Journal: Oncotarget

Article Title: N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

doi: 10.18632/oncotarget.6827

Figure Lengend Snippet: ( A ) Retrovirally overexpressed Bcl-2 in A549 cells was examined by western blot. ( B ) Cell viability was measured 48 hours after C12 or actinomycin D (ActD) exposure. ( C ) Caspase-3/7 activities were determined following 2 hour exposure to C12 and 24 hour exposure to actinomycin D. ( D ) C12's effect on mitochondrial membrane potential is independent of Bcl-2. A549-vector and A549-Bcl-2-overexpressing cells were loaded with JC1, and its fluorescence was measured using imaging microscopy during the treatment with 100 μM C12 and 5 μM FCCP. Typical results from three independent experiments are shown. ( E ) C12 caused equivalent depolarization of mitochondrial potential in vector and Bcl-2-overexpressing A549 cells. All data are shown as mean ± standard deviation of three independent experiments. Asterisks indicate P < 0.05 (*); ns, no significant by student's unpaired t test.

Article Snippet: NSCLC cell line A549 cells were obtained from ATCC, which were produced from adenocarcinomic alveolar basal epithelial cells of a lung cancer patient [ ].

Techniques: Western Blot, Membrane, Plasmid Preparation, Fluorescence, Imaging, Microscopy, Standard Deviation

Journal: Oncotarget

Article Title: N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

doi: 10.18632/oncotarget.6827

Figure Lengend Snippet: ( A – B ) Growth of A549-vector (A) and A549-Bcl-2-overexpressing tumors (B) in athymic nude mice treated with the vehicle control or C12 (15 mg/kg/day). Data are shown as mean ± standard deviation of tumor volumes of 8 animals in either vehicle control (DMSO) or C12-treated groups. ( C ) Apoptotic cells in tumor sections were identified by TUNEL assay. Representative images of tumor sections from DMSO- and C12-treated mice are shown. Scale bar, 60 μm. ( D ) Summary of the data shown in (C). More apoptotic cells were detected in tumors of C12-treated mice than those of vehicle control mice. Mean ± standard deviation of three independent tumor sections. For all the data, asterisk indicates P < 0.05 (*) by student's unpaired t test.

Article Snippet: NSCLC cell line A549 cells were obtained from ATCC, which were produced from adenocarcinomic alveolar basal epithelial cells of a lung cancer patient [ ].

Techniques: Plasmid Preparation, Control, Standard Deviation, TUNEL Assay

Journal: Oncotarget

Article Title: N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

doi: 10.18632/oncotarget.6827

Figure Lengend Snippet: ( A ) Expression of PON2 in NSCLC tissue specimens and corresponding adjacent normal tissues from 11 patients were evaluated by western blot. Samples 1–4, 6, 8, 9, 11 were from adenocarcinoma patients, whereas samples 5, 7, 10 were from squamous cell carcinoma patients. T, tumor; N, normal. ( B ) The intensities of bands in (A) were quantified using ImageJ software (NIH). To normalize loading variation, the relative levels of PON2 were calculated by dividing the PON2 value into the corresponding value for actin. The data were shown as a ratio of PON2 levels in a tumor tissue sample versus its corresponding normal tissue, and the value bigger than 1 indicates that PON2 expression is increased in tumor tissues. Differential expression of PON2 in tumor versus normal tissues is significant with the value of “P” smaller than 0.01 as calculated by student's paired t test. ( C ) The expression of PON2 and PON3 in primary HBE cells and their transformed counterparts was determined by western blot.

Article Snippet: NSCLC cell line A549 cells were obtained from ATCC, which were produced from adenocarcinomic alveolar basal epithelial cells of a lung cancer patient [ ].

Techniques: Expressing, Western Blot, Software, Quantitative Proteomics, Transformation Assay

Journal: Oncotarget

Article Title: N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

doi: 10.18632/oncotarget.6827

Figure Lengend Snippet: ( A ) PON2 expression in A549 cells was stably reduced by shRNA. The expression levels of PON2 were determined by western blot. ( B ) C12 induced less apoptosis in A549 cells with reduced PON2 expression than in control vector cells, which is opposite to the effects of actinomycin D or tunicamycin. Cell death was assessed after 32 hour incubation. ( C ) Upon treatment with different doses of C12 for 32 hours, less caspase-3/7 activation was detected in cells with reduced PON2 expression than control vector cells. ( D ) Actinomycin D or tunicamycin induced more caspase-3/7 activation in A549 cells with higher PON2 expression following 48 hour treatment. (E-F) Growth of A549-vector tumors ( E ) and A549 tumors with reduced PON2 expression ( F ) in athymic nude mice treated with vehicle control or C12 (15 mg/kg/day). Data are mean ± standard deviation of tumor volumes of 7 animals in each group. Asterisks indicate P values of < 0.05 (*) by student's unpaired t test. ( G ) Stable reduction of PON2 expression in NCI-H1299 cells was evaluated by western blot. ( H ) C12 induced less cell death in NCI-H1299 cells with reduced PON2 following 24 hour treatment. ( I ) More cell death was detected in NCI-H1299 cells with lower PON2 expression following 24 hour exposure of actinomycin D. ( J ) Less apoptosis was detected in NCI-H1299 cells with reduced PON2 expression than control vector cells induced by C12, which is opposite to the effect of actinomycin D. Cell death was assessed after 24 hour incubation. All data shown are mean ± standard deviation of three independent experiments performed in triplicate. Asterisks indicate P values of < 0.05 (*) or < 0.01 (**) by student's unpaired t test.

Article Snippet: NSCLC cell line A549 cells were obtained from ATCC, which were produced from adenocarcinomic alveolar basal epithelial cells of a lung cancer patient [ ].

Techniques: Expressing, Stable Transfection, shRNA, Western Blot, Control, Plasmid Preparation, Incubation, Activation Assay, Standard Deviation

Journal: Oncotarget

Article Title: N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

doi: 10.18632/oncotarget.6827

Figure Lengend Snippet: ( A ) Human or murine PON3 cDNA was stably overexpressed in A549 cells with reduced PON2 expression by retroviral infection. Expression levels of PON3 were determined by western blot. ( B – C ) After treating with different doses of C12 for 24 hours, more cell death (B) and caspase-3/7 activation (C) were detected in PON2-knockdown A549 cells with increased mouse and human PON3 expression comparing to vector control and parental cells. ( D ) Stable overexpression of human or murine PON3 in PON2-knockdown NCI-H1299 cells was examined by western blot. ( E – F ) C12 induced more cell death (E) and caspase-3/7 activation (F) in PON2-knockdown NCI-H1299 cells expressing mouse and human PON3 after 24 hours treatment. All data shown are mean ± standard deviation of three independent experiments performed in triplicate. Asterisks indicate P values of < 0.05 (*) or < 0.01 (**) by student's unpaired t test.

Article Snippet: NSCLC cell line A549 cells were obtained from ATCC, which were produced from adenocarcinomic alveolar basal epithelial cells of a lung cancer patient [ ].

Techniques: Stable Transfection, Expressing, Retroviral, Infection, Western Blot, Activation Assay, Knockdown, Plasmid Preparation, Control, Over Expression, Standard Deviation

Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: miR‐100 inhibited NSCLC cell proliferation. ( a ) qRT‐PCR was used to measure miR‐100 expressions in NSCLC cells. ( b , c ) miR‐100 overexpression or inhibition was confirmed by qRT‐PCR. ( d , e ) MTT assays were performed to detect the functions of miR‐100 in NSCLC cell proliferation. * P < 0.05, ** P < 0.01, *** P < 0.001 (d: NC and miR‐100 mimics; e: NC and miR‐100 inhibitor).

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Quantitative RT-PCR, Over Expression, Inhibition

Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: miR‐100 suppressed the cell invasion and migration abilities of NSCLC cells. ( a , b ) The impacts of miR‐100 restoration on NSCLC cell invasion and migration were determined using transwell assays. ( c , d ) Transwell assay was performed to determine invasion and migration capacities of miR‐100 suppressed NSCLC cells. ** P < 0.01, *** P < 0.001.

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Migration, Transwell Assay

Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: HOXA1 was a direct target of miR‐100 in NSCLC cells. ( a )The WT and MUT binding sites of miR‐100 on HOXA1 3′‐UTR. ( b ) Dual‐luciferase reporter assay was used to confirm the association between miR‐100 and HOXA1 in NSCLC cells ( NC and miR‐100 mimics). ( c , d ) Regulatory effects of miR‐100 on HOXA1 expressions in NSCLC cells. * P < 0.05, ** P < 0.01.

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Binding Assay, Luciferase, Reporter Assay

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 1. A549 cell growth inhibition by soy isoflavones and radiation. A, Soy dose titration. Cells were treated for 4 days with soy at concen- tration of 0, 10, 25, 50, 75, and 100 M, and viable cells were counted. B, Radiation dose titra- tion. Cells were irradiated with doses of 1, 2, 3, 5, 6, and 8 Gy, and viable cells were counted. Bars represent the mean number of cells of duplicate wells calculated as the percentage of control cells SE. *p 0.001 relative to control group.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Inhibition, Titration, Irradiation, Control

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 2. Survival fraction of A549 cells treated with soy and/or radiation. A, Survival of cells treated with soy. A549 cells were treated for 72 hours with soy at concentrations of 5, 7.5, 10, 15, 20, 25, and 30 M and plated in a clono- genic assay. B, Survival of cells treated with radia- tion. A549 cells were irradiated with different doses of 1 to 10 Gy and plated in a clonogenic assay. Inset: the radiation dose survival curve is fitted by a nonlinear least-square regression ac- cording to the linear-quadratic formula (SAS JMPV7). C, Survival of cells treated with soy and radiation. A549 cells were pretreated with soy at 5, 7.5, and 10 M for 72 hours, then irradiated at 3 Gy and plated in a clonogenic assay in the pres- ence of the same concentration of soy. Inset in (C) shows the products of SF(S) and SF(R), which are the survival fraction of soy treatment at 5, 7.5, and 10 M multiplied by the survival fraction of 3 Gy radiation SF(R). These products are greater than the survival fraction of cells treated with combined soy and radiation [SF(S R)] at each concentration of soy tested. In panels A, B, and C, bars represent the mean survival fraction of tripli- cate wells SE. *p 0.001 represent significance of each treatment group relative to control group.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Irradiation, Clonogenic Assay, Concentration Assay, Control

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 3. Increased DNA double-strand breaks in A549 cells treated with soy and radiation. A549 cells were treated with soy at 30 M for 24 hours then irradiated with 3 Gy and tested at 1 hour and 24 hours after radiation, corre- sponding to 24 and 48 hours of soy treatment, respectively. Control cells (con), cells treated with soy (soy), cells treated with radiation (Rad), and both combined (Soy Rad) were processed for immunostaining of -H2AX to detect DNA double-strand breaks (DSBs). A, -H2AX staining at 1 hour after radiation or 24 hours after soy treatment. B, -H2AX staining at 24 hours after radiation or 48 hours after soy treatment.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Irradiation, Control, Immunostaining, Staining

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 4. Effect of soy and radiation on HIF-1 and -H2AX in A549 cells. Cells were either untreated (Con for control) or pretreated with 30 M soy, 3 Gy radiation (Rad), or combined (Soy Rad). Cells were pretreated with soy for 24 hours, and then cells were irradiated. At 1 hour or 24 hours post radiation, corresponding to 24 and 48 hours soy treatment, cells were processed for Western blot analysis to detect -H2AX and HIF-1. -Actin protein levels served as a loading control.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Control, Irradiation, Western Blot

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 5. Pretreatment with soy inhibits APE-1/Ref-1 expression and HIF-1 and NF-B DNA-binding activity induced by radiation. A549 cells were pretreated with 30 M soy for 72 hours then irradiated at 3 Gy. At 5 hours after radiation, nuclear extracts were subjected to Western blot analysis or electrophoretic mobility shift assay (EMSA). Untreated control (Con), soy alone (Soy), radiation 3 Gy (Rad), and soy combined with radiation (Soy Rad). A, Nuclear expression of APE1/Ref-1. Nuclear extracts (10 g) were run on 10% Western gel and blotted against APE-1/Ref-1 Ab. B, HIF-1 DNA-binding activity. Nuclear extracts were incubated with IRDye-700-labeled HIF-1 oligonucleotide and subjected to EMSA. C, NF-B DNA-binding activ- ity. Nuclear extracts were incubated with IRDye-700-labeled NF-B oligonucleotide and subjected to EMSA. In panels A, B, and C, data are also presented as the mean integrated density value (I.D.V.) of the band/g protein loaded (SE). *p 0.05 and ** p 0.001 represent significance of each treatment group relative to control group. Dotted lines and p values in I.D.V. diagrams show the difference between cells treated with radiation soy versus radiation only. Western blot for retinoblas- toma (Rb) protein in the nuclear extract was performed as an internal loading control.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Expressing, Binding Assay, Activity Assay, Irradiation, Western Blot, Electrophoretic Mobility Shift Assay, Control, Incubation, Labeling

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 6. Effect of APE1/Ref-1 inhibitors on -H2AX ex- pression in A549 cells. A549 cells were pretreated with E3330 (50 M), methoxyamine (20 mM), or soy isoflavones (30 M) for 24 hours and then irradiated at 3 Gy. Cells were further incubated for 1 hour and 24 hours after radiation and then processed for -H2AX immunostaining. Control cells (Control), cells treated with radiation (Rad) and both combined (Soy Rad), and cells treated with E3330 and radiation (E3330 Rad) or methoxyamine and radiation (Methox Rad) were processed for immunostaining of -H2AX to detect DNA double-strand breaks (DSBs). A, Mean number of -H2AX foci per cell. B, Mean percent of -H2AX-positive cells per field. In both (A) and (B) panels, bars represent the mean values SD calculated from three different fields.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Irradiation, Incubation, Immunostaining, Control

Journal: Journal of Thoracic Oncology

Article Title: Soy Isoflavones Augment Radiation Effect by Inhibiting APE1/Ref-1 DNA Repair Activity in Non-small Cell Lung Cancer

doi: 10.1097/jto.0b013e31821034ae

Figure Lengend Snippet: FIGURE 7. Effect of APE1/Ref-1 in- hibitors on A549 cell growth. A549 cells were pretreated either with E3330 (25 M) or methoxyamine (10 mM), or soy isoflavones (15 M) or in combination (E3330 soy or Me- thox soy) for 24 hours and then irradiated with 3 Gy. Cells were fur- ther incubated, and viable cells were counted at 3 days after radiation. Sin- gle treatment and combined treat- ments of E3330, methoxyamine, and soy isoflavones are also shown. Bars represent the mean number of cells of duplicate wells calculated as the per- centage of control cells SE. Dotted lines and p values show the difference between cells treated with single and combined treatments; *p 0.05 and **p 0.005.

Article Snippet: The human non-small cell lung carcinoma (NSCLC) A549 and H1650 cell lines were purchased from ATCC (Manassas, VA) and grown in F-12K culture medium (CM) containing 7% heat-inactivated fetal bovine serum with supplements.

Techniques: Irradiation, Incubation, 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: Oncotarget

Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

doi: 10.18632/oncotarget.21781

Figure Lengend Snippet: (A-C) Immunofluorescence (IF) analysis of A549 cells relative to the unstained controls (A: only secondary antibody) without membrane permeabilization (B) shows positive Shh (green) and nuclear staining (blue, DAPI). White arrows: positive membranous Shh staining in relatively few cells. (C) IF analysis of A549 cells with membrane permeabilization (Triton X-100) shows positive Shh (green) and nuclear staining (blue, DAPI) in a majority of the cells probed. (D) Flow cytometric analysis of A549 cells without membrane permeabilization probed for Shh (0.18%). (E) Flow cytometric analysis of A549 cells with membrane permeabilization (Tween 20) probed for Shh (70.12%). (F) IF analysis of sorted A549 cells without membrane permeabilization shows strong positive membranous Shh staining (green, white arrows) in Shh+ cells and low/no staining in Shh- cells/controls without the primary antibody. Red: membranous staining (lipophilic dye); blue: nuclear staining (DAPI). (G) Shh gene expression analysis by ddPCR in A549 Shh+ and Shh- cells. (H) Percentage of Shh+ cells (%, mean ±SD) in several NSCLC cell lines.

Article Snippet: All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Membrane, Staining, Gene Expression

Journal: Oncotarget

Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

doi: 10.18632/oncotarget.21781

Figure Lengend Snippet: (A) Immunoblot of supernatants from non-transfected Shh-sorted A549 cells probed for the Sonic Hedgehog (Shh) protein, with secreted MMP2 as a loading control. (B) Schematic representation of Shh constructs showing the sizes and locations of N-term HA and C-term FLAG tags stably expressed in NSCLC cells. (C) Immunofluorescence analysis of H838 cells showing cytosolic and membrane staining of N-term, C-term, wild-type Shh and C198A Shh constructs probed for the presence of HA (red) and FLAG (yellow). (D) NSCLC cell lines (A549 and H838) used in (C), analyzed for increases in viability (MTS assay) relative to the vector control after 4 days ( ** p<0.01). (E) Supernatants from NSCLC cells were applied to parental cells and analyzed as in D ( ** p<0.01).

Article Snippet: All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Transfection, Control, Construct, Stable Transfection, Immunofluorescence, Membrane, Staining, MTS Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

doi: 10.18632/oncotarget.21781

Figure Lengend Snippet: (A) Correlation between the percentage of Shh+ cells (%) and cisplatin IC 50 in NSCLC cell lines. (B) Percentage of Shh+ cells (%) in A549 cells treated with cisplatin (1 mM, 72h). †dead cells; ‡Shh- cells; ₮Shh+ cells (5% of live cells). (C) Percentage of Shh+ cells (%) in A549 cells treated by cisplatin (600 μM and 1 mM, 72h). * p<0.05; ** p<0.01, compared to baseline. (D) Shh expression level (qRT-PCR) in A549 cells treated with cisplatin (600 μM and 1 mM, 72h) (normalized to PBS-treated cells, log-scale). ** p<0.01, compared to PBS. (E) Percentage of Shh+ cells (%) in A549 cells treated with GDC0449 (40 μM, 72h). †dead cells; ‡Shh- cells; ₮Shh+ cells (0%). (F) Shh expression level (qRT-PCR) in A549 cells treated with GDC0449 (20 μM and 40 μM, 72h) (normalized to DMSO-treated cells). ** p<0.01, compared to DMSO. (G) MTS assay of A549 cells treated with DSMO or GDC0449 (40 μM, 72h). ** p<0.01, compared to day 0. (H) Percentage of Shh+ cells (%) in A549 cells treated with cisplatin and DMSO/GDC0449 (40 μM, 72h) or docetaxel and DMSO/GDC0449 (40 μM, 72h). ** p<0.01, compared to cisplatin/docetaxel and DMSO. (I) Percentage of Shh+ cells (%) in A549 xenograft model treated with cisplatin (CPT, 10 mg/kg, IV weekly), followed by vehicle or GDC0449 (20 mg/kg, IP daily). * p<0.05.

Article Snippet: All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, MTS Assay