF-κBp65 was detected in the nucleus at 24 h, 48 h, and 72 h. Cy3 labled IκBα siRNA (red) was observed in the cytoplasm. Cell nuclei were counterstained with DAPI (blue).

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Fluorescence, Microscopy