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: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 1 Elevated miR-762 expression is associated with gefitinib resistance in NSCLC cells. a RT-qPCR analysis of miR-762 expression in different NSCLC cells. Relative expression levels of miR-762 were obtained in each sample by normalization of the expressions of miR-762 to that of the U6 snRNA signal. For presentation of data, expression levels of miR-762 from NuLi-1 cells were taken as 100% and the others were normalized accordingly. Each value is a mean ± S.E.M. from three independent experiments. Different superscript letters denote groups that are statistically different (P < 0.05). b The IC50 value (inhibitory concentration to produce 50% cell death) following a 48-h exposure to gefitinib was determined in PC-9 and PC-9/GR cells using MTT assay. c The IC50 value following a 48-h exposure to gefitinib was determined in A549 and A549/GR cells using MTT assay. d-e Characterization of miR-762 expression in different NSCLC cells using RT-qPCR. The value indicates the relative expression levels of miR-762 in the cells (PC-9/PC-9/GR and A549/A549/GR cells) at different batches of gefitinib resistant induction

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, MTT Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 2 IL-6 serves as a potential upstream regulator of miR-762 induction in NSCLC cells. a-b Characterization of expression levels of different cytokines in different NSCLC cells using RT-qPCR. Each value is a mean ± S.E.M. from three independent experiments. c A549 cells were incubated with different cytokines, including IL-1α (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) and IL-8 (50 ng/ml) for 24 h, followed by RT-qPCR analysis of miR-762 expression. d A549 cells were stimulated with different doses of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression. e A549 cells were stimulated with 10 ng/ml of IL-6 for different durations as indicated, followed by RT-qPCR analysis of miR-762 expression. f A549 cells were stimulated with 10 ng/ml of IL-6, in the presence or absence or co-treatment with 5 μM of WP1066, for 24 h, followed by RT-qPCR analysis of miR-762 expression. Inhibition of STAT3 activation was verified using immunoblotting analysis of pSTAT3 expression (upper panel). g A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA. 48 h later, knockdown of STAT3 in A549 cells was validated using immunoblotting. h 48 h after siRNA treatment, A549 cells were stimulated with 10 ng/ml of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Incubation, Inhibition, Activation Assay, Western Blot, Transfection, Knockdown

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 3 miR-762 upregulation desensitizes NSCLC cells to gefitinib treatment. a 48 h after transfection with miR-762 inhibitors or negative controls (NC), PC-9/GR and A549/GR cells were subjected to RT-qPCR analysis of miR-762 expression. b PC-9/GR and A549/GR cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). c Cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). d In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and ** P < 0.01 when comparing Inhibitors + vehicle to Inhibitors + gefitinib). e 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9 and A549 cells were subjected to RT-qPCR analysis of miR-762 expression. f PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). g PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). h In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and **P < 0.01 when comparing Mimics + vehicle to Mimics + gefitinib)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 4 ABR serves as the direct target of miR-762 in NSCLC cells. a Prediction of putative target genes of miR-762 by Target scan and miRDB programs. b PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by immunoblotting analysis of ABR expression. c PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by RT-qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). d PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. e PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by RT- qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). f Predicted miR-762 binding sites in the 3′-UTR of ABR gene. g miR-762 mimics/Mimics- NC (25 pmol/well) and pGL3-ABR 3’UTR-Luc reporter (0.25 μg/well), together with 0.001 μg of the Renilla luciferase reporter (Promega, Beijing, China), were co-transfected into subconfluent proliferating NIH/3 T3 cells for 24 h, followed by measurement of the relative luciferase activity (*P < 0.05)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

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

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 5 ABR overexpression alleviates miR-762-induced gefitinib resistance. a PC-9 and A549 cells that stably expressed the exogenous ABR was established as described in Materials and methods. PC-9/ABR and A549/ABR cells were transiently transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. b 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm. c 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell apoptosis was assayed using an ApoStrand™ ELISA Apoptosis Detection Kit at 405 nm. Different superscript letters denote groups that are statistically different (P < 0.05). d 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were subjected to a xenograft model to measure the in vivo gefitinib sensitivity, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 when comparing Mimics + gefitinib + vector to Mimics + gefitinib + pCMV3-ABR)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Over Expression, Stable Transfection, Transfection, Western Blot, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Plasmid Preparation

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

Journal: Cells

Article Title: Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients

doi: 10.3390/cells9112409

Figure Lengend Snippet: SNVs around rDNA promoter region in 20 human cancer cell lines.

Article Snippet: The human LUAC cell line A549, colon adenocarcinoma cell lines HCT116 and DLD-1, gastric adenocarcinoma cell lines Kato III and MKN45, Burkitt lymphoma cell line Raji, urinary bladder carcinoma cell line T24, hepatoblastoma cell line HepG2, and cervical carcinoma cell line HeLa were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan).

Techniques:

Journal: Cells

Article Title: Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients

doi: 10.3390/cells9112409

Figure Lengend Snippet: Recurrence-free survival stratified by ( A ) presence or absence of germline single-nucleotide variants (SNVs) at any site at positions +1 to +100 in the rDNA promoter region (presence versus wild type (WT)) in 82 lung adenocarcinoma (LUAC) patients, and ( B ) germline SNV at positions +1 to +100 in the rDNA promoter region (presence versus WT) in Stage I LUAC excluding MIA ( n = 42), and ( C ) in Stage I/II LUAC excluding MIA ( n = 60).

Article Snippet: The human LUAC cell line A549, colon adenocarcinoma cell lines HCT116 and DLD-1, gastric adenocarcinoma cell lines Kato III and MKN45, Burkitt lymphoma cell line Raji, urinary bladder carcinoma cell line T24, hepatoblastoma cell line HepG2, and cervical carcinoma cell line HeLa were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan).

Techniques:

Journal: Cells

Article Title: Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients

doi: 10.3390/cells9112409

Figure Lengend Snippet: Overall survival stratified by ( A ) germline single-nucleotide variants (SNVs) at any site at positions +1 to +100 in the rDNA promoter region (presence versus wild type (WT)) in 82 lung adenocarcinoma (LUAC) patients, and ( B ) germline SNV at positions +1 to +100 in the rDNA promoter region (presence versus WT) in Stage I LUAC excluding MIA ( n = 42), and ( C ) in Stage I/II LUAC excluding MIA ( n = 60).

Article Snippet: The human LUAC cell line A549, colon adenocarcinoma cell lines HCT116 and DLD-1, gastric adenocarcinoma cell lines Kato III and MKN45, Burkitt lymphoma cell line Raji, urinary bladder carcinoma cell line T24, hepatoblastoma cell line HepG2, and cervical carcinoma cell line HeLa were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan).

Techniques: