Journal: The FASEB Journal

Article Title: The deubiquitinating enzyme USP48 stabilizes TRAF2 and reduces E-cadherin-mediated adherens junctions

doi: 10.1096/fj.201700415RR

Figure Lengend Snippet: Knockdown of USP48 or inhibition of TNIK and JNK promotes epithelial barrier integrity. A) Scheme showing principle of ECIS system. B) Knockdown of USP48 increases TEER in A549 cells. A549 cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA. After transfection, changes in TEER at 0 to 36 h were measured with ECIS. Data are presented as means ± sd (n = 4). C) Knockdown of USP48 increases TEER in Beas2B cells. Beas2B cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA. After transfection, changes in TEER at 48 h were measured with ECIS. Data are presented as means ± sd (n = 4). P < 0.01 compared to control siRNA–transfected cells. The rest of the cells were analyzed by USP48, TRAF2, and β-actin immunoblotting. D) Inhibition of TNIK or JNK increases TEER. Beas2B cells cultured on ECIS gold electrodes were treated with DMSO (10 μM), KY-05009 (10 μM), or SP600125 (10 μM), and changes in TEER at 0 to 36 h were measured with ECIS. Data are presented as means ± sd, n = 4. E) Knockdown of USP48 promotes LPA-increased TEER. Human bronchial epithelial cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA and incubated for 3 d. Cells were then challenged with LPA (0.5 μM), and changes in TEER were measured with ECIS. F) Scheme showing GSK3β phosphorylates and activates USP48, thus stabilizing TRAF2 and influencing TNIK/JNK-mediated E-cadherin expression and epithelial barrier integrity.

Article Snippet: Human lung epithelial cells [Beas2B and human bronchial epithelial cells; American Type Culture Collection (ATCC), Manassas, VA, USA] and murine lung epithelial 12 (MLE12) cells (ATCC) were cultured with medium supplemented with hydrocortisone, insulin, transferrin, estrogen, selenium, 10% fetal bovine serum, and antibiotics at 37°C in 5% CO 2 incubator.

Techniques: Knockdown, Inhibition, Cell Culture, Transfection, Control, Western Blot, Incubation, Expressing

Journal: Endocrinology

Article Title: Dexamethasone and tumor necrosis factor-alpha act together to induce the cellular inhibitor of apoptosis-2 gene and prevent apoptosis in a variety of cell types.

doi: 10.1210/en.2002-220188

Figure Lengend Snippet: FIG. 2. Dexamethasone and TNF- act in an additive or more than additive manner to drive transcription from the human c-IAP2 promoter. A549 cells were transfected with IAP(947–54)-LUC and treated for 24 h with the indicated concentrations of dexamethasone (A and B), dexamethasone with RU486 (B), or dexamethasone with TNF- (A, 0 ng/ml, closed square; 0.01 ng/ml, open circle; 0.1 ng/ml, inverted closed triangle; 1 ng/ml, open square, and 10 ng/ml, closed circle). Cells were lysed, and relative light units (RLU) were measured. Representative experiments from three separate assays are shown.

Article Snippet: A549 human lung epithelial cells and A172 human glioblastoma cells were obtained from ATCC (Manassas, VA) and cultured in DMEM containing 10% FBS. (Life Technologies, Inc., Gaithersburg, MD) at 37 C with 5% CO2 in a humidified incubator.

Techniques: Transfection

Journal: Endocrinology

Article Title: Dexamethasone and tumor necrosis factor-alpha act together to induce the cellular inhibitor of apoptosis-2 gene and prevent apoptosis in a variety of cell types.

doi: 10.1210/en.2002-220188

Figure Lengend Snippet: FIG. 4. Dexamethasone and TNF- act together to abrogate apoptosis in human lung A549 cells. A549 cells were treated for 24 h with TNF- (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. The ADP to ATP ratio was calculated and expressed as a percent (A), and caspase 8 activity was measured by absorbance of a cleaved colorimetric product at 405 nm (B). Data shown represent the average of three separate experiments.

Article Snippet: A549 human lung epithelial cells and A172 human glioblastoma cells were obtained from ATCC (Manassas, VA) and cultured in DMEM containing 10% FBS. (Life Technologies, Inc., Gaithersburg, MD) at 37 C with 5% CO2 in a humidified incubator.

Techniques: Activity Assay

Journal: Endocrinology

Article Title: Dexamethasone and tumor necrosis factor-alpha act together to induce the cellular inhibitor of apoptosis-2 gene and prevent apoptosis in a variety of cell types.

doi: 10.1210/en.2002-220188

Figure Lengend Snippet: FIG. 3. Human cIAP-2 promoter responsiveness to dexamethasone is conferred by a GRE at –513. A549 cells were transfected with IAP(395– 54)-LUC (A) or IAP(947–54mutGRE)-LUC (B) and treated for 24 h with TNF- (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. Cells were lysed, and relative light units (RLU) were measured. Representative experiments from three separate assays are shown.

Article Snippet: A549 human lung epithelial cells and A172 human glioblastoma cells were obtained from ATCC (Manassas, VA) and cultured in DMEM containing 10% FBS. (Life Technologies, Inc., Gaithersburg, MD) at 37 C with 5% CO2 in a humidified incubator.

Techniques: Transfection

Journal: PLoS Pathogens

Article Title: DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection

doi: 10.1371/journal.ppat.1007125

Figure Lengend Snippet: A. A549 cells were transfected for 60h with targeting siRNAs specific for DDX3 or scrambled siRNA controls followed by infection with LCMV Cl13 (M.O.I. 0.005). Viral titers in cell culture supernatants harvested 24 and 72 h.p.i. are shown. B. A549 cells were transfected for 24h with plasmids encoding LCMV-NP-HA, LASV-NP-HA or HA-USP14, or infected with 3rLCMV-HA-GFP, lysed and immunoprecipitated with anti-HA agarose beads (IP HA); eluates were analyzed by Immunoblot (IB). Immunoblots with anti-DDX3 and anti-GAPDH (load control) Abs were performed in input samples. C. LCMV infected A549 cells (M.O.I 1) were stained for DDX3 (green), LCMV NP (red) and DAPI and processed for Confocal Microscopy. Graphs on the right represent overlapping NP and DDX3 fluorescence intensities. D. Schematics of the genome of rLCMV-NP-HA. White: ORFs of viral proteins. Pink: HA tag. Black: viral untranslated regions. E. A549 cells were infected with rLCMV-NP-HA or 3rLCMV-HA-GFP for 24h, lysed in buffer containing RNAseA 0.1 mg/ml, immunoprecipitated (IP) and analyzed by Immunoblot (IB). (>) indicates NP-HA cleaved band. F. Viral titers in supernatants from DDX3 ko-1, DDX3 ko-2, WT-pCas9 (control) and WT A549 cells infected with LCMV Cl13 (M.O.I. 0.5) were quantified at 24, 48 and 72 h.p.i. G. DDX3 ko-1 and WT A549 cells transduced with RV expressing DDX3 or empty-RV were infected with LCMV Cl13 (M.O.I. 0.5) for 24h and viral RNA levels ( lcmvgp) were determined relative to gapdh by RT-qPCR and represented as relative fold expression. (B & E) Numbers on the right: MW (kDa). Data are representative of 2 ( A, E and G ) or 4 ( B, C and F ) independent experiments. * p<0.05, ** p<0.01, ***p<0.001. Stars colors represent: DDX3 vs. Scr1 (blue) or Scr2 (black) ( A ) and WT A549 vs DDX3 ko-1 (red) or vs DDX3 ko-2 (black) ( F ).

Article Snippet: A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza).

Techniques: Transfection, Infection, Cell Culture, Immunoprecipitation, Western Blot, Control, Staining, Confocal Microscopy, Fluorescence, Transduction, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection

doi: 10.1371/journal.ppat.1007125

Figure Lengend Snippet: A to C. DDX3 ko-1, DDX3 ko-2 and WT A549 cells were infected with LASV (strain Josiah) at low M.O.I. (0.01, 0.05 and 0.1) for 48h or at high M.O.I. (0.5 and 3) for 24h. A-B. Cells were stained with Hoechst and anti-LASV antibodies, for confocal microscopy. A. Representative images are shown for infections at M.O.I. = 0.1. B . Percentage of infected cells were calculated by high-content quantitative image-based analysis. Left panel: GP + cells at 48 h.p.i.; Right panel: NP + cells at 24 h.p.i. C. Absolute copy numbers of viral RNA were determined in tissue culture supernatants by qRT-PCR. Representative results are shown for infections at M.O.I. = 0.01. D. DDX3 ko-1, DDX3 ko-2 and WT A549 cells were transduced with empty-RV (EV-RV) or RV encoding DDX3 (DDX3-RV) prior to LASV infection and then processed as in B . All data are representative of 2 independent experiments. * p<0.05, ** p<0.01, ***p<0.001. Star colors represent: WT-A549 vs. DDX3 ko-1 (red) or vs. DDX3 ko-2 (black) ( B ) and DDX3 ko-1+EV-RV vs DDX3 ko-1+DDX3-RV (red) or DDX3 ko-2+EV-RV vs DDX3 ko-2+DDX3-RV (black) ( D ).

Article Snippet: A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza).

Techniques: Infection, Staining, Confocal Microscopy, Quantitative RT-PCR, Transduction

Journal: PLoS Pathogens

Article Title: DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection

doi: 10.1371/journal.ppat.1007125

Figure Lengend Snippet: DDX3 ko-1, DDX3 ko-2 and WT A549 cells were infected with LCMV Cl13 (M.O.I. 0.5) for the indicated times. Total RNA in cell lysates was extracted and normalized interferon transcript levels ( ifnb/gapdh) determined by qRT-PCR and represented as relative fold expression. B. DDX3 ko-1 and WT A549 cells were transduced with empty-RV (EV-RV) or RV encoding DDX3 (DDX3-RV) before infection, and processed as in A for quantification of ifnb/gapdh transcripts via qRT-PCR ( B, left panel) or determination of bioactive IFN-I levels in cell culture supernatants at indicated h.p.i. ( B, right panel). C. DDX3 ko-1 and WT A549 cells were pre-incubated for 2 h and infected with LCMV Cl13 (M.O.I. 0.5) in the presence of anti-IFNAR mAb (IFNAR Ab, solid lines) or Isotype control (Iso Ab, broken lines), which were left for the remaining of the culture. Viral titers were determined at indicated h.p.i. D . Translation assay performed in HEK-293T cells treated with DDX3 or scrambled (Scr) siRNA. E. Minireplicon assay performed in DDX3 ko-1 or WT A549 cells. F and G. DDX3 ko-1 or WT cells were transfected with 0.4 μg of empty plasmid, plasmid expressing DDX3 ( F-G ) or the indicated point-mutant DDX3 ( G ) and used for minireplicon assay. 100% value was given to WT A549 transfected with empty plasmid. Data are representative of 2 ( A-C & E-F ) or 3 ( D & G ) independent experiments. * p<0.05, ** p<0.01, ***p<0.001. Stars represent: DDX3ko vs WT (blue) or DDX3 ko at the indicated h.p.i. versus DDX3 ko at time = 0 (red) ( A ), DDX3 ko-EV vs WT-EV (black) or vs DDX3 ko-RV-DDX3 (red) ( B ), DDX3 ko-IFNAR vs WT-IFNAR (black) or vs DDX3 ko-Isotype (red) ( C ).

Article Snippet: A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza).

Techniques: Infection, Quantitative RT-PCR, Expressing, Transduction, Cell Culture, Incubation, Control, Transfection, Plasmid Preparation, Mutagenesis

Journal: PLoS Pathogens

Article Title: DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection

doi: 10.1371/journal.ppat.1007125

Figure Lengend Snippet: A. A549 cells were transfected with plasmid encoding JUNV NP-HA for 24h, lysed and immunoprecipitated with anti-HA agarose beads (IP HA). Eluates (upper panel) or input samples (load control, lower panel) were analyzed by Immunoblotting (IB) with anti-DDX3 or anti-GAPDH, respectively. B-I. DDX3 ko-1, DDX3 ko-2 and WT A549 cells were infected with JUNV Candid 1 ( B-F ) or Romero ( G-I ) strains at the indicated M.O.I. for 24h. Cells were stained with anti-GP antibodies and Hoechst, for confocal microscopy. Representative images for infected cells are shown ( D ). Numbers of infected cells were determined by high-content quantitative image-based analysis ( B - C and G and I ). Normalized viral RNA levels ( junv/gapdh ) represented as relative fold expression ( E and F ) and absolute copy numbers ( H ) of viral RNA in tissue culture supernatants were determined by qRT-PCR. When indicated, ( C, F and I ) DDX3 ko and WT A549 cells were transduced with empty-RV (EV-RV) or RV encoding DDX3 (DDX3-RV) before infection, and processed for confocal microscopy (C and I) or qRT-PCR (F) . Data are representative of 2 independent experiments. * p<0.05, **p<0.01,*** p<0.005, ****p<0.001. Star colors represent: DDX3 ko-EV vs WT-EV (black) or vs DDX3 ko-RV-DDX3 (red) ( C ), WT vs DDX3 ko-1 (red) or DDX3 ko-2 (black) ( E & G ) and DDX3 ko-1 + EV-RV vs DDX3 ko-1 + DDX3-RV (red) or DDX3 ko-2 + EV-RV vs DDX3 ko-2 + DDX3-RV (black) ( I ).

Article Snippet: A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control, Western Blot, Infection, Staining, Confocal Microscopy, Expressing, Quantitative RT-PCR, Transduction

Journal: Molecular Neurobiology

Article Title: Resveratrol Can Attenuate Astrocyte Activation to Treat Spinal Cord Injury by Inhibiting Inflammatory Responses

doi: 10.1007/s12035-021-02509-4

Figure Lengend Snippet: Application of RSV in various diseases

Article Snippet: Staphylococcus aureus , S. aureus / A549 human lung epithelial cells (ATCC CCL 185) , S. aureus strains were cultured in media with RSV/ ATCC CCL 185 were cultured in media with treated strain of S. aureus , RSV effectively inhibited the expression of Hla and alleviated the cell damage of ATCC CCL 185 co-cultured by bacteria. , Anti-apoptosis , [ ] .

Techniques: Capsules, Injection, Expressing, Modification, In Vitro, Transgenic Assay, Cell Culture, Suspension, Saline, Derivative Assay, Functional Assay, Activation Assay, Transfection, Bacteria

Journal: International Journal of Molecular Sciences

Article Title: TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract

doi: 10.3390/ijms232214431

Figure Lengend Snippet: Downregulation of HTRA1 directly activated the TGF-β/Smad signalling in primary HLECs. ( A ) Primary HLECs after LV-HTRA1-RNAi lentivirus transfection, the middle image showed the GFP expression, and the right image showed the mRNA levels of HTRA1 after transfection. ( B , C ) Changes of HTRA1, Smad2/3, p-Smad2/3, TGF-β1 and TGF-β2 gene expression detected by Western blotting (Smad2/3 p > 0.05, p-Smad2/3 p = 0.014, TGF-β1 p = 0.021, TGF-β2 p = 0.039) in primary HLECs collected from PSC eyes after treatment of HTRA1 knockdown ( Htra1 KD). ( D ) Changes of p-Smad2/3 expression detected by Western blotting with HTRA1 knockdown ± TGF-βR1/2 inhibitor treatment (10 ng/mL for 24 h) in primary HLECs (p-Smad2/3 p = 0.042, 0.027, respectively). ( E ) The mRNA levels of α-SMA, PAI-1 and CTGF elevated in HLECs after treatment of HTRA1 knockdown ( p = 0.022, 0.005, and 0.038, respectively). ( F ) The protein levels of FN-1, and α-SMA in HLECs after treatment of HTRA1 knockdown ( p = 0.017 and 0.031, respectively). * p < 0.05, *** p < 0.001.

Article Snippet: Primary HLECs were treated with recombinant human TGF-β1 (7666-MB-005, R&D Systems, Minneapolis, MN, USA) in a concentration gradient of 10 ng/mL for a duration of 24 h. The selective TGF-βR1/2 inhibitors (LY2109761, Selleck, Shangai, China) were added to primary HLECs after lentivirus transfection at a working concentration of 10 μM for 24 h.

Techniques: Transfection, Expressing, Gene Expression, Western Blot, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract

doi: 10.3390/ijms232214431

Figure Lengend Snippet: The proliferation, migration and apoptosis changes of HLECs (SRA 01/04) after treatment of HTRA1 knockdown. ( A ) CCK-8 assay showed promoted growth ability of HLECs after downregulated expression of HTRA1 ( p < 0.001) and was attenuated after treatment of HTRA1 knockdown and the TGF-βR1/2 inhibitor ( p < 0.001). ( B ) Annexin V/7-AAD staining and flow cytometry did not show significant apoptotic cell changes after treatment of HTRA1 knockdown. ( C ) The protein levels of BAX, Bcl2 and cleaved Caspase-3 in HLECs after treatment of HTRA1 knockdown ( p > 0.05). ( D ) The scratch healing assay showed knockdown of HTRA1 promoted the migration of HLECs ( p = 0.022), and was attenuated after treatment of HTRA1 knockdown and the TGF-βR1/2 inhibitor. * p < 0.05, *** p < 0.001.

Article Snippet: Primary HLECs were treated with recombinant human TGF-β1 (7666-MB-005, R&D Systems, Minneapolis, MN, USA) in a concentration gradient of 10 ng/mL for a duration of 24 h. The selective TGF-βR1/2 inhibitors (LY2109761, Selleck, Shangai, China) were added to primary HLECs after lentivirus transfection at a working concentration of 10 μM for 24 h.

Techniques: Migration, Knockdown, CCK-8 Assay, Expressing, Staining, Flow Cytometry

Journal: Cancer Research

Article Title: Cancer-Induced Muscle Wasting Requires p38β MAPK Activation of p300

doi: 10.1158/0008-5472.can-19-3219

Figure Lengend Snippet: Figure 1. Site-specific phosphorylation of p300 on Ser-12 induced by LLC is necessary and sufficient for the activation of C/EBPb and muscle wasting. A, LLC induces p300 phosphorylation on Ser-12 and this reaction is critical to p300-mediated acetylation and activation of C/EBPb. C2C12 myoblasts were transfected with a plasmid- encoding phosphorylation-defective mutant of p300, p300-S12A, or p300-S89A, or empty vector as control in three independent experiments. After differentiation, myotubeswere treated with LCM or conditioned medium of nontumorigenic NL20 cells for 2 hours. Cell lysates were analyzed by Western blotting. B,Overexpression of p300-S12A attenuates muscle weight loss in LLC tumor–bearing mice. TA muscle of LLC tumor–bearing mice was transfected with the p300-S12A–encoding plasmid (n ¼ 5). The contralateral TA muscle was transfected with empty vector as control. After the development of cachexia, TA muscles were collected and weighed on day 21. Overexpression of p300-S12A was confirmed by Western blotting against p300. C, Overexpression of p300-S12A prevents the loss of myofiber mass in LLC tumor–bearing mice. H&E–stained TA cross-sections of B were analyzed for myofiber cross-sectional area. D, Overexpression of p300-S12A in TA muscles attenuates muscle weight loss in KPC tumor–bearing mice (n ¼ 5). E, Overexpression of p300-S12A in TA muscles prevents the loss of myofiber cross- sectional area of KPC tumor–bearing mice (n ¼ 5). F, Overexpression of p300-S12D in TA muscles causes loss of muscle weight in cancer-free mice (n ¼ 5). G, Overexpression of p300-S12D in TA muscles causes loss of myofiber cross-sectional area in cancer-free mice (n ¼ 5). , P < 0.05, a statistically significant difference determined by one-way ANOVA (A), paired Student t test (B, D, and F), or x2 test (C, E, and G).

Article Snippet: Conditionedmedium of nontumorigenic NL20 cells (human lung epithelial cells, ATCC) was used as control.

Techniques: Phospho-proteomics, Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, Control, Western Blot, Over Expression, Muscles, Staining

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of bromfenac on TGF-β2-induced EMT and cell migration in HLEC-B3 cells: the FN (A), MMP2 (B), and α-SMA (C) mRNA expression induced by 10 ng/mL TGF-β2 at 2 h, 12 h, and 24 h, measured by qPCR (*p < 0.05, **p < 0.01 vs TGF-β2 free group); effects of bromfenac in different concentrations on the TGF-β2-induced upregulation of FN (D), MMP2 (E), and α-SMA (F) mRNA expression measured by qPCR (*p < 0.05, **p < 0.01 vs TGF-β2 treated alone group); (G) FN, MMP2, and α-SMA protein expression induced by 10 ng/mL TGF-β2 for different time points detected by Western blot; (H) effects of bromfenac in different concentrations on the TGF-β2-induced upregulation of FN, MMP2, and α-SMA protein expression detected by Western blot. The cells were pre-treated, with or without bromfenac, for 24 h before induced by 10 ng/mL TGF-β2 for 24 h. (I) the wound-healing assay showed the effects of bromfenac in different concentrations on TGF-β2-induced cell migration (scale bar, 500 μm; n = 3; error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Migration, Expressing, Western Blot, Wound Healing Assay

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Immunofluorescence staining of the change of cellular EMT markers: 80 μg/mL bromfenac inhibited the upregulation of FN (A), MMP2 (B), and α-SMA (C) induced by 10 ng/mL TGF-β2 in HLEC-B3 cells (scale bar, 100 μm).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of bromfenac on the TGF-β2-activated ERK/GSK-3β pathway in HLEC-B3. U0126 suppressed TGF-β2-induced up-regulation of the EMT markers FN, MMP2, α-SMA, and Snail. (A) TGF-β2 stimulated the phosphorylation of ERK1/2 and GSK-3β, and (B) bromfenac significantly inhibited phosphorylation. The cells were pre-treated with or without bromfenac for 24 h before induced by 10 ng/mL TGF-β2. (C) TGF-β2 stimulated the phosphorylation of Smad2/3, but (D) bromfenac could not inhibit phosphorylation; and pre-treatment with U0126 for 2 h suppressed the TGF-β2-induced upregulation of FN (E), MMP2 (F), α-SMA (G), and Snail (H) mRNA, and protein expression (I) (*p < 0.05, **p < 0.01 vs TGF-β2 treated alone group, n = 3, error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of U0126 and CHIR-99021 on TGF-β2-induced EMT in HLEC-B3 and the role of Snail in TGF-β2-induced EMT. The cells were treated with 20 nM CHIR-99021 for 24 h and 20 μM U0126 for 2 h before 10 ng/mL TGF-β2 treatment. The mRNA (A, B, C, and D) and protein (E) expression of FN, MMP2, α-SMA, and Snail detected by qPCR and Western blot analysis, respectively (*p < 0.05, **p < 0.01 between groups); (F) the TGF-β2-induced phosphorylation of GSK-3β after treatment with CHIR-99021 and U0126 and evaluated by Western blot analysis. The cells were pre-treated with or without bromfenac for 24 h before induced by 10 ng/mL TGF-β2, and Snail expression (G) was detected by Western blot analysis. The cells were transfected with Snail siRNA or NC siRNA before bromfenac and 10 ng/mL TGF-β2 treatment. The protein (H) and mRNA (I, J, and K) expression of FN, MMP2, and α-SMA detected by qPCR and Western blot analysis, respectively (*p < 0.05, **p < 0.01 between groups). NS represents no significance compared with the Snail siRNA + TGF-β2 treated alone group (n = 3, error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Expressing, Western Blot, Phospho-proteomics, Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A and B, mRNA expression levels of LEDGF/p75 ( black bars ) and Sp1 ( gray bars ) were analyzed by real time PCR. Total RNA was isolated from LECs separated from lenses of human subjects of different age groups and reverse transcribed cDNA was subjected to real time PCR analysis with specific primers as detailed in . Age group 1 (n = 4, 16–26 years); Age group 2 (n = 3, 34–42 years); Age group 3 (n = 7, 52–75 years). n; denotes number of subjects. The data represent the mean ± S.D. from three independent experiments (** p<0.001 ). C, Western analysis of LEDGF/p75 and Sp1 protein using their corresponding specific antibodies. hLECs isolated from eye lenses of 24- and 64-year-old human subjects were cultured as described in . Cellular proteins from confluent cells were extracted, and equivalent amounts were loaded onto SDS-PAGE, transferred to a PVDF membrane and processed for immunoblotting. Western analysis showed the expression levels of LEDGF/p75 ( upper panel) and Sp1 ( middle panel). Lower panel, membrane probed with β-actin antibody as loading/internal control. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Each band of blot was quantified using densitometer shown at the right . Images are representatives from three independent experiments. D and E, Sp1 upregulated expression of LEDGF/p75 protein and mRNA in hLECs in dose dependent fashion. hLECs were transfected with either pCMV-vector or increasing amounts of pCMV-Sp1 (2, 4 and 8 µg) as indicated and described in section. Total Protein and RNA were extracted after 48 h of transfection and were used for Western analysis (D) and real time PCR (E) respectively, using specific probes. D, left , Western analysis data showing the expression levels of LEDGF/p75 ( upper panel) in cells transfected with plasmid encoding Sp1 at different concentrations ( middle panel). Lower panel, membrane probed with β-actin antibody. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Right , Histogram displaying relative protein band density indicated as values ± S.D. of three independent experiments. E, Histogram showing the values (mean ± S.D.) of Sp1 concentration-dependent expression of LEDGF/p75 mRNA (black bars vs gray bars) obtained from three independent experiments (** p <0.001). F, A Sp1 inhibitor, artemisinin, reduced expression of LEDGF/p75 in LECs in dose-dependent manner. Cultured cells were treated with either increasing concentrations of artemisinin (50, 150 and 300 µM) or with vehicle control. Cell lysates were resolved onto SDS-PAGE and analyzed by Western blot for the effects of artemisinin on expression of LEDGF/p75 and Sp1 protein. Relative band density in pixels is shown below the Western blot images (* p <0.01, ** p <.001). β-actin was used as internal control. G and H, Representative immunoblots showing depletion of Sp1 using Sp1 Knockdown assay. Sp1-specific shRNA constructs were transiently (G) and stably (H) transfected as described in section. Protein lysate was prepared and Western analysis was carried out. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1 or LEDGF/p75 or β-actin. Relative band density in pixels is shown below the Western blot images (** p <.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Reverse Transcription, Western Blot, Cell Culture, SDS Page, Membrane, Control, Stripping Membranes, Transfection, Plasmid Preparation, Concentration Assay, Knockdown, shRNA, Construct, Stable Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Left half, diagrams showing the 5′-deletion constructs of LEDGF/p75 promoter linked to CAT reporter gene used for transient transfections. Right half, CAT activity of the LEDGF/p75 promoter deletion constructs and empty CAT vector in hLECs. 5′-deletion mutant constructs and pGFP were cotransfected into hLECs. 48 h later, protein was extracted and CAT activity was measured. CAT activity ( right ) was normalized to GFP readings (O.D.). The data represent the mean ± S.D. from three independent experiments. B, Point mutation analysis showing Sp1 site-dependent transcriptional activity of the LEDGF/p75 gene promoter in hLECs. Left half, schematic representation of Sp1-site-directed mutants of LEDGF/p75 promoter linked to CAT. Right half, CAT activity of the wild-type (WT) and its mutant constructs (Mut-3, Mut-2, Mut-1 and Mut- 1+2+3) and empty CAT vector in hLECs. All data are presented as the mean ± S.D. derived from three independent experiments (* p <0.01, ** p <0.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Construct, Transfection, Activity Assay, Plasmid Preparation, Mutagenesis, Derivative Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Representative gel-shift mobility assays showing Sp1 binding to radiolabeled oligonucleotide probes containing consensus Sp1 sites as indicated. Nuclear extracts isolated from hLECs were incubated with 32 p-labeled probes containing Sp1 binding sites (WT-probes) or their corresponding mutants (Mut probes). Nuclear extracts bound to oligos containing Sp1 sites and yielded to complex, Sp1/DNA (Cm1) (A, lanes 1, 3 and 5). No complex occurred with mutant probes (A, lanes 2, 4, and 6). The oligonucleotide probes of both wild-type and mutated sequence used in assay are shown adjacent to image. B, Gel-shift assay showing the binding of Sp1 in nuclear extract of Sp1 overexpressed with hLECs to 32 p-labeled probes with its site. Nuclear extract isolated from cells transfected with plasmid encoding Sp1 or its corresponding vector was incubated with WT-probe1 or standard control probe (sc-2502; Santa Cruz Biotech). The DNA-protein complex was resolved on a 5% acrylamide gel. A discrete Sp1 expression-dependent DNA-protein complex was observed (B; lanes 1 vs 3) in comparison to vector transfected cells (lane 1), while the mutated probe failed to generate the complex (B, lanes 2 and 4). B, Right (lanes 5 and 6) , depletion of endogenous Sp1 with its specific antibody. Nuclear extracts were incubated with either anti-Sp1 antibody (lane 6) or normal rabbit IgG (lane 5), and recovered nuclear extracts were incubated with the same probes (lanes 5 and 6). Lanes 7 and 8, standard control containing • Sp1 site (sc-2502, Santa Cruz Biotech) or its @ mutant (sc-2503) processed for gel-shift assay using the same nuclear extracts. Extreme right , Depletion assay using anti-Sp3 antibody with nuclear extract showing no change in Sp1/DNA complex (lane 10) and the complex was indistinguishable from Lane 9. Images are representatives from three independent consistent observations.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Gel Shift, Binding Assay, Isolation, Incubation, Labeling, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Control, Acrylamide Gel Assay, Expressing, Comparison, Depletion Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Interrupting Sp1 activity by artemisinin interrupted LEDGF/p75 promoter activity in a concentration-dependent manner. Upper panel, a diagram of the LEDGF/p75 promoter representing three Sp1-binding sites (−170/+35) used for CAT activity. A selective Sp1 inhibitor , artemisinin, reduced the activity of LEDGF/p75 promoter in LECs in dose-dependent fashion. Artemisinin or its diluents (control) were added to culture medium of LEDGF/p75 promoter constructs or empty vector transfected cells monolayer. Cells were disrupted and CAT activities were measured as described in the section. Data are the mean of three experiments, and error bars indicate standard deviation (** p<0.001 ). B and C, Influence of Sp1 overexpression on transcriptional activity of LEDGF/p75 promoter. Plasmid encoding pCAT- LEDGF/p75 (−170/+35) or pCAT-V was cotransfected into hLECs ( B ) and Cos7 cells ( C ) with indicated amounts of pCMV-Sp1. Following CAT assay, CAT values were analyzed and represented as histograms, with Sp1 (black bar) or without Sp1 (open bar) overexpression. Empty CAT vector shows insignificant CAT activity (gray bar). Transfections were carried out as described in and level of Sp1 protein was evaluated using Western analysis (B and C, Upper panel). The data are representative of at least three independent experiments. Each value represents the mean ± S.D. (** p<0.001 ).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Activity Assay, Concentration Assay, Binding Assay, Control, Construct, Plasmid Preparation, Transfection, Standard Deviation, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Repression of LEDGF/p75 transcription by Sp1 Sumoylation. Cells were transfected or cotransfected with pCAT- LEDGF/p75 (pCAT-LED) or pCAT vector (pCAT-V) and/or with increasing amounts of a plasmid encoding Sumo1 (pEGFP-Sumo1) as indicated. Cells were disrupted at predefined times and processed for CAT assay. Data indicate CAT activity in cells overexpressing different amounts of Sumo1 (A, gray bars) and without Sumo1 (black bars). Experiments were performed three times, and data are presented as mean ± S.D. B, ChIP assay coupled with desumoylation and DNA-protein complex dissociation experiments showed that the effect of Sumo1 on the abundance of Sp1 was concentration-dependent. hLECs were transfected with either pCMV-Sp1 alone or cotransfected with pEGFP-Sumo1 or pFLAG-Senp1. ChIP assay was performed in duplicates from each sample with anti-Sp1 or anti-Sp3 antibody or control IgG. Following processing, one set of precipitated samples was submitted for PCR analysis of Sp1 responsive region of LEDGF/p75 promoter (B) as described in . In another set of experiments, DNA bound proteins were eluted with high salt solution, and Western analysis was performed on elutes to measure Sp1 prevalence by anti-Sp1-antibody (C: lane 1, pCMV-Sp1; lane 2, pEGFP-Sumo1; lane 3, pFLAG-Senp1; lane 4, pCMV-Sp1 plus pEGFP-Sumo1; lane 5, pCMV-Sp1 plus pFLAG-Senp1; p-vector). Images shown in the panel (B) are of representatives of Sp1-3 (site 3) region. Similar results were obtained with Sp1-1 (site 1) and Sp1-2 (site 2) in the LEDGF/p75 promoter when ChIP-PCR analysis was done (data not shown). Following stripping of Sp1 immunoblotted membrane, the same membrane was reprobed with Sp3 specific antibody, and no bands were observed (D).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Control, Western Blot, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, hLECs were transfected with either mock, negative control siRNA or LEDGF/p75 siRNA. Following transfection, cell extracts were prepared and expression was examined by Western analysis using Anti-LEDGF/p75 antibody (A). Relative density in pixels is shown on the right . B, Control siRNA (siControl) or LEDGF/p75 siRNA (siLEDGF/p75) transfected cells were seeded in 12-well plates and submitted to UVB exposure as described in . A survival assay-MTS assay was conducted, and data shown are mean ± S.D. values of three independent experiments. ** p<0.001 compared with control siRNA. C, Sp1 overexpression in cells with siRNA LEDGF/p75 conferred resistance against UVB stress. LEDGF/p75 siRNA transfected hLECs were transiently re-transfected with pCMV-Sp1 and then exposed to UVB stress. MTS assay was performed to evaluate vulnerability. * p<0.01 compared with respective controls. D, Sp1 or LEDGF/p75 overexpression in hLECs provided cytoprotection against stress induced by UVB. Cells were cultured and exposed to different doses of UV stress as indicated. Cell viability was analyzed using MTS assay as described in . * * p<0.001 compared with respective controls. Data represent mean ± S.D. from three independent experiments.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Negative Control, Expressing, Western Blot, Control, Clonogenic Cell Survival Assay, MTS Assay, Over Expression, Cell Culture