Journal: Molecular Cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: TGFBI sensitizes NSCLC cells to chemotherapy . A549 cells transfected with TGFBI siRNA plasmid (TGFBIsi) (A), and H1299 cells transfected with TGFBI expression vector (TGFBIve) (B), were exposed to increasing amounts of different cytotoxic agents for 48 h and cell viability was measured. Three independent experiments were performed. Comparisons to analyze statistical differences between control and transfected cells were performed using Student t -test (* p < 0.05; ** p < 0.01). IC 50 for each experimental condition is indicated. (C, D) PARP-1 cleavage was detected by Western blot analysis of total protein extracts derived from A549-TGFBIsi (C) and H1299-TGFBIve (D) transfected cells treated with etoposide for 48 h. One representative experiment out of three is shown.

Article Snippet: To silence TGFBI expression, 1 × 10 6 A549 cells/mL (A549-TGFBIsi) were transfected by electroporation with 10 μg of a commercially available shRNA targeting TGFBI cloned in a pRS plasmid vector (Origene, MD, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Derivative Assay

Journal: Molecular Cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: High concentration of rh-TGFBI protein increases cell death and potentiates NSCLC responses to chemotherapy . Caspase 3/7 activity was measured in A549 (white histograms) and H1299 (black histograms) NSCLC cells seeded onto non coated 96-well plates and further treated with increasing amounts of rh-TGFBI (A), or seeded onto 96 well plates coated with increasing amounts of rh-TGFBI (B), using the caspase 3/7 Glo-assay from Promega. (C and D) Caspase 3/7 activity of 24 h etoposide-treated A549 (5 μM) and H1299 (50 μM) cells seeded onto non coated 96-well plates and further exposed for 24 h to increasing amounts of rh-TGFBI previous to etoposide exposure (C) or seeded onto 96-well plates coated with increasing amounts of rh-TGFBI before exposure to etoposide (D). Statistical analyses were performed using Student t -test to compare caspase 3/7 activity in treated cells to non-treated cells (* p < 0.05; ** p < .01).

Article Snippet: To silence TGFBI expression, 1 × 10 6 A549 cells/mL (A549-TGFBIsi) were transfected by electroporation with 10 μg of a commercially available shRNA targeting TGFBI cloned in a pRS plasmid vector (Origene, MD, USA).

Techniques: Concentration Assay, Activity Assay, Glo Assay

Journal: Molecular Cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: TGFBI influences NSCLC susceptibility to chemotherapy through binding to the αvβ 3 integrin . Adhesion of calcein-labeled A549 (A) and H1299 (B) NSCLC cells to rh-TGFBI coated (1 μg/mL) 96-well plates was evaluated in the presence of antibodies against several integrins. Statistical analyses were performed using Student t -test and comparing treated cells to control cells (** p < 0.01). (C) Flow cytometry analysis of basal αvβ3 integrin expression in A549 cells (dashed histogram), H1299 cells (line histrogram) or isotype control (dotted histogram). (D) Caspase 3/7 detection in A549 and H1299 cells seeded onto non-coated 96 well-plates, pre-incubated for 1 h with anti-αvβ3 monoclonal blocking antibody, exposed to rh-TGFBI (20 μg/mL) for 24 h and further treated with etoposide at the IC 50 (5 μM for A549 cells and 50 μM for H1299 cells) for additional 24 h. Statistical comparisons were performed using Student t -test. ** p < 0.01 for the comparison of the activity control cells versus integrin blocked cells. ## p < 0.01 for the comparison of the activity of etoposide-treated cells versus cells treated in the presence of the anti-integrin blocking antibody.

Article Snippet: To silence TGFBI expression, 1 × 10 6 A549 cells/mL (A549-TGFBIsi) were transfected by electroporation with 10 μg of a commercially available shRNA targeting TGFBI cloned in a pRS plasmid vector (Origene, MD, USA).

Techniques: Binding Assay, Labeling, Flow Cytometry, Expressing, Incubation, Blocking Assay, Activity Assay

Journal: Molecular Cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: Peptides derived from TGFBI mediate NSCLC cells response to chemotherapy and the induction of caspase 8 and caspase 3/7 activation . (A) Western blot detection of proteolytic fragments of TGFBI in H1299 and A549 cells transiently transfected with the TGFBI expression vector. (B) Detection of caspase 3/7 activity in A549 and H1299 cells exposed for 24 h to cell supernatants derived from cultures of A549 and H1299 cells transiently transfected with the TGFBI expression vector. Control : non-treated cells; Control > 3 kDa : NSCLC cells exposed to supernatants from non-transfected cell cultures containing fragments larger than 3 kDa in size; Control < 3 kDa : NSCLC cells exposed to supernatants from non-transfected cell cultures containing fragments smaller than 3 kDa in size; TGFBI > 3 kDa : NSCLC cells exposed to supernatants from TGFBI-transfected cells containing fragments larger than 3 kDa in size; TGFBI < 3 kDa : NSCLC cells exposed to supernatants from TGFBI-transfected cells containing fragments smaller than 3 kDa in size; αvβ3 : cells blocked with anti-αvβ3 blocking antibody 1 h before the addition of the supernatant; Et : Cells exposed to IC 50 etoposide for 24 h. Statistical analyses were performed using Student t -test to compare caspase 3/7 activity in control cells (** p < 0.01) or etoposide treated cells (## p < 0.01) to the caspase activity in cell cultures treated with supernatants. (C) Caspase 8 and (D) caspase 3/7 detection in A549 and H1299 cells maintained for different time-periods in the presence of TGFBI < 3 kDa supernatant. Statistical analyses were performed using Student t -test and the caspase activity of treated cells was compared to that of untreated cells. (* p < 0.05; ** p < 0.01).

Article Snippet: To silence TGFBI expression, 1 × 10 6 A549 cells/mL (A549-TGFBIsi) were transfected by electroporation with 10 μg of a commercially available shRNA targeting TGFBI cloned in a pRS plasmid vector (Origene, MD, USA).

Techniques: Derivative Assay, Activation Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Activity Assay, Blocking Assay

Journal: BMC Cancer

Article Title: Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene

doi: 10.1186/1471-2407-12-309

Figure Lengend Snippet: NSCLC cells express receptor EphA2, Ephrin-A1 and Caludin-2. Plate A : NSCLC cell lines (A549, H2126, H838, H522, H23) express receptor EphA2, ephrin-A1, and claudin-2 . Expression of EphA2, ephrin-A1, and claudin-2 was analyzed by Western blot analysis. Plate B : Western blot analysis of EphA2 protein expression the β-actin was probed to demonstrate equal sample loading. NSCLC cell lines were either transfected with pcDNA-EFNA1 (pcDNA-EFNA1 is vector containing ephrin-A1 construct) or empty vector and receptor EphA2 expression was analyzed. Plate C : A549 cells were activated for 5 minutes to 120 minutes with ephrin-A1. Western blot analysis of phosphorylated EphA2 and Erk1/Erk2 was performed, the β-actin was probed to demonstrate equal sample loading. Data presented is the representative of three separate experiments.

Article Snippet: For the over expression of cdx-2 gene, pcMV6-XL5 was used as an expression vector for cdx-2 and control vector in A549 cells (Origene Technologies, Inc.; Rockville, MD).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Construct

Journal: BMC Cancer

Article Title: Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene

doi: 10.1186/1471-2407-12-309

Figure Lengend Snippet: Ephrin-A1 ligand activation decreases tumor growth in NSCLC. Equal number of A549 cells was seeded in matrigels after various treatments as described and after 7 days of culture tumor growth was recorded by a SPOT digital camera attached to Nikon microscope. The data presented are a single representative of three similar but independent experiments. Magnification = 5 μM.

Article Snippet: For the over expression of cdx-2 gene, pcMV6-XL5 was used as an expression vector for cdx-2 and control vector in A549 cells (Origene Technologies, Inc.; Rockville, MD).

Techniques: Activation Assay, Microscopy

Journal: PLoS ONE

Article Title: The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

doi: 10.1371/journal.pone.0101564

Figure Lengend Snippet: A. and B. Cells were cultured in attached (A) or detached (D) conditions for the indicated times and then subjected to subjected to Cell Death ELISA (A) or western blotting (B) with antibodies against caspase-3, PARP, and B-actin. In A, three independent experiments were performed in triplicates; *, p<0.05 as compared to attached cells (Student's t test). C. and D. Cells were transfected with control- or Bit1 specific siRNAs, and 48 h later, cells were subjected to immunoblotting (C) with antibodies against Bit1 and B-actin to confirm the knockdown of Bit1 expression. In parallel, control- and Bit1- siRNA treated cells were cultured in attached (A) or detached (D) conditions for the indicated times and subjected to Cell Death Elisa (D). E and F. Stable A549 control shRNA and Bit1 shRNA knockdown clones and pools were generated (as described in ) and subjected to western blotting (E) using specific antibodies to Bit1 and B-actin. In F, the control shRNA and Bit1 shRNA pools were cultured in attached (A) or detached (D) conditions for the indicated times and subsequently analysed for apoptosis by Cell Death Elisa. In A, D, and F, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to control cells (Student's t test).

Article Snippet: To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Expressing, shRNA, Clone Assay, Generated

Journal: PLoS ONE

Article Title: The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

doi: 10.1371/journal.pone.0101564

Figure Lengend Snippet: A., B. and C. Cells transfected with vector or Bit1 mito construct were subjected to soft agar assay as described in . The representative colonies are shown in A. The extent of colony formation was quantified by counting the number of visible colonies with a diameter greater than 30 uM (B) and by alamar blue staining and fluorescent assay (C). D, E and F. The stable A549 control shRNA and Bit1shRNA knockdown pools were also subjected to soft agar assay. The representative colonies are visualized in A and quantified by counting the number of visible colonies (E) and alamar blue assay (F). In B, C, E and F, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to corresponding control cells (Student's t test).

Article Snippet: To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene).

Techniques: Transfection, Plasmid Preparation, Construct, Soft Agar Assay, Staining, Fluorescence, shRNA, Alamar Blue Assay

Journal: PLoS ONE

Article Title: The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

doi: 10.1371/journal.pone.0101564

Figure Lengend Snippet: A. and B. A549 cells were transfected with Bit1 mito and/or with GFP-tagged TLE1 construct. 24 h post transfection, the expression of exogenous Bit1 and TLE1 in transfected cells was confirmed by western blotting using specific antibodies to myc and GFP tags. The amount of plasmid transfected into cells was normalized with the vector construct. In parallel, transfected cells were grown in suspension for 48 h and then subjected to Cell Death ELISA assay (B). C. A549 can HOP-92 cells were transfected with Bit1 cyto or GFP-TLE1 construct. 48 h post-transfection, cells were harvested and analyzed for apoptosis by Cell Death Elisa. D. A549 cells were transfected with control- or TLE1 specific siRNAs, and 48 h later, cells were subjected to immunoblotting with antibodies against TLE1 and B-actin to confirm the knockdown of TLE1 expression. E. A549 cells were transfected with vector or Bit1 mito construct, and 24 hr later, the mitochondrial Bit1 treated cells were transfected with control- or TLE1-siRNAs as indicated. Cells were then cultured in suspension for 48 hr and subjected to Cell Death Elisa. In B, C, and E, three independent experiments were performed in triplicates, * indicates p<0.05 by Student's t test.

Article Snippet: To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene).

Techniques: Transfection, Construct, Expressing, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: PLoS ONE

Article Title: The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

doi: 10.1371/journal.pone.0101564

Figure Lengend Snippet: A. and B. A549 cells were transfected with GFP-TLE1 together with N-terminally myc tagged Bit1 construct. The amount of DNA was normalized with the empty vector in each transfection. 24 h post-transfection, cells were harvested and cell extracts were prepared, immunoprecipitated with agarose-immobilized anti-myc, and immunoblotted with anti-AES and anti-myc antibodies (A). In parallel, transfected cells were subjected to nuclear isolation as indicated in the . The resulting nuclear fraction was run on SDS-PAGE and western blotted with an anti-AES antibody. The Histone H2B was used as a nuclear marker (B). C. A549 cells were transfected with vector or GFP-TLE1 construct, and 24 h post-transfection, cells were subjected to immunoprecipitation with agarose-immobilized anti-GFP and western blotting with anti-AES and anti-GFP antibodies.

Article Snippet: To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene).

Techniques: Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Isolation, SDS Page, Western Blot, Marker

Journal: PLoS ONE

Article Title: The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

doi: 10.1371/journal.pone.0101564

Figure Lengend Snippet: A. A549 derived control shRNA and Bit1 shRNA cells (1.0×10 6 ) were injected subcutaneously in 8 week old BALB/c nude mice. Tumors were measured periodically with a calliper on the days after injection as indicated. B. and C. Mice were anesthetized and sacrificed on the day 35 after injection. Subcutaneous tumors were surgically excised, and the tumors were then photographed (the representative tumors are shown in (B) and weighed (C). In D and E, tissue sections of control shRNA and Bit1 shRNA xenografts were subjected to TUNEL assay to detect cellular apoptosis. Representative images of TUNEL stained tumor sections are shown in (C). Arrows indicate apoptotic cells characterized by a brown staining. D. TUNEL positive tumor cells in serial sections of control shRNA and Bit1 shRNA tumors were quantified. In C and E, * indicates p<0.05 as compared to control tumors (Student's t test).

Article Snippet: To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene).

Techniques: Derivative Assay, shRNA, Injection, TUNEL Assay, Staining

Journal: Molecular Oncology

Article Title: CHML is an NRF2 target gene that regulates mTOR function

doi: 10.1002/1878-0261.13194

Figure Lengend Snippet: CHML/ Rep2 is an NRF2 target gene. (A) Sequence and upstream location of putative CHML antioxidant response element (ARE; −1622 ATGACTCAGCA ‐1612 ). Biotinylated wild type (WT; ATGACTCAGCA) or mutant (MT; AACTCTCACGA) ARE‐containing oligonucleotides were incubated with A549 WT or NRF2 knockout (KO) cell lysate and ARE‐bound proteins were pulled down using streptavidin beads. NRF2 protein levels were assessed via immunoblot analysis. GAPDH was used as an internal loading control. (B) ChIP‐PCR of NRF2‐bound DNA immunoprecipitated from A549 WT cells using an anti‐NRF2 antibody. A region of the CHML promoter containing the putative ARE was amplified and compared to an IgG control. (C) A549 WT and NRF2 KO or H1299 WT and KEAP1 KO cells were co‐transfected with 1 µg of a plasmid encoding a Firefly luciferase under the control of either a WT or MT‐ CHML ARE‐driven promoter, as well as 1 µg of a Renilla luciferase plasmid under the control of a universal promoter as an internal control and subjected to a dual luciferase activity assay. Data = mean ± SD. n = 3 per group. * P < 0.05 compared to WT control. Unpaired student’s t‐test. (D‐H) Immunoblot analysis of NRF2, Rep2, and NQO1 protein levels in BEAS‐2B or H1299 WT cells treated with 5 µ m sulforaphane (SF) for 16 h (D‐E), H1299 WT vs. KEAP1 KO cells (F), A549 WT cells treated for 16 h with 40 n m Brusatol (G), or A549 WT vs. NRF2 KO cells (H). (I) CHML mRNA levels in A549 WT vs. NRF2 KO cells. Data = mean ± SD. n = 3 per group. * P < 0.05 compared to A549 WT control. Unpaired student’s t‐test. (J‐L) Immunoblot analysis of NRF2, Rep2, and NQO1 protein levels in MDA‐231 and A375 cells treated with SF for 16 h (J‐K) or A375 cells transfected with 1 µg of an NRF2 plasmid for 24 h (L). All groups for immunoblot analysis were performed in triplicate, and experiments were repeated two times to ensure validity of results.

Article Snippet: For live cell imaging, A549 WT cells transfected with 1 µg of a CHML encoding plasmid (Origene) for 24 h or treated with CHML siRNA for 72 h were then transfected with 1 µg of the mRFP‐GFP‐LC3 tandem fluorescent reporter plasmid using Lipofectamine 3000 according to the manufacturer’s instructions.

Techniques: Sequencing, Mutagenesis, Incubation, Knock-Out, Western Blot, Immunoprecipitation, Amplification, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Molecular Oncology

Article Title: CHML is an NRF2 target gene that regulates mTOR function

doi: 10.1002/1878-0261.13194

Figure Lengend Snippet: CHML/ Rep2 knockdown decreases A549 proliferation and migration. (A) Immunoblot analysis of Rep2 protein levels in A549 WT cells following treatment with 5 n m of either NT or CHML siRNA for 72 h. (B) Representative images and (C) quantification of percent confluence (indicator of cell proliferation) following knockdown. Scale bar = 100 μm. (D) MTT assay for cell viability of A549 cells transfected with 5 n m of either NT or CHML siRNA for 24 h. (E) Representative images of a scratch assay (indicator of cell migration) following Rep2 knockdown and Bru treatment. Scale bar = 200 μm. (F) Quantification of % wound closure over the 24 h brusatol treatment period from (E). Data = mean ± SD. n = 5 per group. * P < 0.05 compared to control siRNA group. Unpaired student’s t‐test. All experiments were repeated two times to ensure validity of results.

Article Snippet: For live cell imaging, A549 WT cells transfected with 1 µg of a CHML encoding plasmid (Origene) for 24 h or treated with CHML siRNA for 72 h were then transfected with 1 µg of the mRFP‐GFP‐LC3 tandem fluorescent reporter plasmid using Lipofectamine 3000 according to the manufacturer’s instructions.

Techniques: Migration, Western Blot, MTT Assay, Transfection, Wound Healing Assay

Journal: Molecular Oncology

Article Title: CHML is an NRF2 target gene that regulates mTOR function

doi: 10.1002/1878-0261.13194

Figure Lengend Snippet: Knockdown of CHML/ Rep2 decreases protein levels without affecting autophagy. (A) Immunoblot analysis of Rep2, p62, and LC3‐I/II protein levels in A549 cells treated with 5 n m of either NT or CHML /Rep2 siRNA for 72 h. (B) RFP‐GFP‐LC3 tandem fluorescence analysis of autophagy flux following siRNA knockdown same as (A). Yellow puncta = autophagosomes, red puncta = autolysosomes. Scale bar = 10 μm. (C) Immunoblot analysis of Rep2, p62, and LC3‐I/II protein levels in A549 cells transfected with 1 µg of either an empty vector (EV) or WT‐ CHML (Rep2 OE) for 24 h. (D) RFP‐GFP‐LC3 tandem fluorescent analysis of autophagy flux following Rep2 overexpression same as (C). Scale bar = 10 μm. (E) Immunoblot analysis of Rep2, LAMP1, Atg7, p62, Snap29, Rab7, and LC3‐I/II protein levels following Rep2 knockdown for 72 h. All groups for immunoblot and immunofluorescence analysis were performed in triplicate, and experiments were repeated two times to ensure validity of results.

Article Snippet: For live cell imaging, A549 WT cells transfected with 1 µg of a CHML encoding plasmid (Origene) for 24 h or treated with CHML siRNA for 72 h were then transfected with 1 µg of the mRFP‐GFP‐LC3 tandem fluorescent reporter plasmid using Lipofectamine 3000 according to the manufacturer’s instructions.

Techniques: Western Blot, Fluorescence, Transfection, Plasmid Preparation, Over Expression, Immunofluorescence

Journal: Molecular Oncology

Article Title: CHML is an NRF2 target gene that regulates mTOR function

doi: 10.1002/1878-0261.13194

Figure Lengend Snippet: Knockdown of CHML/ Rep2 decreases, whereas overexpression increases, mTOR activation. (A) Immunoblot analysis of phosphorylated mTOR (S2448), mTOR, phosphorylated S6K (S371), S6K, phosphorylated S6 (S235/236), and S6 protein levels in A549 cells treated with 5 n m NT or CHML /Rep2 siRNA for 72 h., then amino acid starved for 30 min., and left untreated or treated for 15 min. with 1 m m l ‐arginine (R) to activate the mTOR pathway. (B) Endogenous immunofluorescence of mTOR (red) colocalization with LAMP1‐positive lysosomes (green) following knockdown and l ‐arginine treatment same as (A). Scale bar = 10 μm. (C) Immunoblot analysis of the same proteins assessed in (A) in A549 WT cells transfected with 1 µg of either empty vector or WT‐ CHML for 24 h, then amino acid starved for 30 min., and left untreated or treated for 15 min. with 1 m m l ‐arginine (R). (D) Endogenous immunofluorescence of mTOR (red) colocalization with LAMP1‐positive lysosomes (green) following Rep2 overexpression and l ‐arginine treatment same as (C). Scale bar = 10 μm. All groups for immunoblot and immunofluorescence analysis were performed in triplicate, and experiments were repeated two times to ensure validity of results. (E) RT‐PCR of mTOR , S6KB , S6 , and CHML mRNA levels following Rep2 knockdown (72 h) or overexpression (24 h). Data = mean ± SD. n = 3 per group. * P < 0.05 compared to NT siRNA or EV group. Unpaired student’s t‐test. (F) SILAC determination of protein synthesis following knockdown with CHML /Rep2 siRNA and treatment with heavy arginine or heavy lysine for 24 h. Data = mean ± SD. n = 6 per group. * P < 0.05 compared to NT siRNA group. (G) Immunoblot analysis of mTOR pathway proteins in A549 WT vs. NRF2 KO cells treated with Rep2 siRNA or transfected with a WT‐ CHML plasmid as described above. All groups for immunoblot analysis were performed in triplicate, and experiments were repeated two times to ensure validity of results.

Article Snippet: For live cell imaging, A549 WT cells transfected with 1 µg of a CHML encoding plasmid (Origene) for 24 h or treated with CHML siRNA for 72 h were then transfected with 1 µg of the mRFP‐GFP‐LC3 tandem fluorescent reporter plasmid using Lipofectamine 3000 according to the manufacturer’s instructions.

Techniques: Over Expression, Activation Assay, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction