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: APL Bioengineering

Article Title: A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts

doi: 10.1063/5.0115464

Figure Lengend Snippet: Effects of anti-fibrotic drugs on the growth and invasiveness of A549 cell spheroids in fibrin/Matrigel co-cultured with MRC-5 fibroblasts. (a) Top: confocal fluorescence projection images of typical A549 cell spheroids co-cultured with fibroblasts in fibrin/Matrigel treated with various concentrations of NTD for 72 h. Bottom: projection areas and 1/Circularity values of A549 cell spheroids under the treatment of NTD treatment. (b) Results with the PFD treatment. The data were obtained from three independent experiments. Each dot represents one spheroid. ***, P < 0.001; **, P < 0.01 in comparison with the group without NTD treatment (Dunn's post hoc test).

Article Snippet: We used the A549 (RRID:CVCL_0023) human lung adenocarcinoma cell line, MRC-5 (RRID:CVCL_0440) human lung fibroblast cell line (both from Bioresource Collection and Research Center, Hsinchu, Taiwan), and NHLF cell line (CC-2512, Lonza, Basel, Switzerland) in this study.

Techniques: Cell Culture, Fluorescence, Comparison

Journal: APL Bioengineering

Article Title: A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts

doi: 10.1063/5.0115464

Figure Lengend Snippet: Effects of CDDP and CDDP plus NTD on A549 cell spheroids in fibrin/Matrigel. (a) Experimental procedures. (b) Confocal fluorescence projection images of typical A549 cell spheroids without fibroblasts under the treatments of CDDP with various concentrations for 72 h (top) and with the combination of 0.5 μ M NTD (bottom). (c) Projection areas and (d) 1/Circularity values of A549 cell spheroids under the treatment of CDDP and CDDP+NTD. (e)–(g) are the same as (b)–(d) but with fibroblast co-culture. ***, P < 0.001; **, P < 0.01; *, and P < 0.05 in comparison with the group without any treatment. ###, P < 0.001 between the two groups as indicated. The significance of differences was checked with the Kruskal–Wallis test and Dunn's post hoc test.

Article Snippet: We used the A549 (RRID:CVCL_0023) human lung adenocarcinoma cell line, MRC-5 (RRID:CVCL_0440) human lung fibroblast cell line (both from Bioresource Collection and Research Center, Hsinchu, Taiwan), and NHLF cell line (CC-2512, Lonza, Basel, Switzerland) in this study.

Techniques: Fluorescence, Co-Culture Assay, Comparison

Journal: APL Bioengineering

Article Title: A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts

doi: 10.1063/5.0115464

Figure Lengend Snippet: Effects of anti-fibrotic drugs on the activities of fibroblasts. (a) Confocal fluorescence projection images of fixed MRC-5 fibroblasts and normal human lung fibroblasts (NHLFs) in fibrin/Matrigel. The fibroblasts were in the medium conditioned by A549 cells and treated by NTD or PFD. Only NTD treatment changed cell morphology. Green, F-actin labeled with phalloidin-FITC. Blue, cell nuclei labeled with DAPI. (b) Area variations of fibrin/Matrigel laden with fibroblasts in the A549 cell-conditioned medium. With the MRC-5 fibroblasts, the hydrogel area was decreased by ∼50% after 48 h of culture. With the NHLFs, the hydrogel area was decreased by ∼20% after 120 h of culture. The 0.5 μ M NTD treatment impeded the area shrinkage caused by both types of fibroblasts. In contrast, the 1000 μ M PFD treatment did not show an observable effect. The data presented are from three independent experiments. *, P < 0.05 (Dunn's post hoc test).

Article Snippet: We used the A549 (RRID:CVCL_0023) human lung adenocarcinoma cell line, MRC-5 (RRID:CVCL_0440) human lung fibroblast cell line (both from Bioresource Collection and Research Center, Hsinchu, Taiwan), and NHLF cell line (CC-2512, Lonza, Basel, Switzerland) in this study.

Techniques: Fluorescence, Labeling

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