Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: XPA is phosphorylated in cells on UV irradiation. A, A549 cells were treated with 20 J/m2 UV or mock treated. Total cell lysates were harvested at 4 hours after UV irradiation and then treated with 200 units of CIAP (Promega) for 1 hour at 37°c in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of 50 mmol/L glycerophosphate (G.P.) or mock treated (lanes 1 and 2). The treated cell lysates were then subjected to Western blotting and probed with anti-XPA (a) and anti-RPA32 (b), respectively. c, cells were treated with 20 J/m2 UV or mock treated and total cell lysates were harvested for immunoprecipitation assays with anti-XPA antibody. The immunoprecipitated XPA was treated with CIAP or mock treated and then analyzed by Western blotting with anti-XPA antibody. B, A549 cells were grown overnight in phosphate-depleted medium before irradiation with 20 J/m2 UV. Then, 32P-labeled orthophosphoric acid was added and cells were further incubated for 8 hours before harvest. Immunoprecipitation assay was done with anti-XPA antibody. Immunoprecipitates were separated on SDS-PAGE and radiolabeled proteins were detected (left). Immunoprecipitated endogenous XPA was probed by Western blotting (right). C, cells were UV irradiated or mock treated and then cytoplasmic and nuclear extractions were separated on SDS-PAGE for Western blot analysis using anti-XPA antibody. D, a, cells were irradiated with 20 J/m2 of UV and the cytoplasmic fraction (lane 1) was isolated. The nuclear pellet was then sequentially extracted with buffer of increasing salt concentration (lanes 2-5). NM, nuclear matrix (lane 6). b, nuclear extracts were prepared at indicated times after 20 J/m2 UV treatment of cells. c, cells were irradiated with indicated doses of UV and the nuclear extracts were prepared at 4 hours after UV treatment.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Irradiation, Western Blot, Immunoprecipitation, Labeling, Incubation, SDS Page, Isolation, Concentration Assay

Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: ATR is the major kinase responsible for cellular XPA phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or HeLa cells were transfected with indicated siRNA and then treated with 20 J/m2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Phospho-proteomics, Irradiation, Incubation, Western Blot, Transfection

Journal:

Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

doi: 10.1158/0008-5472.CAN-05-3403

Figure Lengend Snippet: ATR interaction and colocalization with XPA in cells after UV irradiation. A, total cell lysates prepared from UV-treated or mock-treated A549 cells were used for coimmunoprecipitation assays with anti-ATR antibody. Proteins from the immunoprecipitates were detected by Western blotting using anti-XPA and anti-ATR antibodies. As controls, 10% of the total volumes of the whole cellular lysates used for the coimmunoprecipitation were also included. B, top, total cell lysates prepared from A549 cells were used for coimmunoprecipitation assays with anti-XPA antibody; bottom, whole-cell extracts prepared from 2 × 106 cells were mixed with 2 μg of His-XPA and incubated at 4°c for 10 to 14 hours. The XPA-bound proteins were probed by anti-ATR antibody. C, total cellular lysates were incubated with anti-ATR antibodies for 4 to 6 hours, followed by 1-hour incubation with protein A/G-agarose beads. The immunoprecipitates were washed thrice with PBS containing 0.5% NP40 and further incubated with the buffer of high concentration of salt [15 mmol/L Tris-Cl (pH 7.5), 0.6 mol/L NaCl, 0.1% NP40] for 30 minutes at 4°c. After centrifugation and washing, purified His-XPA was added and further incubated in 500 AL of XPA binding buffer [40 mmol/L HEPES-KOH (pH 7.5), 75 mmol/L KCl, 8 mmol/L MgCl2, 1 mmol/L DTT, 5% glycerol and 100 Ag/mL bovine serum albumin, 0.1% NP40] for 4 to 6 hours. The bound proteins were detected by Western blotting with anti-XPA antibody. LC, loading control (20 ng purified His-XPA). D, cells were treated with 20 J/m2 UV or mock treated, followed by 4-hour incubation. After extraction of cytoplasmic proteins with PBS containing 0.5% NP40, cells were fixed and incubated with anti-XPA and anti-ATR antibodies. Cells were then stained with fluorescent dye-linked secondary antibodies and visualized by fluorescence microscopy. b and f, red, anti-ATR stained cells; c and g, green, anti-XPA stained cells; d and h, merged images of the anti-XPA and anti-ATR stained cells; a and e, 4‘,6-diamidino-2-phenylindole-stained nuclei.

Article Snippet: Human lung adenocarcinoma cells A549 were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°c and 5% CO 2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Techniques: Irradiation, Western Blot, Incubation, Concentration Assay, Centrifugation, Purification, Binding Assay, Control, Extraction, Staining, Fluorescence, Microscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: TGFBR2 mediated phosphorylation of BUB1 reduces its interaction with TGFBR2 and SMAD2. (A) IP for Myc and then blotting for TGFBR2 in lysates from A549 cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with HA-TGFBR2. Cells were serum starved and treated with TGF-β (5 ng/mL for 1 h) before harvesting. (B) IP for Myc and then blotting for SMAD2 in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with FL-SMAD2. Cells were serum starved and treated with TGF-β (5 ng/mL for 1 hour) before harvesting.

Article Snippet: The human lung carcinoma cell line A549 and normal kidney cell line HEK293T were obtained from American Type Culture collection (ATCC) maintained in RPMI-1620 or DMEM media respectively supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, and 0.1% penicillin/streptomycin/gentamycin (GIBCO-Invitrogen).

Techniques: Phospho-proteomics, Transfection, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: TGFBR2 mediated phosphorylation of BUB1 is a signaling cue for the activated TGFBR complex disassembly. (A) HEK293T cells were transfected with Myc-BUB1 WT (as positive control) and truncation mutant Myc-BUB1 241–482 along with S318A and S318D truncation mutants. His-TGFBR1 and FL-SMAD2 were co-transfected with BUB1 and cells were starved and treated with TGF-β (5 ng/mL for 1 h) before harvesting. Lysates were subjected to IP with TGFBR1 and blotted for Myc, TGFBR2 and FLAG. Arrow in the IB: SMAD2 blot denotes the FL-SMAD2 specific band. The band below is Heavy Chain of the antibody (Hc). The arrow in the input blot is His-TGFBR1. The His-tag blot in input is reprobed after probing with Myc-tag antibody. The thick bands below His-TGFBR1 (in inputs) are that of Myc-BUB1 truncation mutants. (B) HEK293T cells were transected with non-targeting control (NSS) or BUB1 siRNA along with Myc-BUB1 WT, S318A and S318D mutants, His-TGFBR1 and FL-SMAD2. Cells were starved and treated with TGF-β before harvesting. Lysates were subjected to IP with TGFBR1 and blotted for Myc, TGFBR2 and FLAG. (C) MDA-231-1833 and A549 cells were transfected with Myc-BUB1 241–482 and 241–482 S318A and 241–482 S318D mutants. 24 h post-transfection, cells were serum starved over-night. Cells were treated with 5 ng/mL for an hour before harvesting. The resulting lysates were resolved on SDS–PAGE gels and probed with antibodies against pSMAD2, pSMAD3, Myc-tag and Actin.

Article Snippet: The human lung carcinoma cell line A549 and normal kidney cell line HEK293T were obtained from American Type Culture collection (ATCC) maintained in RPMI-1620 or DMEM media respectively supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, and 0.1% penicillin/streptomycin/gentamycin (GIBCO-Invitrogen).

Techniques: Phospho-proteomics, Transfection, Positive Control, Mutagenesis, Control, SDS Page

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Viability of A549 cells after prolonged (24, 48 and 72 h) exposure to PBDEs at concentrations from 1.5 to 48 μg mL −1 determined with the MTS assay; 0 μg mL −1 represents untreated cells. The results are expressed as percentage of corresponding control, untreated cells and given as means ± SD ( n = 6). The dashed frame highlights a time-dependent effect. & p < 0.05; # p < 0.01; * p < 0.0001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: MTS Assay, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Cytotoxicity curves of BDE-99 and -209 at concentrations from 1.5 to 48 μg mL −1 in A549 cells over 24, 48 and 72 h. The results of the MTS assay are given as means ± SD ( n = 6).

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: MTS Assay

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: IC 50 values of BDE-99 and -209 in A549 cells over 24, 48 and 72 h.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques:

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Prolonged exposure of A549 cells to a house dust sample extract. A549 cells were exposed to ∑PBDE in house dust at concentrations from 0.375 to 24 μg mL −1 for 24, 48 and 72 h, and cell viability was determined with the MTS assay. The results are given as means ± SD ( n = 6). The dashed frame highlights the concentration found in the house dust sample. & p < 0.05; # p < 0.01; vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: MTS Assay, Concentration Assay, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: LOAEL cytotoxicity of BDE-99 (7.5 μg mL −1 ) and BDE-209 (15 μg mL −1 ), alone and combined, in A549 cells treated for 24, 48 and 72 h. The results of the MTS assay are given as means ± SE ( n = 6). # p < 0.01; $ p < 0.001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: MTS Assay, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Levels of LDH release after 4 h exposure of A549 cells to PBDE congeners and house dust sample extract. Triton (0.08%) was used as the positive control. The results are presented as percentage of LDH release and given as means ± SD ( n = 3). # p < 0.01; $ p < 0.001; * p < 0.0001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: Positive Control, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: ROS levels after the 4 h exposure of A549 cells to PBDE congeners, house dust sample extract and H 2 O 2 (100 µM) as positive control. The results of DCF-fluorescence signal measurements are presented as relative fluorescence units (RFU) and given as means ± SD ( n = 6). & p < 0.05; # p < 0.01; $ p < 0.001; * p < 0.0001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: Positive Control, Fluorescence, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: GSH levels after the 4 h exposure of A549 cells to single PBDEs and house dust sample extract at 3, 6 and 12 µg mL −1 and to tBOOH (100 µM) as positive control. The results of MCB-fluorescence measurements are presented as relative fluorescence units (RFU) and given as means ± SD ( n = 6). & p < 0.05; # p < 0.01; $ p < 0.001; * p < 0.0001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: Positive Control, Fluorescence, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Mitochondrial membrane potential after 4 h exposure of A549 cells to single BDE congeners and house dust sample extract at 3, 6 and 12 µg mL −1 and CCCP (50 µM) as positive control. The results are presented as relative fluorescence units (RFU) and given as means ± SD ( n = 6). & p < 0.5, # p < 0.01, * p < 0.001 vs. untreated control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: Membrane, Positive Control, Fluorescence, Control

Journal: Toxics

Article Title: PBDEs Found in House Dust Impact Human Lung Epithelial Cell Homeostasis

doi: 10.3390/toxics10020097

Figure Lengend Snippet: Percentage of different stages of apoptosis of A549 cells after 4 h exposure to single PBDE congener and house dust sample extract at 12 µg mL −1 and PFA (0.08%, v / v ) as a positive control. The results are presented as percentage of total apoptotic cells compared to vehicle control (2.4% methanol) and given as means ± SD ( n = 6). * p < 0.001 vs. control.

Article Snippet: The certified human carcinoma alveolar basal epithelial A549 cell line (ECACC 86012804) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) cell bank.

Techniques: Positive Control, Control

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Increased Cx26 is positively correlated with gefitinib resistance in NSCLC cells. ( a ) Differential expression of Cx26, Cx31.1, Cx32, and Cx43 in different gefitinib-sensitive NSCLC cell lines was determined by RT-PCR. ( b and c ) High level of Cx26 in gefitinib-insensitive A549 and H1299 cells than that in gefitinib-sensitive HCC827 and PC9 cells was detected by RT-PCR and western blotting. GAPDH or β -actin was used as internal loading control

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 induces acquired gefitinib resistance in NSCLC cells via GJIC-independent manner. ( a ) Functional GJIC was detected by parachute assay and no detectable GJIC was found in HCC827 GR, PC9 GR, and their parental cells. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( b ) No enhancement of GJIC in these cells incubated with 10, 20, and 40 μ M of RA (a well-defined GJIC enhancer) for 4, 8, 12, 24, and 48 h, respectively. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( c and d ) Immunofluorescence staining of the cellular localization of Cx26 with or without RA treatment. All scare bars represent 50 μ m

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Functional Assay, Fluorescence, Incubation, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 and PI3K/Akt pathway functionally interplay to promote EMT and gefitinib resistance in NSCLC cells. ( a and b ) Effect of LY294002 or Akt overexpression on Cx26 expression in HCC827, PC9, and their GR cells was determined by western blotting. ( c ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on cell morphology changes in HCC827 and PC9 cells. Original magnification, × 400. ( d – f ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on the expression of EMT markers (E-cadherin, vimentin, and slug), cell migration, and invasion, as well as cell sensitivity to gefitinib in HCC827 and PC9 cells, respectively. Error bars are mean±S.D. from four independent experiments, ** P <0.01 versus vector group. # P <0.05 and ## P <0.01 versus Akt-overexpressing group

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Western Blot, Migration, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection

doi: 10.1371/journal.pone.0126437

Figure Lengend Snippet: Panel A : E . coli strains were incubated with epithelial cells for 30 min at 37°C. Following this, cells were washed to remove unbound bacteria, lysed, diluted, and spread onto agar plates to calculate the number of bound bacteria. The results are expressed as the mean percentage (± standard error) of inoculated bacteria attached to A549 cells. The values in parentheses show the actual percentage. Panel B : E . coli strains were cultured in the wells of PVC microplates, stained with crystal violet, washed with deionized water, and the wells were photographed. The arrow shows biofilm formation, which was quantitated by extracting crystal violet with methanol and measuring absorbance at 570 nm. The results are shown in parentheses and are expressed as the mean (± standard error) absorbance. Both panels : The asterisks indicate that the increase in adherence and biofilm formation of E . coli carrying pBpaB, compared to E . coli harboring pBHR1ΔDra, is statistically significant ( P values < 0.05, Mann-Whitney test).

Article Snippet: The cell lines A549 (human type II alveolar epithelium; ATCC CCL85) and J774A.1 (murine macrophages; ATCC TIB-67) were cultured as described elsewhere [ , ].

Techniques: Incubation, Bacteria, Cell Culture, Staining, MANN-WHITNEY